Metabolic shift toward ketosis in asocial cavefish increases social-like affinity

Iwashita M, Tran A, Garcia M, Cashon J, Burbano D, Salgado V, Hasegawa M, Balmilero-Unciano, Politan K, Wong M, Lee R, Yoshizawa M

BMC Biology. 2023 Oct 16

Social affinity and collective behavior are nearly ubiquitous in the animal kingdom, but many lineages feature evolutionarily asocial species. These solitary species may have evolved to conserve energy in food-sparse environments. However, the mechanism by which metabolic shifts regulate social affinity is not well investigated. In this study, we used the Mexican tetra (Astyanax mexicanus), which features riverine sighted surface (surface fish) and cave-dwelling populations (cavefish), to address the impact of metabolic shifts on asociality and other cave-associated behaviors in cavefish, including repetitive turning, sleeplessness, swimming longer distances, and enhanced foraging behavior. After 1 month of ketosis-inducing ketogenic diet feeding, asocial cavefish exhibited significantly higher social affinity, whereas social affinity regressed in cavefish fed the standard diet. The ketogenic diet also reduced repetitive turning and swimming in cavefish. No major behavioral shifts were found regarding sleeplessness and foraging behavior, suggesting that other evolved behaviors are not largely regulated by ketosis. We further examined the effects of the ketogenic diet via supplementation with exogenous ketone bodies, revealing that ketone bodies are pivotal molecules positively associated with social affinity. Our study indicated that fish that evolved to be asocial remain capable of exhibiting social affinity under ketosis, possibly linking the seasonal food availability and sociality.

Environmental microbes promote phenotypic plasticity in reproduction and sleep behaviour

Téfit MA, Budiman T, Dupriest A, Yew JY

Molecular Ecology. 2023 Sep 29

Use of MICRO, MGAL, and InSTAR Core Facilities

The microbiome has been hypothesized as a driving force of phenotypic variation in host organisms that is capable of extending metabolic processes, altering development and in some cases, conferring novel functions that are critical for survival. Only a few studies have directly shown a causal role for the environmental microbiome in altering host phenotypic features. To assess the extent to which environmental microbes induce variation in host life-history traits and behaviour, we inoculated axenic Drosophila melanogaster with microbes isolated from drosophilid populations collected from two different field sites and generated two populations with distinct bacterial and fungal profiles. We show that microbes isolated from environmental sites with modest abiotic differences induce large variation in host reproduction, fatty acid levels, stress tolerance and sleep behaviour. Importantly, clearing microbes from each experimental population removed the phenotypic differences. The results support the causal role of environmental microbes as drivers of host phenotypic plasticity and potentially, rapid adaptation and evolution.

Post-Diapause DNA Replication during Oogenesis in a Capital-Breeding Copepod

Monell KJ, Roncalli V, Hopcroft RR, Hartline DK, Lenz PH

Integrative Organismal Biology. 2023 Jun 12

Use of MICRO/BEMF Core Facility

In high-latitude environments where seasonal changes include periods of harsh conditions, many arthropods enter diapause, a period of dormancy that is hormonally regulated. Diapause is characterized by very low metabolism, resistance to environmental stress, and developmental arrest. It allows an organism to optimize the timing of reproduction by synchronizing offspring growth and development with periods of high food availability. In species that enter dormancy as pre-adults or adults, termination of diapause is marked by the resumption of physiological processes, an increase in metabolic rates and once transitioned into adulthood for females, the initiation of oogenesis. In many cases, individuals start feeding again and newly acquired resources become available to fuel egg production. However, in the subarctic capital-breeding copepod Neocalanus flemingeri, feeding is decoupled from oogenesis. Thus, optimizing reproduction limited by fixed resources such that all eggs are of high quality and fully-provisioned, requires regulation of the number of oocytes. However, it is unknown if and how this copepod limits oocyte formation. In this study, the phase in oocyte production by post-diapause females that involved DNA replication in the ovary and oviducts was examined using incubation in 5-Ethynyl-2'-deoxyuridine (EdU). Both oogonia and oocytes incorporated EdU, with the number of EdU-labeled cells peaking at 72 hours following diapause termination. Cell labeling with EdU remained high for two weeks, decreasing thereafter with no labeling detected by four weeks post diapause, and three to four weeks before spawning of the first clutch of eggs. The results suggest that oogenesis is sequential in N. flemingeri with formation of new oocytes starting within 24 hours of diapause termination and limited to the first few weeks. Lipid consumption during diapause was minimal and relatively modest initially. This early phase in the reproductive program precedes mid-oogenesis and vitellogenesis 2, when oocytes increase in size and accumulate yolk and lipid reserves. By limiting DNA replication to the initial phase, the females effectively separate oocyte production from oocyte provisioning. A sequential oogenesis is unlike the income-breeder strategy of most copepods in which oocytes at all stages of maturation are found concurrently in the reproductive structures.

Geology and land use shape nitrogen and sulfur cycling groundwater microbial communities in Pacific Island aquifers

Watson SJ, Arisdakessian C, Petelo M, Keliipuleole K, Tachera DK, Okuhata BK, Dulai H, Frank KL

ISME Communications. 2023 Jun 07

Resource-constrained island populations have thrived in Hawai'i for over a millennium, but now face aggressive new challenges to fundamental resources, including the security and sustainability of water resources. Characterizing the microbial community in groundwater ecosystems is a powerful approach to infer changes from human impacts due to land management in hydrogeological complex aquifers. In this study, we investigate how geology and land management influence geochemistry, microbial diversity and metabolic functions. We sampled a total of 19 wells over 2-years across the Hualālai watershed of Kona, Hawai'i analyzing geochemistry, and microbial communities by 16S rRNA amplicon sequencing. Geochemical analysis revealed significantly higher sulfate along the northwest volcanic rift zone, and high nitrogen (N) correlated with high on-site sewage disposal systems (OSDS) density. A total of 12,973 Amplicon Sequence Variants (ASV) were identified in 220 samples, including 865 ASVs classified as putative N and sulfur (S) cyclers. The N and S cyclers were dominated by a putative S-oxidizer coupled to complete denitrification (Acinetobacter), significantly enriched up to 4-times comparatively amongst samples grouped by geochemistry. The significant presence of Acinetobacter infers the bioremediation potential of volcanic groundwater for microbial-driven coupled S-oxidation and denitrification providing an ecosystem service for island populations dependent upon groundwater aquifers.

Herbivorous fish microbiome adaptations to sulfated dietary polysaccharides

Podell S, Oliver A, Wegley Kelly L, et al.

Applied and Environmental Microbiology. 2023 May 03

Use of MGAL Core Facility

Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three different species of macro-algivorous Hawaiian kyphosids have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Assembled metagenomes from terrestrial ruminant microbiota containing carbohydrate-utilizing bacterial taxa closely related to those found in kyphosid fish, including Alistipes-related Bacteroidota and Clostridia-related Bacillota, were used as negative controls to identify shared features common to both marine and non-marine saccharolytic intestinal bacteria. This strategy provided the statistical power necessary to distinguish macro-algivorous digestion-specific activities from more generalized aspects of carbohydrate metabolism. Gene co-localization patterns of enriched digestive enzyme families on assembled contigs were used to identify polysaccharide utilization locus associations, and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides, providing foundational resources for future investigations into suppression of coral reef macroalgal overgrowth, fish host physiology, the use of macroalgal feedstocks in terrestrial and aquaculture animal feeds, and the bioconversion of macroalgae biomass into value-added commercial fuel and chemical products.

Genomics data analysis via spectral shape and topology

Amézquita EJ, Nasrin F, Storey KM, Yoshizawa M

PLOS ONE. 2023 Apr 26

Mapper, a topological algorithm, is frequently used as an exploratory tool to build a graphical representation of data. This representation can help to gain a better understanding of the intrinsic shape of high-dimensional genomic data and to retain information that may be lost using standard dimension-reduction algorithms. We propose a novel workflow to process and analyze RNA-seq data from tumor and healthy subjects integrating Mapper, differential gene expression, and spectral shape analysis. Precisely, we show that a Gaussian mixture approximation method can be used to produce graphical structures that successfully separate tumor and healthy subjects, and produce two subgroups of tumor subjects. A further analysis using DESeq2, a popular tool for the detection of differentially expressed genes, shows that these two subgroups of tumor cells bear two distinct gene regulations, suggesting two discrete paths for forming lung cancer, which could not be highlighted by other popular clustering methods, including t-distributed stochastic neighbor embedding (t-SNE). Although Mapper shows promise in analyzing high-dimensional data, tools to statistically analyze Mapper graphical structures are limited in the existing literature. In this paper, we develop a scoring method using heat kernel signatures that provides an empirical setting for statistical inferences such as hypothesis testing, sensitivity analysis, and correlation analysis.

Genomic determination of breeding systems and trans-specific evolution of HD MAT genes in suilloid fungi

Ke YH, Branco S, Bazzicalupo AL, Nguyen NH, et al.

Genetics. 2023 Apr 18

Studying the signatures of evolution can help to understand genetic processes. Here we demonstrate how the existence of balancing selection can be used to identify the breeding systems of fungi from genomic data. The breeding systems of fungi are controlled by self-incompatibility loci that determine mating types between potential mating partners, resulting in strong balancing selection at the loci. Within the fungal phylum Basidiomycota, two such self-incompatibility loci, namely HD MAT locus and P/R MAT locus, control mating types of gametes. Loss of function at one or both MAT loci results in different breeding systems and relaxes the MAT locus from balancing selection. By investigating the signatures of balancing selection at MAT loci, one can infer a species' breeding system without culture-based studies. Nevertheless, the extreme sequence divergence among MAT alleles imposes challenges for retrieving full variants from both alleles when using the conventional read-mapping method. Therefore, we employed a combination of read-mapping and local de novo assembly to construct haplotypes of HD MAT alleles from genomes in suilloid fungi (genera Suillus and Rhizopogon). Genealogy and pairwise divergence of HD MAT alleles showed that the origins of mating types predate the split between these two closely related genera. High sequence divergence, trans-specific polymorphism, and the deeply diverging genealogy confirm the long-term functionality and multiallelic status of HD MAT locus in suilloid fungi. This work highlights a genomics approach to studying breeding systems regardless of the culturability of organisms based on the interplay between evolution and genetics.

Effect of acetic acid bacteria colonization on oviposition and feeding site choice in Drosophila suzukii and its related species

Sato A, Yew JY, Takahashi A

bioRxiv. 2023 Mar 23 - Preprint

Use of MGAL Core Facility

Oviposition site choice has a large impact on offspring performance. Unlike other vinegar flies that colonize decaying fruits, Drosophila suzukii lay eggs into hard ripening fruits by using their enlarged and serrated ovipositors (oviscapts). This behavior has an advantage over other species by providing access to the host fruit earlier and avoiding competition. However, the larvae are not fully adapted to a low-protein diet, and the availability of intact healthy fruits is seasonally restricted. Thus, to investigate oviposition site preference for microbial growth in this species, we conducted an oviposition assay using single species of commensal Drosophila acetic acid bacteria, Acetobacter and Gluconobacter. The oviposition site preferences for media with or without bacterial growth were quantified in multiple strains of D. suzukii and its closely related species, D. subpulchrella and D. biarmipes, and a typical fermenting-fruit consumer, D. melanogaster. Our comparisons demonstrated a continuous degree of preference for sites with Acetobacter growth both within and across species, suggesting that the niche separation is notable but not complete. The preference for Gluconobacter showed large variations among replicates and no clear differences between the strains. In addition, the lack of interspecific differences in feeding site preference for Acetobacter-containing media implies that the interspecific divergence in oviposition site preference occurred independently from the feeding site preference. Our oviposition assays measuring the preference of multiple strains from each fly species for acetic acid bacteria growth revealed intrinsic properties of shared resource usage among these fruit fly species.

Fungal Hyphosphere Microbiomes Are Distinct from Surrounding Substrates and Show Consistent Association Patterns

Nguyen NH

Microbiology Spectrum. 2023 Mar 20

Mat-forming fungi are common in forest and grassland soils across the world, where their activity contributes to important soil ecological processes. These fungi maintain dominance through aggressive and abundant hyphae that modify their internal physical and chemical environments and through these modifications select for what appears to be a suite of mycophilic bacteria. Here, the bacteria associated with the fungal mats of Leucopaxillus gentianeus and Leucopaxillus albissimus from western North America are compared to adjacent nonmat substrates. Within the mats, the bacterial richness and diversity were significantly reduced, and the community composition was significantly different. The bacterial community structure between the two fungal hosts was marginally significant and indicated a shared set of bacterial associates. The genera Burkholderia, Streptomyces, Bacillus, Paenibacillus, and Mycobacterium were significantly abundant within the fungal mats and represent core members of these hypha-rich environments. Comparison with the literature from fungal mat studies worldwide showed that these genera are common and often significantly found within fungal mats, further reinforcing the concept of a mycophilic bacterial guild. These genera are incorporated into a synthesis discussion in the context of our current understanding of the nature of fungal-bacterial interactions and the potential outcomes of these interactions in soil nutrient cycling, plant productivity, and human health. IMPORTANCE Fungi and bacteria are the most abundant and diverse organisms in soils (perhaps more so than any other habitat on earth), and together these microorganisms contribute to broad soil ecosystem processes. There is a suite of bacteria that appears consistently within the physical space called the hyphosphere, the area of influence surrounding fungal hyphae. How these bacteria are selected for, how they are maintained, and what broader ecological functions they perform are subjects of interest in this relatively new field-the cross-kingdom interactions between fungi and bacteria. Understanding their cooccurrence and their interactions can open new realms of understanding in soil ecological processes with global consequences.

A Small Contribution to a Large System: The Leptin Receptor Complex

Simien JM, Orellana GE, Phan HTN, ...Haglund E

The Journal of Physical Chemistry B. 2023 Mar 13

Obesity is a classified epidemic, increasing the risk of secondary diseases such as diabetes, inflammation, cardiovascular disease, and cancer. The pleiotropic hormone leptin is the proposed link for the gut-brain axis controlling nutritional status and energy expenditure. Research into leptin signaling provides great promise toward discovering therapeutics for obesity and its related diseases targeting leptin and its cognate leptin receptor (LEP-R). The molecular basis underlying the human leptin receptor complex assembly remains obscure, due to the lack of structural information regarding the biologically active complex. In this work, we investigate the proposed receptor binding sites in human leptin utilizing designed antagonist proteins combined with AlphaFold predictions. Our results show that binding site I has a more intricate role in the active signaling complex than previously described. We hypothesize that the hydrophobic patch in this region engages a third receptor forming a higher-order complex, or a new LEP-R binding site inducing allosteric rearrangement.

Exploring Taxonomic and Functional Microbiome of Hawaiian Stream and Spring Irrigation Water Systems Using Illumina and Oxford Nanopore Sequencing Platforms

Klair D, Dobhal S, Ahmed A, Hassan ZUI, Uyeda J, Silva J, Wang K, Kim S, Alvarez AM, Arif M

Frontiers in Microbiology. 2023 Feb 17

Irrigation water is a common source of contamination that carries plant and foodborne human pathogens and provides a niche for proliferation and survival of microbes in agricultural settings. Bacterial communities and their functions in irrigation water were investigated by analyzing samples from wetland taro farms on Oahu, Hawaii using different DNA sequencing platforms. Irrigation water samples (stream, spring, and storage tank water) were collected from North, East, and West sides of Oahu and subjected to high quality DNA isolation, library preparation and sequencing of the V3-V4 region, full length 16S rRNA, and shotgun metagenome sequencing using Illumina iSeq100, Oxford Nanopore MinION and Illumina NovaSeq, respectively. Illumina reads provided the most comprehensive taxonomic classification at the phylum level where Proteobacteria was identified as the most abundant phylum in the stream source and associated water samples from wetland taro fields. Cyanobacteria was also a dominant phylum in samples from tank and spring water, whereas Bacteroidetes were most abundant in wetland taro fields irrigated with spring water. However, over 50% of the valid short amplicon reads remained unclassified and inconclusive at the species level. In contrast, Oxford Nanopore MinION was a better choice for microbe classification at the genus and species levels as indicated by samples sequenced for full length 16S rRNA. No reliable taxonomic classification results were obtained while using shotgun metagenome data. In functional analyzes, only 12% of the genes were shared by two consortia and 95 antibiotic resistant genes (ARGs) were detected with variable relative abundance. Full descriptions of microbial communities and their functions are essential for the development of better water management strategies aimed to produce safer fresh produce and to protect plant, animal, human and environmental health. Quantitative comparisons illustrated the importance of selecting the appropriate analytical method depending on the level of taxonomic delineation sought in each microbiome.

Mass mortality of collector urchins Tripneustes gratilla in Hawai`i

Work TM, Dagenais J, Rameyer B, Breeden R, Weatherby TM

Diseases of Aquatic Organisms. 2023 Feb 02

Use of MICRO/BEMF Core Facility

As grazers, sea urchins are keystone species in tropical marine ecosystems, and their loss can have important ecological ramifications. Die-offs of urchins are frequently described, but their causes are often unclear, in part because systematic examinations of animal tissues at gross and microscopic level are not done. In some areas, urchins are being employed to control invasive marine algae. Here, we describe the pathology of a mortality event in Tripneustes gratilla in Hawai`i where urchins were translocated to control invasive algae. Although we did not determine the cause of the mortality event, our investigation indicates that animals died from in flammation of the test and epidermal ulceration, followed by inability to maintain coelomic fluid volume, colonization of coelomic fluid by opportunists (diatom, algae), and inappetence. Parasites, bacteria, fungi, and viruses were not evident as a primary cause of death. Pathology was suggestive of a toxin or other environmental cause such as lack of food, possibilities that could be pursued in future investigations. These findings highlight the need for caution and additional tools to better assess health when translocating marine invertebrates to ensure maximal biosecurity.

Evolution of left-right asymmetry in the sensory system and foraging behavior during adaptation to food-sparse cave environments

Fernandes VFL, Glaser Y, Iwashita M, Yoshizawa M

BMC Biology. 2022 Dec 27

Laterality in relation to behavior and sensory systems is found commonly in a variety of animal taxa. Despite the advantages conferred by laterality (e.g., the startle response and complex motor activities), little is known about the evolution of laterality and its plasticity in response to ecological demands. In the present study, a comparative study model, the Mexican tetra (Astyanax mexicanus), composed of two morphotypes, i.e., riverine surface fish and cave-dwelling cavefish, was used to address the relationship between environment and laterality. The use of a machine learning-based fish posture detection system and sensory ablation revealed that the left cranial lateral line significantly supports one type of foraging behavior, i.e., vibration attraction behavior, in one cave population. Additionally, left-right asymmetric approaches toward a vibrating rod became symmetrical after fasting in one cave population but not in the other populations. Based on these findings, we propose a model explaining how the observed sensory laterality and behavioral shift could help adaptation in terms of the tradeoff in energy gain and loss during foraging according to differences in food availability among caves.

A charge-free and membrane-free hybrid capacitive mixing system for salinity gradient energy harvesting

Yang B, Yu J, Ma T

Journal of Materials Chemistry A. 2022 Dec 20

Use of MICRO/BEMF Core Facility

Capacitive mixing (CapMix) is a promising technology to harvest salinity gradient energy (SGE) by generating electricity through the potential difference owing to electrolyte salinity change at the electrode/electrolyte interface. However, existing CapMix methods suffer from the high cost of ion-exchange membranes, additional energy investment, and limited energy density and cycle life. Herein, we propose a charge-free and membrane-free hybrid CapMix system consisting of ethylenediamine-modified activated carbon (EDA-YP80F) as a capacitive anode and a Prussian blue analog, iron hexacyanoferrate (FeHCF), as a battery-type cathode. The selective incorporation/absorption of Na+ ions and Cl− ions into FeHCF and EDA-YP80F achieves a membrane-free operation. The matching potentials of FeHCF and EDA-YP80F enable reversible discharges to a final voltage of zero, eliminating the energy investment by external power sources. Without an external power source and membranes, the FeHCF/EDA-YP80F full cell achieves an average power density of 110 mW m−2 and energy density of 117 J m−2 over 150 cycles under the 1–0.01 M NaCl salinity gradient. Under a high salinity gradient involving hypersaline solutions, i.e., 5–0.01 M NaCl, a remarkably high average power density of 218 mW m−2 and energy density of 305 J m−2 are demonstrated over 50 cycles.

Root exudate composition from different plant species influences the growth of rhizosphere bacteria

Dhungana I, Kantar MB, Nguyen NH

Rhizosphere. 2022 Dec 17

Plant roots release exudates that fuel microbial activities and can structure rhizosphere microbial communities, but how different plant species use their root exudate to potentially select for different soil microbes in the rhizosphere is not well understood. Here, we investigated how root exudate from plants of three diverging lineages, Lactuca sativa (lettuce), Brassica juncea (mustard cabbage), and Zea mays (maize) influence the growth of their own rhizosphere bacteria (host) and those from other plant species (non-host) in growth bioassays. We found that on the community level, lettuce rhizosphere bacteria grew better in non-host exudate, but mustard cabbage and maize rhizosphere bacteria grew similarly well in both host and non-host exudate. However, individual bacteria taxa showed strong preferences for exudate from different plant species. The bacterial growth patterns were independent of C and N quantity, suggesting that certain exometabolic compounds may drive the growth patterns. Our results demonstrate that root exudate from a given plant species have the potential to stimulate or suppress soil bacteria and hint at a mechanism that different plant species use to select for their specific suite of rhizosphere bacteria. These findings contribute to our broader understanding of how root exudate composition could be a mechanism that plants use to select for distinct microbial communities in the rhizosphere.

Globally-distributed microbial eukaryotes exhibit endemism at deep-sea hydrothermal vents

Hu SK, Smith AR, Anderson RE, ... Frank KL, et al.

Molecular Ecology. 2022 Oct 27

Single-celled microbial eukaryotes inhabit deep-sea hydrothermal vent environments and play critical ecological roles in the vent-associated microbial food web. 18S rRNA amplicon sequencing of diffuse venting fluids from four geographically- and geochemically-distinct hydrothermal vent fields was applied to investigate community diversity patterns among protistan assemblages. The four vent fields include Axial Seamount at the Juan de Fuca Ridge, Sea Cliff and Apollo at the Gorda Ridge, all in the NE Pacific Ocean, and Piccard and Von Damm at the Mid-Cayman Rise in the Caribbean Sea. We describe species diversity patterns with respect to hydrothermal vent field and sample type, identify putative vent endemic microbial eukaryotes, and test how vent fluid geochemistry may influence microbial community diversity. At a semi-global scale, microbial eukaryotic communities at deep-sea vents were composed of similar proportions of dinoflagellates, ciliates, Rhizaria, and stramenopiles. Individual vent fields supported distinct and highly diverse assemblages of protists that included potentially endemic or novel vent-associated strains. These findings represent a census of deep-sea hydrothermal vent protistan communities. Protistan diversity, which is shaped by the hydrothermal vent environment at a local scale, ultimately influences the vent-associated microbial food web and the broader deep-sea carbon cycle.

Consequences of PDGFRα+ fibroblast reduction in adult murine hearts

Kuwabara JT, Hara A, Bhutada S, et al.

eLife. 2022 Sep 23

Use of MICRO/BEMF Core Facility

Fibroblasts produce the majority of collagen in the heart and are thought to regulate extracellular matrix (ECM) turnover. Although fibrosis accompanies many cardiac pathologies and is generally deleterious, the role of fibroblasts in maintaining the basal ECM network and in fibrosis in vivo is poorly understood. We genetically ablated fibroblasts in mice to evaluate the impact on homeostasis of adult ECM and cardiac function after injury. Fibroblast-ablated mice demonstrated a substantive reduction in cardiac fibroblasts, but fibrillar collagen and the ECM proteome were not overtly altered when evaluated by quantitative mass spectrometry and N-terminomics. However, the distribution and quantity of collagen VI, microfibrillar collagen that forms an open network with the basement membrane, was reduced. In fibroblast-ablated mice, cardiac function was better preserved following angiotensin II/phenylephrine (AngII/PE)-induced fibrosis and myocardial infarction (MI). Analysis of cardiomyocyte function demonstrated altered sarcomere shortening and slowed calcium decline in both uninjured and AngII/PE-infused fibroblast-ablated mice. After MI, the residual resident fibroblasts responded to injury, albeit with reduced proliferation and numbers immediately after injury. These results indicate that the adult mouse heart tolerates a significant degree of fibroblast loss with a potentially beneficial impact on cardiac function after injury. The cardioprotective effect of controlled fibroblast reduction may have therapeutic value in heart disease.

Elevation of Clavibacter michiganensis subsp. californiensis to species level as Clavibacter californiensis sp. nov., merging and re-classification of Clavibacter michiganensis subsp. chilensis and Clavibacter michiganensis subsp. phaseoli as Clavibacter phaseoli sp. nov. based on complete genome in silico analyses

Arizala D, Dobhal S, Alvarez AM, Arif M

International Journal of Systematic and Evolutionary Microbiology. 2022 Sep 15

The Gram-positive genus Clavibacter is currently divided into seven species (Clavibacter michiganensis, Clavibacter nebraskensis, Clavibacter capsici, Clavibacter sepedonicus, Clavibacter tessellarius, Clavibacter insidiosus and Clavibacter zhangzhiyongii) and three subspecies (C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli). Recent studies have indicated that the taxonomic rank of the subspecies must be re-evaluated. In this research, we assessed the taxonomic position of the three C. michiganensis subspecies and clarified the taxonomic nomenclature of other 75 Clavibacter strains. The complete genomes of the type strains of the three Clavibacter subspecies, the type strain of C. tessellarius and C. nebraskensis A6096 were sequenced using PacBio RSII technology. Application of whole-genome-based computational approaches such as average nucleotide identity (ANI), digital DNA-DNA hybridization, multi-locus sequence analysis of seven housekeeping genes (acnA, atpD, bipA, icdA, mtlD, recA and rpoB), a phylogenomic tree reconstructed from 1 028 core genes, and ANI-based phylogeny provided sufficient justification for raising C. michiganensis subsp. californiensis to the species level. These results led us to propose the establishment of Clavibacter californiensis sp. nov. as a species with its type strain C55T (=CFBP 8216T=ATCC BAA-2691T). Moreover, the orthologous and in silico dot plot analyses, along with the above described bioinformatic strategies, revealed a high degree of similarity between C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli. Based on these analyses, we propose that both subspecies be combined into a single taxon and elevated to the species level as Clavibacter phaseoli sp. nov., with LPPA 982T (= CECT 8144T= LMG 27667T) as the type strain.

Enhancing the electrical properties of graphite nanoflake through gamma-ray irradiation

Nguyen AT, Lee Y, Nguyen PQH, Dera P, Yoon S, Lee W

Scientific Reports. 2022 Sep 01

Use of MICRO/BEMF Core Facility

Understanding changes in material properties through external stimuli is critical to validating the expected performance of materials as well as engineering material properties in a controlled manner. Here, we investigate a change in the c-axis electrical properties of graphite nanoflakes (GnFs) induced by gamma-ray irradiation, using conductive probe atomic force microscopy (CP-AFM). The fundamentals behind the change in their electrical properties are elucidated by analyzing the interlayer spacing, graphitization, and morphology. An increase in gamma-ray irradiation dose for GnFs leads to an exponential increase in the electrical conductance and a gradual decrease in the interlayer spacing, while accompanying indistinguishable changes in their morphology. Our experimental results suggest that the c-axis electrical conductance enhancement of GnFs with gamma-ray irradiation might be attributed to a reduction in interlayer spacing, though the created defects may also play a role. This study demonstrates that gamma-ray irradiation can be a promising route to tailor the electrical properties of GnFs.

Sperm degradation after vasectomy follows a sperm chromatin fragmentation-dependent mechanism causing DNA breaks in the toroid linker regions

Ribas-Maynou J, Nguyen H, Valle R, et al.

Molecular Human Reproduction. 2022 Aug 29

Use of MICRO/BEMF Core Facility

Vasectomy is a widely used surgical technique creating an obstructive azoospermia. Although sperm cannot be ejaculated, the testis maintains sperm production in vasectomized males. The continuous accumulation of sperm deposited in the epididymis and the vas deferens fraction necessarily need to be degraded and eliminated. While the elimination process is carried out by granulomas that form after vasectomy, the detailed mechanisms of sperm degradation are still not known. The aim was to assess whether sperm chromatin fragmentation (SCF), a mechanism that degrades the entire sperm genome at the toroid linker regions (TLRs), is activated after vasectomy in sperm cells. We vasectomized mice and evaluated the presence of TLR-specific double-strand breaks through pulsed-field gel electrophoresis and the Comet assay at 1, 2 and 3 weeks after surgery. Results for DNA damage (Olive tail moment) at single-cell level showed an increase of double-strand breaks after vasectomy for vas deferens sperm after 1, 2 and 3 weeks postvasectomy (21.78 ± 2.29; 19.71 ± 1.79 and 32.59 ± 1.81, respectively), compared to mock surgery (7.04 ± 1.03; 10.10 ± 1.29 and 8.64 ± 0.85, respectively; P < 0.001). Similar findings were obtained for cauda epididymis sperm (P < 0.001), but not for caput epididymis (P > 0.05). Pulsed-field gel electrophoresis showed the presence of double-stranded breaks between 15 and 145 kb, indicating that DNA breaks were produced mainly in the sperm TLRs. Results presented here suggest that SCF is a mechanism activated in vas deferens after vasectomy to degrade sperm DNA when they cannot be ejaculated, preventing their function.

Niclosamide-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis

Sutar Y, Nabeela S, Singh S, et al.

PLoS Biology. 2022 Aug 17

Use of MICRO/BEMF Core Facility

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.

A ridge-to-reef ecosystem microbial census reveals environmental reservoirs for animal and plant microbiomes

Amend AS, Swift SOI, Darcy JL, et al.

PNAS. 2022 Aug 16

Use of MGAL Core Facility

Microbes are found in nearly every habitat and organism on the planet, where they are critical to host health, fitness, and metabolism. In most organisms, few microbes are inherited at birth; instead, acquiring microbiomes generally involves complicated interactions between the environment, hosts, and symbionts. Despite the criticality of microbiome acquisition, we know little about where hosts’ microbes reside when not in or on hosts of interest. Because microbes span a continuum ranging from generalists associating with multiple hosts and habitats to specialists with narrower host ranges, identifying potential sources of microbial diversity that can contribute to the microbiomes of unrelated hosts is a gap in our understanding of microbiome assembly. Microbial dispersal attenuates with distance, so identifying sources and sinks requires data from microbiomes that are contemporary and near enough for potential microbial transmission. Here, we characterize microbiomes across adjacent terrestrial and aquatic hosts and habitats throughout an entire watershed, showing that the most species-poor microbiomes are partial subsets of the most species-rich and that microbiomes of plants and animals are nested within those of their environments. Furthermore, we show that the host and habitat range of a microbe within a single ecosystem predicts its global distribution, a relationship with implications for global microbial assembly processes. Thus, the tendency for microbes to occupy multiple habitats and unrelated hosts enables persistent microbiomes, even when host populations are disjunct. Our whole-watershed census demonstrates how a nested distribution of microbes, following the trophic hierarchies of hosts, can shape microbial acquisition.

Chapter 4 - Physics-based impedance spectroscopy characterization of operating PEM fuel cells

Reshetenko T, Kulikovsky A

Advanced Nanomaterials and Their Applications in Renewable Energy (Second Edition). 2022 Aug 05

Use of MICRO/BEMF Core Facility

Polymer electrolyte membrane fuel cell impedance models based on transient mass and charge conservation equations are developed. The models are fitted to experimental impedance spectra of the segmented high- and low-Pt cells operating in the range of current densities between 0.1 and 1.0 A cm−2. The functional cell layers' fitting oxygen and proton transport parameters are discussed. We show how the models help to identify the peaks in the distribution of relaxation times spectra.

Towards a revision of the bamboo corals (Octocorallia): Part 5, new genera and species of Keratoisididae from the Tasmanian deep sea

Lapointe A, Watling L

Zootaxa. 2022 Jul 21

Use of MICRO/BEMF Core Facility

Four new genera and six new species of deep-sea bamboo corals (Family: Keratoisididae) from the seamounts off the eastern and southern coast of Tasmania are described and illustrated. In situ high-definition images from remotely operated vehicles, compound microscopy, and Scanning Electron Microscopy (SEM) of 29 specimens collected at depths of 729 to 3950 m were used to provide data on distinguishing morphological characters. The species described include two new species in the genus Keratoisis, two species in the newly described genus Adinisis, and one species each in the new genera Tanyostea and Dokidisis. The species Keratoisis magnifica and Lepidisis solitaria are re-assigned to the newly described genus Onkoisis due to the shape and design of the polyp arrangement and sclerites, which differ considerably from that of the type species of Keratoisis.

Genome Sequence of Arthrobacter globiformis Phage KeAlii from Hawaiʻi

Chong RA, UHM SEA-PHAGES Students, Donachie SP, Reed FA, Porter ML

Microbiology Resource Announcements. 2022 Jul 20

Use of MICRO/BEMF Core Facility

Here, we report the genome sequence of bacteriophage KeAlii, a Siphoviridae that infects Arthrobacter globiformis strain B-2979, from Honolulu, Hawaiʻi. The 41,850-bp genome contains 66 predicted protein-coding genes and 1 gene that encodes a tRNA for tryptophan. Genome comparisons suggest KeAlii is closely related to actinobacteriophage Adolin.

Development of a GFP biosensor reporter for the unfolded protein response-signaling pathway in plants: incorporation of the bZIP60 intron into the GFP gene

Carrillo R, Christopher DA

Plant Signaling & Behavior. 2022 Jul 20

Use of MICRO/BEMF Core Facility

The ability to measure the activation of the unfolded protein response (UPR) in plants is important when they are exposed to stressful environments. To this end, we developed a unique and versatile biosensor-reporter system to indicate the activation of UPR in living plant cells. The small cytoplasmically spliced intron from the bZIP60 locus was incorporated into the 5’ end of the GFP gene, creating the 35S::bZIP60 intron:GFP construct. When this construct is transiently expressed in Arabidopsis protoplasts, the presence of the bZIP60 intron prevents GFP mRNA from being translated under non-UPR conditions. However, when UPR is activated, the IRE1 kinase/ribonuclease splices this intron from the GFP mRNA and its translation proceeds, generating GFP fluorescence. We demonstrated the utility of the system in Arabidopsis leaf protoplasts treated with DTT, which is a chemical inducer of UPR, followed by visualization and quantification using confocal microscopy. The 35S::bZIP60 intron:GFP construct was also expressed in protoplasts from an overexpressor line containing the coding sequence for the UPR-induced, protein folding chaperone, protein disulfide isomerase-9 (PDI9). PDI9 also influences the strength of the UPR signaling pathway. Protoplasts from WT and PDI9 overexpressor plants treated with DTT exhibited significantly higher GFP fluorescence relative to untreated protoplasts, indicating that the bZIP60 intron was spliced from the GFP mRNA in response to activation of UPR. RT-PCR further confirmed the higher induction of PDI9 and bZIP60 (total and spliced) mRNA levels in DTT-treated protoplasts relative to controls. This system can be adapted for monitoring crop stress and for basic studies dissecting the UPR signaling pathway.

Possible role of left–right asymmetry in the sensory system and behavior during adaptation to food-sparse cave environments

Fernandes VFL, Glaser Y, Iwashita M, Yoshizawa M

bioRxiv. 2022 Jul 12 - Preprint

Laterality in relation to behavior and sensory systems is found commonly in a variety of animal taxa. Despite the advantages conferred by laterality (e.g., the startle response and complex motor activities), little is known about the evolution of laterality and its plasticity in response to ecological demands. In the present study, a comparative study model, the Mexican tetra (Astyanax mexicanus), composed of two morphotypes, i.e., riverine surface fish and caved-welling cavefish, was used to address the relationship between environment and laterality. The use of a machine learning-based fish posture detection system and sensory ablation revealed that the left cranial lateral line significantly supports one type of foraging behavior, i.e., vibration attraction behavior, in one cave population. Additionally, left–right asymmetric approaches toward a vibrating rod became symmetrical after fasting in one cave population but not in the other populations. Based on these findings, we propose a model explaining how the observed sensory laterality and behavioral shift could help adaptations in terms of the tradeoff in energy gain and loss during foraging according to differences in food availability among caves.

In situ lipid profiling of insect pheromone glands by direct analysis in real time mass spectrometry

Cetraro N, Yew JY

Analyst. 2022 Jul 12

Use of MGAL Core Facility

Lipid pheromones play a significant role in the behavior and ecology of many insects. The characterization of pheromone structures is a significant challenge due to their low abundance and ephemeral nature. Here we present a method for the analysis of lipid molecules from single pheromone glands of Drosophila melanogaster (fruit fly) using Direct Analysis in Real Time mass spectrometry (DART MS). Our results reveal that DART MS analysis of single tissues generates reproducible, species-specific lipid profiles comprised of fatty acids, wax esters, diacylglycerides and triacylglycerides. In addition, the ion source temperature and application of a solvent wash can cause significant qualitative and quantitative changes in the mass spectral profile. Lastly, we show that untargeted chemical fingerprinting of the gland can be used to accurately categorize species according to phylogenetic subgroup or genotype. Collectively, our findings indicate that DART MS is a rapid and powerful method for characterizing a broad range of lipids in tissues with minimal preparation. The application of direct tissue DART MS will expand the "secretome" of molecules produced by pheromone glands. In addition to its direct relevance to chemical ecology, the method could potentially be used in pharmaceutical studies for the screening and detection of tissue-specific drug metabolites.

The C-MĀIKI Gateway: A Modern Science Platform for Analyzing Microbiome Data

Cleveland S, Arisdakessian C, Nelson C, Belcaid M, Frank K, Jacobs G

Practice and Experience in Advanced Research Computing. 2022 Jul 08

In collaboration with the Center for Microbiome Analysis throughIsland Knowledge and Investigations (C-M ̄AIKI), the Hawaii EP-SCoR Ike Wai project and the Hawaii Data Science Institute, anew science gateway, the C-M ̄AIKI gateway, was developed tosupport modern, interoperable and scalable microbiome data anal-ysis. This gateway provides a web-based interface for accessinghigh-performance computing resources and storage to enable andsupport reproducible microbiome data analysis. The C-M ̄AIKI gate-way is accelerating the analysis of microbiome data for Hawaiithrough ease of use and centralized infrastructure.

Case Report: Angiostrongylus cantonensis Infection Presenting as Small Fiber Neuropathy

Yates J, Devere T, Sakurai-Burton S, Santi B, McAllister C, Frank K

The American Journal of Tropical Medicine and Hygiene. 2022 Jul 05

Angiostrongylus cantonensis is an emerging parasite that is the most common cause of eosinophilic meningitis worldwide. Human infection typically presents with headache, neck stiffness, and paresthesia. We report a case of a woman with PCR positive A. cantonensis infection who presented with symptoms of small fiber neuropathy (SFN) but no headache. SFN was confirmed by skin biopsy. After failing standard medications for neuropathy, she was treated with intravenous lidocaine with considerable improvement. However, she required medications for 1 year to treat her chronic neuropathy. Infection by A. cantonensis should be added to the list of causes of SFN, and its potential to cause chronic sequelae should be appreciated.

Menthol in electronic cigarettes causes biophysical inhibition of pulmonary surfactant

Xu L, Yang Y, Simien JM, Kang C, Li G, Xu X, Haglund E, Sun R, Zuo YY

American Journal of Physiology-Lung Cellular and Molecular Physiology. 2022 Jun 28

With an increasing prevalence of electronic cigarette (e-cigarette) use, especially among youth, there is an urgent need to better understand the biological risks and pathophysiology of health conditions related to e-cigarettes. A majority of e-cigarette aerosols are in the submicron size and would deposit in the alveolar region of the lung, where they must first interact with the endogenous pulmonary surfactant. To date, little is known whether e-cigarette aerosols have an adverse impact on the pulmonary surfactant. We have systematically studied the effect of individual e-cigarette ingredients on an animal-derived clinical surfactant preparation, bovine lipid extract surfactant, using a combination of biophysical and analytical techniques, including in vitro biophysical simulations using constrained drop surfactometry, molecular imaging with atomic force microscopy, chemical assays using carbon nuclear magnetic resonance and circular dichroism, and in silico molecular dynamics simulations. All data collectively suggest that flavorings used in e-cigarettes, especially menthol, play a predominant role in inhibiting the biophysical function of the surfactant. The mechanism of biophysical inhibition appears to involve menthol interactions with both phospholipids and hydrophobic proteins of the natural surfactant. These results provide novel insights into the understanding of the health impact of e-cigarettes and may contribute to better regulation of e-cigarette products.

A new species of Neostenoptera Meunier (Diptera: Cecidomyiidae: Winnertziinae) from Hawai‘i

Plakidas JD, Nguyen NH, Ferro ML

Insecta Mundi. 2022 Jun 24

A new paedogenetic midge (Diptera: Cecidomyiidae: Winnertziinae: Heteropezini) from O‘ahu Island, Hawai‘i, Neostenoptera hawaiiensis Plakidas, Nguyen, and Ferro, new species, is described and illus­trated. A key to all species in the genus is provided. Specimens were emergent from deadwood gathered at Waimea Arboretum and Botanical Garden. Neostenoptera appalachiensis Plakidas and Ferro were collected from the same set of samples in Hawai‘i, and additional specimens are reported from Georgia and South Carolina, three new state records. The discovery of two paedogenetic midges in Hawai‘i poses a unique set of questions as to their possible mode of arrival on an island ecosystem. We briefly address the possibility that both species are simply “hitchhikers” that went undetected at ports of entry.

Biofinder detects biological remains in Green River fish fossils from Eocene epoch at video speed

Misra AK, Rowley SJ, Zhou J, et al.

Scientific Reports. 2022 Jun 17

Use of MICRO/BEMF Core Facility

The “Search for life”, which may be extinct or extant on other planetary bodies is one of the major goals of NASA planetary exploration missions. Finding such evidence of biological residue in a vast planetary landscape is an enormous challenge. We have developed a highly sensitive instrument, the “Compact Color Biofinder”, which can locate minute amounts of biological material in a large area at video speed from a standoff distance. Here we demonstrate the efficacy of the Biofinder to detect fossils that still possess strong bio-fluorescence signals from a collection of samples. Fluorescence images taken by the Biofinder instrument show that all Knightia spp. fish fossils analysed from the Green River formation (Eocene, 56.0–33.9 Mya) still contain considerable amounts of biological residues. The biofluorescence images support the fact that organic matter has been well preserved in the Green River formation, and thus, not diagenetically replaced (replaced by minerals) over such a significant timescale. We further corroborated results from the Biofinder fluorescence imagery through Raman and attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopies, scanning electron microscopy, energy dispersive X-ray spectroscopy (SEM–EDS), and fluorescence lifetime imaging microscopy (FLIM). Our findings confirm once more that biological residues can survive millions of years, and that using biofluorescence imaging effectively detects these trace residues in real time. We anticipate that fluorescence imaging will be critical in future NASA missions to detect organics and the existence of life on other planetary bodies.

“RSSC-Lineage Multiplex PCR” assay detects and differentiates Ralstonia solanacearum, R. pseudosolanacearum, R. syzygii and the R3bv2 subgroup

Paudel S, Dobhal S, Lowe-Power T, Schlub RL, Hu J, Allen C, Alvarez AM, Arif M

Research Square. 2022 May 26 - Preprint

Bacterial wilt strains in the Ralstonia solanacearum species complex (RSSC) pose serious threats to economically important crops worldwide. In 2014, Safni and co-workers proposed the reclassification of RSSC into three genomospecies: R. solanacearum (Rsol), R. pseudosolanacearum (Rpseu), and R. syzygii (Rsyz). The revision requires the proper identification of strains for diagnostic and epidemiological studies. In response, we developed an inexpensive and user-friendly “RSSC-Lineage Multiplex PCR” that effectively detects plant pathogenic Ralstonia strains in general and also distinguishes between Rpseu, Rsol, Rsyz, and the high-security Select Agent “race 3 biovar 2” subgroup of Rsol, also known as the phylotype IIB-1 potato brown rot pandemic lineage. Genomes were retrieved from the NCBI GenBank database and screened for unique gene regions using OrthoMCL and other comparative genomic approaches. Specific primers were designed for each genomospecies, Ralstonia in general and for “race 3 biovar 2”. AT-rich flaps were added at 5’ position of each primer to optimize the reaction thermodynamics. The specificity was tested in silico using the NCBI GenBank genome database and an in-house database. In vitro specificity and accuracy of the tool was validated with 110 representative Ralstonia strains and 24 strains from other genera. The assay is highly specific, generating neither false positives nor false negatives. Primer set detection limits ranged from 10- to 100-pg. The assay also detected and differentiated strains from naturally and artificially inoculated plant hosts. This tool is highly specific, reliable, and economical for culture characterization, diagnostics, surveys, quarantine decisions, and epidemiological studies.

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region

DeLude A, Wells R, Boomla S, ... Arif M

Research Square. 2022 May 26 - Preprint

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae), causing soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera, and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with 10-fold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18–20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Fine scale transitions of the microbiota and metabolome along the gastrointestinal tract of herbivorous fishes

Sparagon WJ, Gentry EC, Minich JJ, et al.

Animal Microbiome. 2022 May 23

Use of MGAL Core Facility

Gut microorganisms aid in the digestion of food by providing exogenous metabolic pathways to break down organic compounds. An integration of longitudinal microbial and chemical data is necessary to illuminate how gut microorganisms supplement the energetic and nutritional requirements of animals. Although mammalian gut systems are well-studied in this capacity, the role of microbes in the breakdown and utilization of recalcitrant marine macroalgae in herbivorous fish is relatively understudied and an emerging priority for bioproduct extraction. Here we use a comprehensive survey of the marine herbivorous fish gut microbial ecosystem via parallel 16S rRNA gene amplicon profiling (microbiota) and untargeted tandem mass spectrometry (metabolomes) to demonstrate consistent transitions among 8 gut subsections across five fish of the genus of Kyphosus.

Bacterial lipopolysaccharide induces settlement and metamorphosis in a marine larva

Freckelton ML, Nedved BT, Cai Y, Cao S, Turano H, Alegado RA, Hadfield MG

PNAS. 2022 Apr 25

Use of MICRO/BEMF Core Facility

How larvae of the many phyla of marine invertebrates find places appropriate for settlement, metamorphosis, growth, and reproduction is an enduring question in marine science. Biofilm-induced metamorphosis has been observed in marine invertebrate larvae from nearly every major marine phylum. Despite the widespread nature of this phenomenon, the mechanism of induction remains poorly understood. The serpulid polychaete Hydroides elegans is a well established model for investigating bacteria-induced larval development. A broad range of biofilm bacterial species elicit larval metamorphosis in H. elegans via at least two mechanisms, including outer membrane vesicles (OMVs) and complexes of phage-tail bacteriocins. We investigated the interaction between larvae of H. elegans and the inductive bacterium Cellulophaga lytica, which produces an abundance of OMVs but not phage-tail bacteriocins. We asked whether the OMVs of C. lytica induce larval settlement due to cell membrane components or through delivery of specific cargo. Employing a biochemical structure–function approach with a strong ecological focus, the cells and OMVs produced by C. lytica were interrogated to determine the class of the inductive compounds. Here, we report that larvae of H. elegans are induced to metamorphose by lipopolysaccharide produced by C. lytica. The widespread prevalence of lipopolysaccharide and its associated taxonomic and structural variability suggest it may be a broadly employed cue for bacterially induced larval settlement of marine invertebrates.

A Single Application of Compost Can Leave Lasting Impacts on Soil Microbial Community Structure and Alter Cross-Domain Interaction Networks

Heisey S, Ryals R, McClellan Maaz T, Nguyen NH

Frontiers in Soil Science. 2022 Apr 05

Our current understanding suggests that nutrient management strategies applied to agricultural soils over multiple years are required to cause major and stable shifts in soil microbial communities. However, some studies suggest that agricultural soils can benefit even from sporadic, single additions of organic matter. Here we investigate how single additions of high-quality organic matter can cause significant shifts in microbial soil communities over multiple cropping cycles. We grew radishes in a tropical Oxisol soil for six crop cycles after a single application of a high-nitrogen compost or urea. At planting and before biomass harvest, we sampled soils influenced by the radish rhizosphere and sequenced bacterial and archaeal 16S and fungal ITS rDNA marker genes. We measured microbial richness and diversity, community composition and structure, and constructed correlation networks to predict cross-domain microbial interactions. We found that a single application of compost, compared to urea or control, resulted in a persistent improved plant biomass response and led to sustained changes in the soil microbial community throughout the duration of the 227-day study. Compost altered the structure of both the fungal and prokaryotic microbial communities, introduced new microorganisms that persisted in the resident soil system, and altered soil microbial correlation network structure and hub taxa. In contrast, fertilization with urea did not significantly alter the structure of soil microbial communities compared to the control but reduced network complexity and altered hub taxa. This study highlights the significant impacts that high-quality organic matter fertilization can exert on agricultural soil microbiomes and adds to the growing body of knowledge on using organic fertilizers as a way to steer the soil microbiome toward a healthier soil.

Design and implementation of a cohort-based undergraduate research experience in the agricultural sciences

McClellan Maaz T, Nguyen NH, Del Valle Echevarria AR, et al.

Natural Sciences Education. 2022 Mar 08

Science, technology, engineering, and mathematics (STEM) education is consistently seen as a top priority; however, STEM programs often suffer from low retention. Students who start in STEM degree programs often lose interest or face obstacles that cause them to leave. Here, we describe a non-traditional approach meant to encourage a range of students to pursue or continue their STEM education. Active learning approaches have long been touted to stimulate long-term interest and prepare students for a career in natural science. We provide a case study of an interdisciplinary, cohort-centric, mentor-guided summer research internship. By establishing an understanding of how science is currently conducted in agriculture, through several faculty mentors, the program allowed students to embrace their core interests while being able to fit into larger interdisciplinary narratives which provided motivation to continue their path in STEM education.

Editorial: Genome-Wide Analyses of Pectobacterium and Dickeya Species

Arif M, Czajkowski R, Chapman TA

Frontiers in Plant Science. 2022 Mar 03

Pectobacterium and Dickeya, emerging pathogens and key genera included in the Soft Rot Pectobacteriaceae family (SRP; formerly known as pectinolytic Erwinia spp.) (Adeolu et al., 2016), are among the top 10 bacterial plant pathogens that limit crop yields and threaten global food security worldwide (Mansfield et al., 2012). Species within both genera are globally distributed (Mansfield et al., 2012; Ma et al., 2019; Boluk et al., 2021) and cause significant damage to both monocots and dicots, particularly to potato with potential global losses in production (Agrios, 2006), and an estimated cost of US$50–100 million annually in vegetables, fruits, and ornamental plants (Perombelon and Kelman, 1980; Pérombelon, 2002; Ma et al., 2007). Species in both genera have been isolated from infected plant tissues, soil, and water (Glasner et al., 2008; Hugouvieux-Cotte-Pattat et al., 2019; Oulghazi et al., 2019) as well as from alternative non-agricultural plant hosts (Fikowicz-Krosko et al., 2017; Fikowicz-Krosko and Czajkowski, 2018). The pectinolytic bacteria rapidly adapt to new hosts, raising serious concerns for potential damage to new crops (Boluk et al., 2020, 2021; Klair et al., 2021).

Refining real-time predictions of Vibrio vulnificus concentrations in a tropical urban estuary by incorporating dissolved organic matter dynamics

Bullington JA, Golder AR, Steward GF, et al.

Science of The Total Environment. 2022 Feb 24

Use of MGAL Core Facility

The south shore of O'ahu, Hawai'i is one of the most visited coastal tourism areas in the United States with some of the highest instances of recreational waterborne disease. A population of the pathogenic bacterium Vibrio vulnificus lives in the estuarine Ala Wai Canal in Honolulu which surrounds the heavily populated tourism center of Waikīkī. We developed a statistical model to predict V. vulnificus dynamics in this system using environmental measurements from moored oceanographic and atmospheric sensors in real time. During a year-long investigation, we analyzed water from 9 sampling events at 3 depths and 8 sites along the canal (n = 213) for 36 biogeochemical variables and V. vulnificus concentration using quantitative polymerase chain reaction (qPCR) of the hemolysin A gene (vvhA). The best multiple linear regression model of V. vulnificus concentration, explaining 80% of variation, included only six predictors: 5-day average rainfall preceding water sampling, daily maximum air temperature, water temperature, nitrate plus nitrite, and two metrics of humic dissolved organic matter (DOM). We show how real-time predictions of V. vulnificus concentration can be made using these models applied to the time series of water quality measurements from the Pacific Islands Ocean Observing System (PacIOOS) as well as the PacIOOS plume model based on the Waikīkī Regional Ocean Modeling System (ROMS) products. These applications highlight the importance of including DOM variables in predictive modeling of V. vulnificus and the influence of rain events in elevating nearshore concentrations of V. vulnificus. Long-term climate model projections of locally downscaled monthly rainfall and air temperature were used to predict an overall increase in V. vulnificus concentration of approximately 2- to 3-fold by 2100. Improving these predictive models of microbial populations is critical for management of waterborne pathogen risk exposure, particularly in the wake of a changing global climate.

Development of a multiplex TaqMan qPCR targeting unique genomic regions for the specific and sensitive detection of Pectobacterium species and P. parmentieri

Arizala D, Dobhal S, Babler B, Crockford AB, Rioux RA, Alvarez AM, Arif M

Journal of Applied Microbiology. 2022 Feb 19

Aim: The newly defined species Pectobacterium parmentieri has emerged as an aggressive pathogen that causes soft rot and blackleg diseases on potato and has been widely disseminated across the globe, jeopardizing the productivity and potato food safety. The implementation of a fast and accurate detection tool is imperative to control, monitor and prevent further spread of these pathogens. The objective of this work was to develop a specific and sensitive multiplex TaqMan qPCR to detect P. parmentieri and distinguish it from all known Pectobacterium species. A universal internal control was included to enhance the reliability of the assay.

Independent host- and bacterium-based determinants protect a model symbiosis from phage predation

Lynch JB, Bennett BD, Merrill BD, Ruby EG, Hryckowian AJ

Cell Reports. 2022 Feb 15

Use of MICRO/BEMF Core Facility

Bacteriophages (phages) are diverse and abundant constituents of microbial communities worldwide, capable of modulating bacterial populations in diverse ways. Here, we describe the phage HNL01, which infects the marine bacterium Vibrio fischeri. We use culture-based approaches to demonstrate that mutations in the exopolysaccharide locus of V. fischeri render this bacterium resistant to infection by HNL01, highlighting the extracellular matrix as a key determinant of HNL01 infection. Additionally, using the natural symbiosis between V. fischeri and the squid Euprymna scolopes, we show that, during colonization, V. fischeri is protected from phages present in the ambient seawater. Taken together, these findings shed light on independent yet synergistic host- and bacterium-based strategies for resisting symbiosis-disrupting phage predation, and we present important implications for understanding these strategies in the context of diverse host-associated microbial ecosystems.

Overlooked and widespread pennate diatom-diazotroph symbioses in the sea

Schvarcz CR, Wilson ST, Caffin M, et al.

Nature Communications. 2022 Feb 10

Use of MICRO/BEMF Core Facility

Persistent nitrogen depletion in sunlit open ocean waters provides a favorable ecological niche for nitrogen-fixing (diazotrophic) cyanobacteria, some of which associate symbiotically with eukaryotic algae. All known marine examples of these symbioses have involved either centric diatom or haptophyte hosts. We report here the discovery and characterization of two distinct marine pennate diatom-diazotroph symbioses, which until now had only been observed in freshwater environments. Rhopalodiaceae diatoms Epithemia pelagica sp. nov. and Epithemia catenata sp. nov. were isolated repeatedly from the subtropical North Pacific Ocean, and analysis of sequence libraries reveals a global distribution. These symbioses likely escaped attention because the endosymbionts lack fluorescent photopigments, have nifH gene sequences similar to those of free-living unicellular cyanobacteria, and are lost in nitrogen-replete medium. Marine Rhopalodiaceae-diazotroph symbioses are a previously overlooked but widespread source of bioavailable nitrogen in marine habitats and provide new, easily cultured model organisms for the study of organelle evolution.

Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

Li Q, Edwards KF, Schvarcz CR, Steward GF

The ISME Journal. 2022 Feb 10

Use of MICRO/BEMF Core Facility

Small eukaryotic phytoplankton are major contributors to global primary production and marine biogeochemical cycles. Many taxa are thought to be mixotrophic, but quantitative studies of phagotrophy exist for very few. In addition, little is known about consumers of Prochlorococcus, the abundant cyanobacterium at the base of oligotrophic ocean food webs. Here we describe thirty-nine new phytoplankton isolates from the North Pacific Subtropical Gyre (Station ALOHA), all flagellates ~2–5 µm diameter, and we quantify their ability to graze Prochlorococcus. The mixotrophs are from diverse classes (dictyochophytes, haptophytes, chrysophytes, bolidophytes, a dinoflagellate, and a chlorarachniophyte), many from previously uncultured clades. Grazing ability varied substantially, with specific clearance rate (volume cleared per body volume) varying over ten-fold across isolates and six-fold across genera. Slower grazers tended to create more biovolume per prey biovolume consumed. Using qPCR we found that the haptophyte Chrysochromulina was most abundant among the isolated mixotrophs at Station ALOHA, with 76–250 cells mL−1 across depths in the upper euphotic zone (5–100 m). Our results show that within a single ecosystem the phototrophs that ingest bacteria come from many branches of the eukaryotic tree, and are functionally diverse, indicating a broad range of strategies along the spectrum from phototrophy to phagotrophy.

Pectobacterium colocasium sp. nov. isolated from taro (Colocasia esculenta)

Klair D, Arizala D, Dobhal S, Boluk G, Alvarez AM, Arif M

bioRxiv. 2022 Feb 09 - Preprint

Pectobacterium, agenus comprising gram-negative, pectinolytic phytopathogens, is responsible for economic losses in a wide host range of plants. In this study, the bacterial strains PL152T and PL155 were isolated from taro corms in Hawai’i in 2018, and characterized using genomic and biochemical assays. The Next Generation Sequencing technologies, Oxford Nanopore MinION and Illumina NovaSeq, were used for whole genome sequencing of the PL152T strain. Short and long reads were assembled using the Unicycler tool accessible at the bioinformatic resource center, and PATRIC (PathoSystems Resource Integration Center) was used to generate a more accurate and reliable “hybrid” assembly. The 16S rRNA analysis of PL152T with type strains of other known Pectobacterium species showed a close relationship with P. fontis. Multi-locus sequence analysis using nine housekeeping genes (dnaA, gapA, gyrB, recA, dnaN, rpoS, mdh, rpoA and dnaK) differentiated strain PL152T from other species of Pectobacterium and formed a unique and well-defined clade. The concurrent results of average nucleotide identity (ANI) and digital DNA-DNA hybridization, with calculated values lower than 95 and 70%, respectively, supported the delineation of a novel bacterial species. Here, we proposed Pectobacterium colocasium, strain PL152T (=ICMP 24362T; LMG 32536 T) and PL155 as a novel species in the genus Pectobacterium.

Genomic Comparisons and Phenotypic Diversity of Dickeya zeae Strains Causing Bacterial Soft Rot of Banana in China

Zhang J, Arif M, Shen H, Sun D, Pu X, Hu J, Lin B, Yang Q

Frontiers in Plant Science. 2022 Feb 09

Bacterial soft rot of banana, caused by Dickeya zeae, is spreading rapidly in important banana growing areas in China and seriously threatens banana production. In this study, we sequenced the high-quality complete genomes of three typical banana strains, MS1 (size: 4,831,702-bp; genome coverages: 538x), MS_2014 (size: 4,740,000-bp; genome coverages: 586x) and MS_2018 (size: 4,787,201-bp; genome coverages: 583x), isolated in 2009, 2014, and 2018, respectively. To determine their genomic and phenotypic diversity with respect to their hosts of origin, they were compared with other D. zeae strains, including another representative banana strain MS2 from China. The sequenced strains were similar in utilization of carbon source and chemical substrates, and general genomic features of GC content, and tRNA and rRNA regions. They were also conserved in most virulence determinants, including gene-encoding secretion systems, plant cell wall degrading enzymes, and exopolysaccharides. We further explored their genomic diversity in the predicted genomic islands (GIs). These GIs were rich in integrases and transposases, where some genomic dissimilarity was observed in the flagellar gene cluster and several secondary metabolite gene clusters. Different constituents of core biosynthetic modules were found within the bacteriocin and aryl polyene (APE) pigment gene clusters, and the strains from banana showed different phenotypes with respect to antibiosis effects and colony pigmentation. Additionally, clustered regularly interspaced short palindromic repeat (CRISPR) and prophage elements, such as type I-F and III-A CRISPR arrays and an intact prophage of MS1-P5, contributed to bacterial diversity. Phylogenetic tree analysis and genome-genome nucleotide comparison confirmed the genomic divergence among the strains isolated from banana. Considering these characteristics, MS2 and MS_2014 probably diverged later than MS1, while MS_2018 was different and more similar to foreign strains isolated from other hosts in several characteristics. Strain MS_2018 caused severe symptoms on banana varieties previously considered moderately resistant or moderately susceptible, including varieties of Cavendish (Musa AAA) and Plantain (Musa ABB). Our study of genomic and phenotypic diversity raises public attention to the risk of spreading new pathogenic variants within banana growing regions and supports development of predictive strategies for disease control.

Dickeya colocasiae sp. nov. isolated from wetland taro, Colocasia esculentum

Boluk G, Dobhal S, Arizala D, Alvarez AM, Arif M

bioRxiv. 2022 Jan 15 - Preprint

Bacterial pathogens identified as Dickeya sp. have recently been associated with a corm rot of wetland taro on Oahu, Hawaii, but the species designation of these strains was unclear. A Gram-negative, pectinolytic bacterial strain PL65T isolated from an infected taro corm was subjected to polyphasic analysis to determine its genomic and phenotypic characteristics. Multi-locus sequence analyses (MLSA) based on five housekeeping genes (dnaA, gapA, gyrB, atpD, and purA) revealed that Dickeya zeae and D. oryzae, were the closest relatives. Phylogenetic analysis based on 463 core gene sequences clearly showed two potentially new species within Dickeya oryzae. In silico DNA– DNA hybridization value of strain PL65T with 12 Type strains of Dickeya species was <68%. Average nucleotide identity (ANI) analysis revealed that PL65T was at the margin of the species delineation cut-off values with a 96% ANI value. The metabolic profile of strain PL65T using BIOLOG differentiated it from the type strains of all other known species of Dickeya. Based on the results of genome-to-genome comparisons and phenotypic data presented in this report, we propose establishment of a new species, Dickeya colocasiae sp. nov. with strain PL65T as the type strain (ICMP 24361T).

Quantitative Determination of the Hydrophobicity of Nanoparticles

Li G, Cao Z, Ho KKHY, Zuo YY

Analytical Chemistry. 2022 Jan 14

Use of MICRO/BEMF Core Facility

The hydrophobicity of nanoparticles (NPs) is one of the most important physicochemical properties that determines their agglomeration state under various environmental conditions. When studying nano-bio interactions, it is found that the hydrophobicity of NPs plays a predominant role in mediating the biological response and toxicity of the NPs. Although many methods have been developed to qualitatively or quantitatively determine hydrophobicity, there is not yet a scientific consensus on the standard of characterizing the hydrophobicity of NPs. We have developed a novel optical method, called the maximum particle dispersion (MPD), for quantitatively characterizing the hydrophobicity of NPs. The principle of measurement of the MPD method lies in the control of the aggregation state of the NPs via manipulating the van der Waals interactions between NPs across a dispersion liquid. We have scrutinized the mechanism of the MPD method using a combination of dynamic light scattering and atomic force microscopy and further verified the MPD method using a completely independent dye adsorption method. The MPD method demonstrated great promise to be developed into an easy-to-use and cost-effective method for quantitatively characterizing the hydrophobicity of NPs.

Relative Dye Adsorption Method for Determining the Hydrophobicity of Nanoparticles

Li G, Ho KKHY, Zuo YY

The Journal of Physical Chemistry C. 2021 Dec 29

Use of MICRO/BEMF Core Facility

The hydrophobicity of nanoparticles (NPs) is a crucial physicochemical property that determines the agglomeration state of NPs under various environmental and biological conditions. It plays a predominant role in determining the toxicity and environmental, health, and safety impact of NPs. However, to date, there is not yet a well-accepted standard method for characterizing the hydrophobicity of NPs. Here, we developed a relative dye adsorption method for determining the hydrophobicity of NPs. This method is modified from the traditional dye partitioning method that uses either the hydrophobic dye, rose bengal (RB), or the hydrophilic dye, Nile blue (NB). By studying the partitioning quotient for both RB and NB, the relative dye adsorption method eliminates the uncertainty introduced by estimating the surface area of NPs dispersed in liquid phases. We have demonstrated the applicability and accuracy of this method by comparing them to the hydrophobicity of NPs determined with the maximum particle dispersion method. It is concluded that the relative dye adsorption method can be used as a more reliable technique than RB or NB partitioning for determining the hydrophobicity of NPs.

Community RNA-Seq: multi-kingdom responses to living versus decaying roots in soil

Nuccio EE, Nguyen NH, et al.

ISME Communications. 2021 Dec 06

Roots are a primary source of organic carbon input in most soils. The consumption of living and detrital root inputs involves multi-trophic processes and multiple kingdoms of microbial life, but typical microbial ecology studies focus on only one or two major lineages. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis (“community RNA-Seq”) and simultaneously assess the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua. Plants were grown in 13CO2-labeled microcosms amended with 15N-root litter to identify the preferences of rhizosphere organisms for root exudates (13C) versus decaying root biomass (15N) using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this group’s more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving complex interactions, and our methodology can be used to track the trajectory of carbon through multi-kingdom soil food webs.

BAP1 forms a trimer with HMGB1 and HDAC1 that modulates gene × environment interaction with asbestos

Novelli F, Bononi A, Wang Q, Bai F, Patergnani S, Kricek F, Haglund E, et al.

PNAS. 2021 Nov 23

Carriers of heterozygous germline BAP1 mutations (BAP1+/−) are affected by the “BAP1 cancer syndrome.” Although they can develop almost any cancer type, they are unusually susceptible to asbestos carcinogenesis and mesothelioma. Here we investigate why among all carcinogens, BAP1 mutations cooperate with asbestos. Asbestos carcinogenesis and mesothelioma have been linked to a chronic inflammatory process promoted by the extracellular release of the high-mobility group box 1 protein (HMGB1). We report that BAP1+/− cells secrete increased amounts of HMGB1, and that BAP1+/− carriers have detectable serum levels of acetylated HMGB1 that further increase when they develop mesothelioma. We linked these findings to our discovery that BAP1 forms a trimeric protein complex with HMGB1 and with histone deacetylase 1 (HDAC1) that modulates HMGB1 acetylation and its release. Reduced BAP1 levels caused increased ubiquitylation and degradation of HDAC1, leading to increased acetylation of HMGB1 and its active secretion that in turn promoted mesothelial cell transformation.

Hiding in plain sight - platelets, the silent carriers of HIV-1

Baumer Y, Weatherby TM, Mitchell BI, et al.

Platelets. 2021 Nov 17

Use of MICRO/BEMF Core Facility

There are approximately 38 million people globally living with Human immunodeficiency virus 1 (HIV-1) and given the tremendous success of combination antiretroviral therapy (cART) this has dramatically reduced mortality and morbidity with prevention benefits. However, HIV-1 persists during cART within the human body and re-appears upon cART interruption. This HIV-1 reservoir remains a barrier to cure with cellular sites of viral persistence not fully understood. In this study we provide evidence corroborating a recently published article in STM demonstrating the role of platelets as a novel cellular disseminator of HIV-1 particles in the setting of viral suppression. Using classical transmission electron microscopy with and without immunogold labeling, we visualize HIV-1 in both platelets and monocytes in cART suppressed HIV donors. Our study suggests that due to the close proximity of platelets and monocytes an alternative life cycle of HIV-1 cycling within monocytes and platelets without the need of active replication under cART occurs. Our findings are supported by the lack of detectable HIV-1 particles in platelets derived from HIV uninfected donors or the 'Berlin' patient suggesting that platelets may serve as an underappreciated hidden bearer for HIV-1 and should be considered in HIV remission studies and trials.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil

Domingo R, Perez C, Klair D, ... Arif M

Scientific Reports. 2021 Nov 09

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

Viral-Like Particles Are Associated With Endosymbiont Pathology in Florida Corals Affected by Stony Coral Tissue Loss Disease

Work TM, Weatherby TM, Landsberg JH, Kiryu Y, Cook SM, Peters EC

Frontiers in Marine Science. 2021 Nov 05

Use of MICRO/BEMF Core Facility

Stony coral tissue loss disease (SCTLD) was first documented in 2014 near the Port of Miami, Florida, and has since spread north and south along Florida’s Coral Reef, killing large numbers of more than 20 species of coral and leading to the functional extinction of at least one species, Dendrogyra cylindrus. SCTLD is assumed to be caused by bacteria based on presence of different molecular assemblages of bacteria in lesioned compared to apparently healthy tissues, its apparent spread among colonies, and cessation of spread of lesions in individual colonies treated with antibiotics. However, light microscopic examination of tissues of corals affected with SCTLD has not shown bacteria associated with tissue death. Rather, microscopy shows dead and dying coral cells and symbiotic dinoflagellates (endosymbionts) indicating a breakdown of host cell and endosymbiont symbiosis. It is unclear whether host cells die first leading to death of endosymbionts or vice versa. Based on microscopy, hypotheses as to possible causes of SCTLD include infectious agents not visible at the light microscopy level or toxicosis, perhaps originating from endosymbionts. To clarify this, we examined corals affected with SCTLD and apparently healthy corals using transmission electron microscopy. Endosymbionts in SCTLD-affected and apparently healthy corals consistently had varying degrees of pathology associated with elongated particles compatible in morphology with filamentous positive single-stranded RNA viruses of plants termed anisometric viral-like particles (AVLP). There was apparent progression from early to late replication of AVLP in the cytoplasm of endosymbionts adjacent to or at times within chloroplasts, with morphologic changes in chloroplasts consistent with those seen in plant cells infected by viruses. Coral host cell pathology appeared limited to massive proliferation and lysis of mucus cells. Based on these findings, we hypothesize that SCTLD is a viral disease of endosymbionts leading to coral host death. Efforts to confirm the presence of a virus associated with SCTLD through other means would be appropriate. These include showing the presence of a virus through molecular assays such as deep sequencing, attempts to grow this virus in the laboratory through culture of endosymbionts, localization of virus in tissue sections using immunohistochemistry or in situ hybridization, and experimental infection of known-virus-negative corals to replicate disease at the gross and microscopic level.

Transcriptional acclimation and spatial differentiation characterize drought response by the ectomycorrhizal fungus Suillus pungens

Erlandson SR, Margis R, Ramirez A, Nguyen N, et al.

The New Phytologist. 2021 Nov 02

In this study, we used RNA-sequencing (RNA-Seq) to measuretranscriptional changes in the fungusSuillus pungens(SP) exposedto experimental drought. We grewSPin symbiosis withPinusmuricata(PM) in a growth chamber where we manipulated bothlong-term (chronic) and short-term (acute) soil moisture andsequenced messenger RNA (mRNA) extracted from both ectomy-corrhizal roots and extraradical mycelium (ERM). Our primaryaims were to: (1) demonstrate the utility of RNA-Seq to quantifyresponse to global change stressors for EMF; (2) identify biologicalfunctions involved in drought response by comparing geneexpression across moisture treatments and in different organs ofEMF; and (3) estimate the potential for fungal acclimation bycomparing gene expression between chronic and acute droughtstress treatments.

Wild gut microbiomes reveal individuals, species, and location as drivers of variation in two critically endangered Hawaiian honeycreepers

Costantini MS, Medeiros MCI, Crampton LH, Reed FA

PeerJ. 2021 Oct 28

The gut microbiome of animals is an important component that has strong influence on the health, fitness, and behavior of its host. Most research in the microbiome field has focused on human populations and commercially important species. However, researchers are now considering the link between endangered species conservation and the microbiome. In Hawaiʻi, several threats (e.g., avian malaria and habitat loss) have caused widespread population declines of Hawaiian honeycreepers (subfamily: Carduelinae). These threats can have a significant effect on the avian gut microbiome and may even lead to disruption of microbial function. However, the gut microbiome of honeycreeper in the wild has yet to be explored.

Repression precedes independent evolutionary gains of a highly specific gene expression pattern

Pu J, Wang Z, Cong H, Chin JSR, Justen J, Finet C, Yew JY, Chung H

Cell Reports. 2021 Oct 26

Use of MGAL Core Facility

Highly specific expression patterns can be caused by the overlapping activities of activator and repressor sequences in enhancers. However, few studies illuminate how these sequences evolve in the origin of new enhancers. Here, we show that expression of the bond gene in the semicircular wall epithelium (swe) of the Drosophila melanogaster male ejaculatory bulb (EB) is controlled by an enhancer consisting of an activator region that requires Abdominal-B driving expression in the entire EB and a repressor region that restricts this expression to the EB swe. Although this expression pattern is independently gained in the distantly related Scaptodrosophila lebanonensis and does not require Abdominal-B, we show that functionally similar repressor sequences are present in Scaptodrosophila and also in species that do not express bond in the EB. We suggest that during enhancer evolution, repressor sequences can precede the evolution of activator sequences and may lead to similar but independently evolved expression patterns.

Mosquito Microbiome Diversity Varies Along a Landscape-Scale Moisture Gradient

Medeiros MCI, Seabourn PS, Rollins RL, Yoneishi NM

Microbial Ecology. 2021 Oct 06

Microorganisms live in close association with metazoan hosts and form symbiotic microbiotas that modulate host biology. Although the function of host-associated microbiomes may change with composition, hosts within a population can exhibit high turnover in microbiome composition among individuals. However, environmental drivers of this variation are inadequately described. Here, we test the hypothesis that this diversity among the microbiomes of Aedes albopictus (a mosquito disease vector) is associated with the local climate and land-use patterns on the high Pacific island of O 'ahu, Hawai 'i. Our principal finding demonstrates that the relative abundance of several bacterial symbionts in the Ae. albopictus microbiome varies in response to a landscape-scale moisture gradient, resulting in the turnover of the mosquito microbiome composition across the landscape. However, we find no evidence that mosquito microbiome diversity is tied to an index of urbanization. This result has implications toward understanding the assembly of host-associated microbiomes, especially during an era of rampant global climate change.

Toward a Revision of the Bamboo Corals: Part 2, Untangling the Genus Lepidisis (Octocorallia: Isididae)

Watling L, France SC

Bulletin of the Peabody Museum of Natural History. 2021 Oct 05

Use of MICRO/BEMF Core Facility

The genus Lepidisis was established by Verrill in 1883 for three species collected from the continental slope in the western North Atlantic Ocean. Over the intervening years, several species were assigned to this genus using conflicting characters taken from Verrill's original description. Using what remains of Verrill's syntype specimens and a new specimen collected from off of the coast of the New England region, USA, we show that Verrill made a serious mistake in his description of the type species, and that mistake has caused some of the confusion surrounding this genus. We redescribe the type species, then use that description to evaluate the other species currently assigned to the genus.

Formation of a colloidal band via pH-dependent electrokinetics

Doan VS, Shin S

Electrophoresis. 2021 Oct 04

Use of MICRO/BEMF Core Facility

Electroosmosis on nonuniformly charged surfaces often gives rise to intriguing flow behaviors, which can be utilized in applications such as mixing processes and designing micromotors. Here, we demonstrate nonuniform electroosmosis induced by electrochemical reactions. Water electrolysis creates pH gradients near the electrodes that cause a spatiotemporal change in the wall zeta potential, leading to nonuniform electroosmosis. Such nonuniform EOFs induce multiple vortices, which promote the continuous accumulation of particles that subsequently form a colloidal band. The band develops vertically into a "wall" of particles that spans from the bottom to the top surface of the chamber. Such a flow-driven colloidal band can be potentially used in colloidal self-assembly and separation processes irrespective of the particle surface properties. For instance, we demonstrate these vortices can promote rapid segregation of soft colloids such as oil droplets and fat globules.

Social-like responses are inducible in the asocial and blind Mexican cavefish despite the continued exhibition of strong repetitive behavior

Iwashita M, Yoshizawa M

eLife. 2021 Sep 20

Social behavior is a hallmark of complex animal systems; however, some species appear to have secondarily lost this social ability. In these non-social species, whether social abilities are permanently lost or suppressed is unclear. The blind cavefish Astyanax mexicanus is known to be asocial. Here, we reveal that cavefish exhibited social-like interactions in familiar environments but suppressed these interactions in stress-associated unfamiliar environments. Furthermore, the level of suppression in sociality was positively correlated with that of stereotypic repetitive behavior, as seen in mammals. Treatment with a human antipsychotic drug targeting the dopaminergic system induced social-like interactions in cavefish, even in unfamiliar environments, while reducing repetitive behavior. Overall, these results suggest that the antagonistic association between repetitive and social-like behaviors is deeply shared from teleosts through mammals.

Bacterial Quorum-Sensing Regulation Induces Morphological Change in a Key Host Tissue during the Euprymna scolopes-Vibrio fischeri Symbiosis

Essock-Burns T, Bennett BD, Arencibia D, Moriano-Gutierrez S, Medeiros M, McFall-Ngai MJ, Ruby EG

mBio. 2021 Sep 28

Use of MICRO/BEMF Core Facility

Microbes colonize the apical surfaces of polarized epithelia in nearly all animal taxa. In one example, the luminous bacterium Vibrio fischeri enters, grows to a dense population within, and persists for months inside, the light-emitting organ of the squid Euprymna scolopes. Crucial to the symbiont’s success after entry is the ability to trigger the constriction of a host tissue region (the “bottleneck”) at the entrance to the colonization site. Bottleneck constriction begins at about the same time as bioluminescence, which is induced in V. fischeri through an autoinduction process called quorum sensing. Here, we asked the following questions: (i) Are the quorum signals that induce symbiont bioluminescence also involved in triggering the constriction? (ii) Does improper signaling of constriction affect the normal maintenance of the symbiont population? We manipulated the presence of three factors, the two V. fischeri quorum signal synthases, AinS and LuxI, the transcriptional regulator LuxR, and light emission itself, and found that the major factor triggering and maintaining bottleneck constriction is an as yet unknown effector(s) regulated by LuxIR. Treating the animal with chemical inhibitors of actin polymerization reopened the bottlenecks, recapitulating the host’s response to quorum-sensing defective symbionts, as well as suggesting that actin polymerization is the primary mechanism underlying constriction. Finally, we found that these host responses to the presence of symbionts changed as a function of tissue maturation. Taken together, this work broadens our concept of how quorum sensing can regulate host development, thereby allowing bacteria to maintain long-term tissue associations.

Fungal Community Shift Along Steep Environmental Gradients from Geothermal Soils in Yellowstone National Park

Bazzicalupo AL, Erlandson S, Branine M, Ratz M, Ruffing L, Nguyen NH, Branco S

Microbial Ecology. 2021 Sep 01

Geothermal soils offer unique insight into the way extreme environmental factors shape communities of organisms. However, little is known about the fungi growing in these environments and in particular how localized steep abiotic gradients affect fungal diversity. We used metabarcoding to characterize soil fungi surrounding a hot spring-fed thermal creek with water up to 84 °C and pH 10 in Yellowstone National Park. We found a significant association between fungal communities and soil variable principal components, and we identify the key trends in co-varying soil variables that explain the variation in fungal community. Saprotrophic and ectomycorrhizal fungi community profiles followed, and were significantly associated with, different soil variable principal components, highlighting potential differences in the factors that structure these different fungal trophic guilds. In addition, in vitro growth experiments in four target fungal species revealed a wide range of tolerances to pH levels but not to heat. Overall, our results documenting turnover in fungal species within a few hundred meters suggest many co-varying environmental factors structure the diverse fungal communities found in the soils of Yellowstone National Park.

Plasticity in the grazing ecophysiology of Florenciella (Dichtyochophyceae), a mixotrophic nanoflagellate that consumes Prochlorococcus and other bacteria

Li Q, Edwards KF, Schvarcz CR, Selph KE, Steward GF

Limnology and Oceanography. 2021 Aug 31

Use of MICRO/BEMF Core Facility

Mixotrophic nanoflagellates can account for more than half of the bacterivory in the sunlit ocean, yet very little is known about their ecophysiology. Here, we characterize the grazing ecology of an open-ocean mixotroph in the genus Florenciella (class Dictyochophyceae). Members of this class were indirectly implicated as major consumers of Prochlorococcus and Synechococcus in the oligotrophic North Pacific Subtropical Gyre, but their phagotrophic capabilities have never been investigated. Our studies showed that Florenciella readily consumed Prochlorococcus, Synechococcus, and heterotrophic bacteria, and that the ingested prey relieved nutrient limitations on growth. Florenciella grew faster (3 d−1) in nitrogen-deplete medium given sufficient live Synechococcus, than in nitrogen-replete K medium (2 d−1), but it did not grow in continuous darkness. Grazing rates were substantially higher under nutrient limitation and showed a hint of diel variability, with rates tending to be highest near the end of the light period. An apparent trade-off between the maximum clearance rate (5 nL Florenciella−1 h−1) and the maximum ingestion rate (up to ∼ 10 prey cells Florenciella−1 h−1) across experiments suggests that grazing behavior may also vary in response to prey concentration. If the observed grazing rates are representative of other open-ocean mixotrophs, their collective activity could account for a significant fraction of the daily cyanobacterial mortality. This study provides essential parameters for understanding the grazing ecology of a common marine mixotroph and the first characterization of mixotrophic nanoflagellate functional responses when feeding on unicellular cyanobacteria, the dominant marine primary producers in the oligotrophic ocean.

Evaluation of Gene Knockouts by CRISPR as Potential Targets for the Genetic Engineering of the Mosquito Culex quinquefasciatus

Feng X, Kambic L, Nishimoto JHK, Reed FA, Denton JA, Sutton JT, Gantz VM

The CRISPR Journal. 2021 Aug 16

Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people and, separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations. Yet, research into Cx. quinquefasciatus has been lagging compared to other disease vectors. Here, we use this tool to disrupt a set of five pigmentation genes in Cx. quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Cx. quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia, a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Cx. quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily scored transgenesis marker.

Genomic and Phenotypic Biology of Novel Strains of Dickeya zeae Isolated From Pineapple and Taro in Hawaii: Insights Into Genome Plasticity, Pathogenicity, and Virulence Determinants

Boluk G, Arizala D, Dobhal S, Zhang J, Hu J, Alvarez AM, Arif M

Frontiers in Plant Science. 2021 Aug 11

Dickeya zeae, a bacterial plant pathogen of the family Pectobacteriaceae, is responsible for a wide range of diseases on potato, maize, rice, banana, pineapple, taro, and ornamentals and significantly reduces crop production. D. zeae causes the soft rot of taro (Colocasia esculenta) and the heart rot of pineapple (Ananas comosus). In this study, we used Pacific Biosciences single-molecule real-time (SMRT) sequencing to sequence two high-quality complete genomes of novel strains of D. zeae: PL65 (size: 4.74997 MB; depth: 701x; GC: 53.6%) and A5410 (size: 4.7792 MB; depth: 558x; GC: 53.5%) isolated from economically important Hawaiian crops, taro, and pineapple, respectively. Additional complete genomes of D. zeae representing three additional hosts (philodendron, rice, and banana) and other species used for a taxonomic comparison were retrieved from the NCBI GenBank genome database. Genomic analyses indicated the truncated type III and IV secretion systems (T3SS and T4SS) in the taro strain, which only harbored one and two genes of T3SS and T4SS, respectively, and showed high heterogeneity in the type VI secretion system (T6SS). Unlike strain EC1, which was isolated from rice and recently reclassified as D. oryzae, neither the genome PL65 nor A5410 harbors the zeamine biosynthesis gene cluster, which plays a key role in virulence of other Dickeya species. The percentages of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between the two genomes were 94.47 and 57.00, respectively. In this study, we compared the major virulence factors [plant cell wall-degrading extracellular enzymes and protease (Prt)] produced by D. zeae strains and evaluated the virulence on taro corms and pineapple leaves. Both strains produced Prts, pectate lyases (Pels), and cellulases but no significant quantitative differences were observed (p > 0.05) between the strains. All the strains produced symptoms on taro corms and pineapple leaves, but the strain PL65 produced symptoms more rapidly than others. Our study highlights the genetic constituents of pathogenicity determinants and genomic heterogeneity that will help to understand the virulence mechanisms and aggressiveness of this plant pathogen.

A shift to shorter cuticular hydrocarbons accompanies sexual isolation among Drosophila americana group populations

Davis JS, Pearcy MJ, Yew JY, Moyle LC

Evolution Letters. 2021 Jul 17

Use of MGAL Core Facility

Because sensory signals often evolve rapidly, they could be instrumental in the emergence of reproductive isolation between species. However, pinpointing their specific contribution to isolating barriers, and the mechanisms underlying their divergence, remains challenging. Here, we demonstrate sexual isolation due to divergence in chemical signals between two populations of Drosophila americana (SC and NE) and one population of D. novamexicana, and dissect its underlying phenotypic and genetic mechanisms. Mating trials revealed strong sexual isolation between Drosophila novamexicana males and SC Drosophila americana females, as well as more moderate bi-directional isolation between D. americana populations. Mating behavior data indicate SC D. americana males have the highest courtship efficiency and, unlike males of the other populations, are accepted by females of all species. Quantification of cuticular hydrocarbon (CHC) profiles-chemosensory signals that are used for species recognition and mate finding in Drosophila-shows that the SC D. americana population differs from the other populations primarily on the basis of compound carbon chain-length. Moreover, manipulation of male CHC composition via heterospecific perfuming-specifically perfuming D. novamexicana males with SC D. americana males-abolishes their sexual isolation from these D. americana females. Of a set of candidates, a single gene-elongase CG17821-had patterns of gene expression consistent with a role in CHC differences between species. Sequence comparisons indicate D. novamexicana and our Nebraska (NE) D. americana population share a derived CG17821 truncation mutation that could also contribute to their shared "short" CHC phenotype. Together, these data suggest an evolutionary model for the origin and spread of this allele and its consequences for CHC divergence and sexual isolation in this group.

Variable coverage in an Autocidal Gravid Ovitrap intervention impacts efficacy of Aedes aegypti control

Juarez JG, Chaves LF, Garcia-Luna SM, Martin E, Badillo-Vargas I, Medeiros MCI, Hamer GL

The Journal of Applied Ecology. 2021 Jul 13

Control of the arboviral disease vector Aedes aegypti has shown variable levels of efficacy around the globe. We evaluated an Autocidal Gravid Ovitrap (AGO) intervention as a stand‐alone control tool for population suppression of A. aegypti in US communities bordering Mexico. We conducted a cluster randomized crossover trial with weekly mosquito surveillance of sentinel households from July 2017 to December 2018. The intervention took place from August to December of both years. Multilevel models (generalized linear and additive mixed models) were used to analyse the changes in population abundance of female A. aegypti. We observed that female populations were being suppressed 77% (2018) and four times lower outdoor female abundance when AGO coverage (number of intervention AGO traps that surrounded a sentinel home) was high (2.7 AGOs/house). However, we also observed that areas with low intervention AGO coverage resulted in no difference (2017) or slightly higher abundance compared to the control. These results suggest that coverage rate might play a critical role on how populations of female A. aegypti are being modulated in the field. The lack of larval source habitat reduction and the short duration of the intervention period might have limited the A. aegypti population suppression observed in this study. Synthesis and applications. The mosquito, A. aegypti, is a public health concern in most tropical and subtropical regions. With the rise of insecticide resistance, the evaluation of non‐chemical tools has become pivotal in the fight against arboviral disease transmission. Our study shows that the AGO intervention, as a stand‐alone control tool, is limited by its coverage in human settlements. Vector control programmes should consider, that if the target coverage rate is not achieved, measures will be ineffective unless coupled with other control approaches. Although our multilevel modelling was focused on A. aegypti and the AGO, the approach can be applied to other mosquito vector species.

The slippery nature of ectomycorrhizal host specificity: Suillus fungi associated with novel pinoid ( Picea) and abietoid ( Abies) hosts

Pérez-Pazos E, Certano A, Gagne J, Lebeuf R, Siegel N, Nguyen N, Kennedy PG

Mycologia. 2021 Jul 08

Suillus is among the best-known examples of an ectomycorrhizal (ECM) fungal genus that demonstrates a high degree of host specificity. Currently recognized host genera of Suillus include Larix, Pinus, and Pseudotsuga, which all belong to the pinoid clade of the family Pinaceae. Intriguingly, Suillus sporocarps have been sporadically collected in forests in which known hosts from these genera are locally absent. To determine the capacity of Suillus to associate with alternative hosts in both the pinoid and abietoid clades of Pinaceae, we examined the host associations of two Suillus species (S. punctatipes and S. glandulosus) through field-based root tip sampling and seedling bioassays. Root tip collections underneath Suillus sporocarps were molecularly identified (fungi: nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS barcode]; plant: trnL) to assess the association with multiple hosts. The bioassays contained both single- and two-species treatments, including a primary (Larix or Pseudotsuga) and a secondary (Picea, Pinus, or Abies) host. For the S. punctatipes bioassay, an additional treatment in which the primary host was removed after 8 mo was included to assess the effect of primary host presence on longer-term ECM colonization. The field-based results confirmed that Suillus fungi were able to associate with Abies and Tsuga hosts, representing novel host genera for this genus. In the bioassays, colonization on the primary hosts was detected in both single- and two-species treatments, but no colonization was present when Picea and Abies hosts were grown alone. Removal of a primary host had no effect on percent ECM colonization, suggesting that primary hosts are not necessary for sustaining Suillus colonization once they are successfully established on secondary hosts. Collectively, our results indicate that host specificity is more flexible in this genus than previously acknowledged and help to explain the presence of Suillus in forests where recognized hosts are not present.

Field-Deployable Recombinase Polymerase Amplification Assay for Specific, Sensitive and Rapid Detection of the US Select Agent and Toxigenic Bacterium, Rathayibacter toxicus

Arif M, Busot GY, Mann R, Rodoni B, Stack JP

Biology (Basel). 2021 Jul 03

Rathayibacter toxicus is a toxigenic bacterial pathogen of several grass species and is responsible for massive livestock deaths in Australia and South Africa. Due to concern for animal health and livestock industries, it was designated a U.S. Select Agent. A rapid, accurate, and sensitive in-field detection method was designed to assist biosecurity surveillance surveys and to support export certification of annual ryegrass hay and seed. Complete genomes from all known R. toxicus populations were explored, unique diagnostic sequences identified, and target-specific primers and a probe for recombinase polymerase amplification (RPA) and endpoint PCR were designed. The RPA reaction ran at 37 °C and a lateral flow device (LFD) was used to visualize the amplified products. To enhance reliability and accuracy, primers and probes were also designed to detect portions of host ITS regions. RPA assay specificity and sensitivity were compared to endpoint PCR using appropriate inclusivity and exclusivity panels. The RPA assay sensitivity (10 fg) was 10 times more sensitive than endpoint PCR with and without a host DNA background. In comparative tests, the RPA assay was unaffected by plant-derived amplification inhibitors, unlike the LAMP and end-point PCR assays. In-field validation of the RPA assay at multiple sites in South Australia confirmed the efficiency, specificity, and applicability of the RPA assay. The RPA assay will support disease management and evidence-based in-field biosecurity decisions.

An ion‐paired moxifloxacin nanosuspension eye drop provides improved prevention and treatment of ocular infection

Josyula A, Omiadze R, Parikh K, et al.

Bioengineering and Translational Medicine. 2021 Jun 22

Use of MICRO/BEMF Core Facility

There are numerous barriers to achieving effective intraocular drug administration, including the mucus layer protecting the ocular surface. For this reason, antibiotic eye drops must be used multiple times per day to prevent and treat ocular infections. Frequent eye drop use is inconvenient for patients, and lack of adherence to prescribed dosing regimens limits treatment efficacy and contributes to antibiotic resistance. Here, we describe an ion‐pairing approach used to create an insoluble moxifloxacin–pamoate (MOX–PAM) complex for formulation into mucus‐penetrating nanosuspension eye drops (MOX–PAM NS). The MOX–PAM NS provided a significant increase in ocular drug absorption, as measured by the area under the curve in cornea tissue and aqueous humor, compared to Vigamox in healthy rats. Prophylactic and treatment efficacy were evaluated in a rat model of ocular Staphylococcus aureus infection. A single drop of MOX–PAM NS was more effective than Vigamox, and completely prevented infection. Once a day dosing with MOX–PAM NS was similar, if not more effective, than three times a day dosing with Vigamox for treating S. aureus infection. The MOX–PAM NS provided increased intraocular antibiotic absorption and improved prevention and treatment of ocular keratitis, and the formulation approach is highly translational and clinically relevant.

Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis

Larrea-Sarmiento A, Stack JP, Alvarez AM, Arif M

Scientific Reports. 2021 Jun 08

Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.

Sea star wasting disease pathology in Pisaster ochraceus shows a basal-to-surface process affecting color phenotypes differently

Work TM, Weatherby TM, DeRito CM, Besemer RM, Hewson I

Diseases of Aquatic Organisms. 2021 Jun 03

Use of MICRO/BEMF Core Facility

Sea star wasting disease (SSWD) refers to a suite of poorly described non-specific clinical signs including abnormal posture, epidermal ulceration, and limb autotomy (sloughing) causing mortalities of over 20 species of sea stars and subsequent ecological shifts throughout the northeastern Pacific. While SSWD is widely assumed to be infectious, with environmental conditions facilitating disease progression, few data exist on cellular changes associated with the disease. This is unfortunate, because such observations could inform mechanisms of disease pathogenesis and host susceptibility. Here, we replicated SSWD by exposing captive Pisaster ochraceus to a suite of non-infectious organic substances and show that development of gross lesions is a basal-to-surface process involving inflammation (e.g. infiltration of coelomocytes) of ossicles and mutable collagenous tissue, leading to epidermal ulceration. Affected sea stars also manifest increases in a heretofore undocumented coelomocyte type, spindle cells, that might be a useful marker of inflammation in this species. Finally, compared to purple morphs, orange P. ochraceus developed more severe lesions but survived longer. Longer-lived, and presumably more visible, severely-lesioned orange sea stars could have important demographic implications in terms of detectability of lesioned animals in the wild and measures of apparent prevalence of disease.

Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis

Dow A, Sule P, O'Donnell TJ, et al.

PLoS Pathogens. 2021 May 14

Use of MICRO/BEMF Core Facility

Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ sequestration is part of “nutritional immunity”, where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.

First Report of Pectobacterium brasiliense causing soft rot on mizuna (Brassica rapa var. japonica) in the United States

Klair D, Silva J, Dario Arizala E, Boluk G, Dobhal S, Ahmad AA, Uyeda J, Alvarez AM, Arif M

Plant Disease. 2021 May 10

Mizuna (Brassica rapa var. japonica), a member of family Brassicaceae, is a leafy vegetable having phenolic and other compounds beneficial to human health, such as natural antioxidants (Khanam et al. 2012). In October 2020, a field of mizuna (variety: Early) on Oahu island was observed having 20-30% diseased plants. Four randomly selected infected mizuna plants, showing the symptoms of wilt and stem rot (Figure 1A-D), were collected and isolations were made to determine the pathogen.

Small sections of infected stems were cut, surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The tissues were macerated in a sterile 1.5 ml centrifuge tube containing 100 μl sterile water-macerated tissues were streaked onto crystal violet pectate medium (CVP) (Hélias et al. 2011) and incubated at 26 ± 2°C for 48 h. Isolated bacterial colonies that formed pits on the CVP plates were re-streaked onto dextrose peptone agar: Peptone (10 g/L), Dextrose (5 g/L) and Agar (17 g/L) (DPA-without tetrazolium chloride; Norman and Alvarez 1989) to obtain purified colonies for DNA isolation using DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA). The two housekeeping genes (dnaA and gapA) were amplified and sequenced following the protocols used by Dobhal et al. (2020) and Boluk et al. (2020), for identity confirmation and phylogenetic analysis. Cleaned PCR products were sent to the GENEWIZ facility (Genewiz, La Jolla, CA) for sequencing of sense and antisense strands. The obtained sequences were aligned, manually edited, and consensus sequences were analyzed with BLASTn using the NCBI GenBank nucleotide and genome databases for identity confirmation. The BLASTn results demonstrated 100% query coverage of all four strains (PL248-PL251); and showed 100% identity of PL248 and PL249, and 99% identity of PL250 and PL251 with Pectobacterium brasiliense. All the sequences were submitted to the NCBI GenBank database under the following accession numbers: dnaA gene MW560271 – MW560274 (PL248 – PL251); and gapA gene MW560275 – MW560278 (PL248 – PL251). Pathogenicity was assessed by artificially inoculating 100 µl bacterial suspension of each strain (PL248 – 1.12x 10⁸ CFU/ml; PL249 – 1.32x 10⁸ CFU/ml; PL 250 – 1.2x 10⁸ CFU/ml and PL251 – 1.15x 10⁸ CFU/ml) onto four-week-old mizuna (variety: Leafy Asian Greens) plants in three replicates, using sterile pipette tips, which was stabbed into stem halfway and wrapped with parafilm. The inoculated plants were well maintained under controlled greenhouse conditions. As negative controls, three plants were inoculated with 100 µl distilled water. Soft rot and wilt symptoms (Figure 1E-H) were observed 24 hours post inoculation. No symptoms were observed on control plants (Figure 1F). All four strains were re-isolated from the inoculated plants and confirmed as P. brasiliense based on resequencing of the dnaA region and 100% homology with the sequences of original strain. In the phylogenetic tree (Figure 2), based on two housekeeping genes (dnaA and gapA), the bacterial strains from mizuna grouped with other P. brasiliense retrieved from the NCBI GenBank database. To our knowledge, this is the first report of P. brasiliense infecting mizuna plants in Hawaii or in the USA and is important because this species is one of the most aggressive pectolytic pathogens in the genus Pectobacterium. Understanding the diversity of different pectolytic phytopathogens is essential to formulating risk mitigation strategies as P. brasiliense could potentially pose a threat to additional vegetable crops, especially the crucifers vegetables (Arizala et al. 2019; Klair et al, 2021).

Restoration of the mycobiome of the endangered Hawaiian mint Phyllostegia kaalaensis increases its resistance to a common powdery mildew

Egan CP, Koko JH, Muir CD, Zahn G, Swift SOI, Amend AS, Hynson NA

Fungal Ecology. 2021 May 01

Use of MICRO/BEMF Core Facility

Beneficial microbes such as plant mutualistic fungi, hold the promise of ameliorating challenges faced in native plant conservation such as disease management. As an alternative to costly chemical pest control, conservation efforts could potentially harness the benefits of plant mutualistic fungi to aid in defense and disease resistance, but there are few tests of this notion. We set out to test the efficacy of controlling a common foliar pathogen, the powdery mildew Neoerysiphe galeopsidis, by inoculating the endangered Hawaiian plant species Phyllostegia kaalaensis with potentially beneficial members of its wild-type mycobiome. We tested whether inoculating plants with above or belowground fungal mutualists, or both, led to increased disease resistance in the host. We found that while all treatments reduced average disease incidence, colonization by the foliar yeast Moesziomyces aphidis was the only treatment to do so significantly. These results provide an exciting new strategy for plant conservation practices.

Reuse and recycle: Integrating aquaculture and agricultural systems to increase production and reduce nutrient pollution

Farrant DN, Frank KL, Larsen AE

Science of The Total Environment. 2021 Apr 20

Integrated agriculture and aquaculture systems (IAAS) allow nutrients, energy, and water to flow throughout the components of the system, increasing the efficiency with which inputs are converted to food. Yet effectively designing an IAAS requires understanding how nutrients accumulate and alter the system's productivity. Here we developed a mechanistic model for nitrogen transport and utilization and parameterized it using the IAAS in He'eia, Hawai'i. Of note, we modeled tidal influence, which extends existing IAAS models that often assume aquaculture in tank enclosures. We simulated the impact of nitrogen loading from three possible land use scenarios across agriculture and development priorities on the productivity of the fishpond downstream. We projected that organic nitrogen and nitrate concentrations parallel the successive increases in nitrogen loading across management strategies. Autotroph and fish densities were predicted to follow similar trends in response to increased nitrogen availability, causing fish harvests to increase from the current land use (25 kg/ha) to the restored agriculture (35 kg/ha) and urban (50 kg/ha) alternatives. While fish harvests were predicted to be highest in the urban scenario, modeled caloric production in the restored scenario from agriculture and aquaculture would sustain 235 people (4.3 people/ha) in the He'eia IAAS, 16 and 125 times more than the current or urban land uses, respectively. Restoring diversified agriculture was also predicted to retain a larger proportion of nitrogen inputs (0.43) than urbanizing the region (0.30), which would reduce nitrogen export to the adjacent Kāne'ohe Bay. Several state variables were notably sensitive to tidal flux rates, highlighting the importance of incorporating tidal dynamics into a coastal IAAS model. This model provides valuable insights for the management of existing coastal IAAS and design of new IAAS in coastal regions to improve the sustainability of future food systems.

Fungal-Bacterial Cooccurrence Patterns Differ between Arbuscular Mycorrhizal Fungi and Nonmycorrhizal Fungi across Soil Niches

Yuan MM, Kakouridis A, Starr E, Nguyen NH, Shi S, Pett-Ridge J, Nuccio E, Zhou J, Firestone M

mBio. 2021 Apr 20

Soil bacteria and fungi are known to form niche-specific communities that differ between actively growing and decaying roots. Yet almost nothing is known about the cross-kingdom interactions that frame these communities and the environmental filtering that defines these potentially friendly or competing neighbors. We explored the temporal and spatial patterns of soil fungal (mycorrhizal and nonmycorrhizal) and bacterial cooccurrence near roots of wild oat grass, Avena fatua, growing in its naturalized soil in a greenhouse experiment. Amplicon sequences of the fungal internal transcribed spacer (ITS) and bacterial 16S rRNA genes from rhizosphere and bulk soils collected at multiple plant growth stages were used to construct covariation-based networks as a step toward identifying fungal-bacterial associations. Corresponding stable-isotope-enabled metagenome-assembled genomes (MAGs) of bacteria identified in cooccurrence networks were used to inform potential mechanisms underlying the observed links. Bacterial-fungal networks were significantly different in rhizosphere versus bulk soils and between arbuscular mycorrhizal fungi (AMF) and nonmycorrhizal fungi. Over 12 weeks of plant growth, nonmycorrhizal fungi formed increasingly complex networks with bacteria in rhizosphere soils, while AMF more frequently formed networks with bacteria in bulk soils. Analysis of network-associated bacterial MAGs suggests that some of the fungal-bacterial links that we identified are potential indicators of bacterial breakdown and consumption of fungal biomass, while others intimate shared ecological niches.

Multiple internal controls enhance reliability for PCR and real time PCR detection of Rathayibacter toxicus

Arif M, Busot GY, Mann R, Rodoni B, Stack JP

Scientific Reports. 2021 Apr 16

Rathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa. A threat to livestock industries globally, the bacterium was designated a U.S. Select Agent. Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns. Whole genomes of three genetic populations of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus to the population level of discrimination. Specificity was confirmed with appropriate inclusivity and exclusivity panels; no cross reactivity was observed. The endpoint multiplex PCR and TaqMan qPCR assays detected 10 fg and 1 fg of genomic DNA, respectively. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls. Assays were validated by blind ring tests with multiple operators in three international laboratories.

Formation and Function of the Primary Tube During Settlement and Metamorphosis of the Marine Polychaete Hydroides elegans (Haswell, 1883) (Serpulidae)

Huggett MJ, Carpizo-Ituarte EJ, Nedved BT, Hadfield MG

The Biological Bulletin. 2021 Apr 14

Use of MICRO/BEMF Core Facility

The serpulid polychaete Hydroides elegans has emerged as a major model organism for studies of marine invertebrate settlement and metamorphosis and for processes involved in marine biofouling. Rapid secretion of an enveloping, membranous, organic primary tube provides settling larvae of H. elegans firm adhesion to a surface and a refuge within which to complete metamorphosis. While this tube is never calcified, it forms the template from which the calcified tube is produced at its anterior end. Examination of scanning and transmission electron micrographs of competent and settling larvae revealed that the tube is secreted from epidermal cells of the three primary segments, with material possibly transported through the larval cuticle via abundant microvilli. The tube is composed of complexly layered fibrous material that has an abundance of the amino acids that characterize the collagenous cuticle of other polychaetes, plus associated carbohydrates. The significance of the dependence on surface bacterial biofilms for stimulating settlement in this species is revealed as a complex interaction between primary tube material, as it is secreted, and the extracellular polymeric substances abundantly produced by biofilm-residing bacteria. This association appears to provide the settling larvae with an adhesion strength similar to that of bacteria in a biofilm and significantly less when larvae settle on a clean surface.

Assessing the Effects of Digestates and Combinations of Digestates and Fertilizer on Yield and Nutrient Use of Brassica juncea (Kai Choy)

Jamison J, Khanal SK, Nguyen NH, Deenik JL

Agronomy. 2021 Mar 09

Anaerobic digestion of organic wastes produces solid residues known as digestates, which have potential as a fertilizer and soil amendment. The majority of research on digestate focuses on their fertilizer value. However, there is a lack of information about additional effects they may have on plant growth, both positive and negative. Understanding the effects of digestate on plant growth is essential to optimizing their use in agriculture and helping close the loop of material and energy balances. This greenhouse study evaluated the effects of two different digestates, a food waste digestate (FWD) and a lignocellulosic biomass digestate (LBD); a liquid fertilizer; and various combinations of fertilizer and digestates on plant growth, nutrient uptake and nutrient use efficiency (NUE) of Brassica juncea (kai choy) plants. It also evaluated potential negative attributes of the digestates, including salinity and possible biohazards. Combinations of LBD and fertilizer performed as well or slightly better than the fertilizer control for most parameters, including aboveground biomass and root length. These same combinations had significantly higher nitrogen use efficiency than the fertilizer control. Inhibitory effects were observed in 100% LBD treatments, likely due to the high electrical conductivity of the media from digestate application. Based on this research, LBD could partially replace mineral fertilizers for kai choy at up to 50% of the target nitrogen rate and may lead to increased plant growth beyond mineral fertilizers. FWD could replace up to 100% of the target nitrogen application, without causing significant negative effects on plant growth. Increasing the use of digestates in agriculture will provide additional incentives for the anaerobic digestion process, as it produces two valuable products: biogas for energy and digestate for fertilizer.

First Report of Bacterial Soft Rot Disease on Pak Choi (Brassica rapa subsp. chinensis) caused by Pectobacterium brasiliense in the United States

Klair D, Boluk G, Silva J, Dario Arizala E, Dobhal S, Arif M

Plant Disease. 2021 Mar 1

Pak choi (Brassica rapa subsp. chinensis) is an important vegetable crop native to China, known for high water content and low caloric value, containing high quality of protein, carbohydrates, fiber, vitamins, minerals, and secondary plant metabolites (Acikgoz, 2016). A pak choi field (8,000 sq. ft.) on Oahu, Hawaii, was visited in May 2020. About 10% plants were infected and showed characteristic symptoms of soft rot, wet lesions, macerated infected stem and necrotic leaves (Figure1A-D); leading to the suspect of one of the most devastating bacterial pathogens within genus Pectobacterium (Boluk et al. 2020; Li et al. 2019; Arizala et al. 2020; Arizala and Arif, 2019).

Four infected plants were collected from the field, and stems were surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The stems were aseptically macerated, streaked on Crystal violet pectate medium (CVP) (Hélias et al. 2011), and incubated for 48 h at 26 ± 2°C. The peculiar morphological characteristic of pectolytic bacterial pathogen, forming pits on CVP, were observed (Meng et al. 2016) (Figure 1E). Purification of bacterial colonies were done by re-streaking of a single colony on dextrose peptone agar (DPA-without tetrazolium chloride; Norman and Alvarez 1989). DNA was isolated from bacterial cultures using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA), respectively. Molecular identification of four strains (PL243-246) were performed by the sequencing region of the housekeeping gene dnaA (chromosomal replication initiation protein) using Pec. dnaA-F1/R1 primer set (Dobhal et al. 2020). The amplified PCR product was enzymatically cleaned using ExoSAP-ITTM (Affymetrix Inc, Santa Clara, CA), and sent for sequencing at the GENEWIZ facility (Genewiz, La Jolla, CA) using both forward and reverse primers. The dnaA gene sequences were aligned using Geneious, and manually edited to remove the errors. The consensus sequences were analyzed with the NCBI BLASTn tool and were deposited in the NCBI GenBank under the accession numbers MT899920-MT899923. The NCBI BLASTn report indicated that all the sequences shared 99-100% identity and query cover with Pectobacterium brasiliense accession numbers MN544627-29. A phylogenetic analysis, using Geneious, was performed with the dnaA sequences representing different Pectobacterium spp., all strains grouped within the clade of P. brasiliense (Figure 2; Arizala et al, 2020). A pathogenicity assay was carried out in three replications on pak choi grown in pots containing commercial pot mixture, and maintained in the controlled-greenhouse (temperature 26-30°C; relative humidity 50-58%). Three-weeks old plant stems were artificially inoculated with 100 µl bacterial suspensions of PL243 (1.3x 10⁸ CFU/ml), PL244 (1.2x 10⁸ CFU/ml), PL 245 (1.2x 10⁸ CFU/ml) and PL246 (1.1x 10⁸CFU/ml); control plants were inoculated with 100 µl of distilled water (Figure 1F). Two days after inoculation, the soft rot and wilting symptoms (Figure 1G-H), similar to the ones observed on the field, were developed for all four strains tested. Bacteria was successfully re-isolated from the inoculated plants; DNA was isolated, amplified, sequenced for dnaA region and analyzed for 100% homology with original strains, to fulfill Koch’s postulates. Based on the molecular characteristics re-isolates were identical to the original strains. To the best of our knowledge, this is the first report of P. brasiliense on pak choi in the USA. Recent reports indicated that the pathogen could potentially pose a threat to cruciferous crops, therefore, highlighting a need to conduct a state-wide survey for pectinolytic bacteria, and implement better management strategies to combat the vegetable crop losses.

Vector-Borne Blood Parasites of the Great-Tailed Grackle (Quiscalus mexicanus) in East-Central Texas, USA

Golnar AJ, Medeiros MCI, Rosenbaum K, Bejcek J, Hamer SA, Hamer GL

Microorganisms. 2021 Feb 27

Great-tailed grackles (Quiscalus mexicanus) have dramatically expanded into North America over the past century. However, little is known about the blood that parasites they support. Here, for the first time, we document an assemblage of trypanosome, haemosporida, and filarial nematodes co-circulating in invasive great-tailed grackles. Between February and July, 2015, 61 individuals were captured in an urban environment of College Station, Texas. Field microscopy and molecular diagnostics indicate that 52% (24/46) were visually infected with filarioid nematodes, 24% (11/46) with avian trypanosomes, and 73% (n = 44/60) with haemosporida parasites, such as Haemoproteus (Parahaemoproteus) and Plasmodium cathemerium. Overall, 87% of great-tailed grackles were infected with blood parasites. Although 50% of individuals hosted parasites from multiple phylum, no patterns of parasite assembly were observed. Results indicate that great-tailed grackles can support a relatively high level of blood parasitism. However, the consequences for avian health remain to be determined.

Improved multiplex TaqMan qPCR assay with universal internal control offers reliable and accurate detection of Clavibacter michiganensis

Ramachandran S, Dobhal S, Alvarez AM, Arif M

Journal of Applied Microbiology. 2021 Feb 23

Aim: Clavibacter michiganensis (Cm) is a seed-borne plant pathogen that significantly reduces tomato production worldwide. Due to repeated outbreaks and rapid spread of the disease, seeds/transplants need to be certified free of the pathogen before planting. To this end, we developed a multiplex TaqMan qPCR assay that can accurately detect Cm in infected samples.

The Eco-Bio-Social Factors That Modulate Aedes aegypti Abundance in South Texas Border Communities

Juarez JG, Garcia-Luna SM, Medeiros MCI, et al.

Insects. 2021 Feb 21

Aedes aegypti control requires dedicated resources that are usually scarce, limiting the reach and sustainability of vector control programs. This generates a need to focus on areas at risk of disease transmission and also understand the factors that might modulate local mosquito abundance. We evaluated the eco-bio-social factors that modulate indoor and outdoor relative abundance of female Ae. aegypti in communities of South Texas. We conducted housing quality and Knowledge Attitudes and Practices surveys in households that were part of a weekly mosquito surveillance program in November of 2017 and 2018. Our results showed widespread knowledge of mosquitoes and Zika virus by our participants. However, less than 35% considered them as serious problems in this region. The presence of window-mounted air conditioning units increased the risk of female mosquito relative abundance indoors. An increase in outdoor relative abundance was associated with larger properties and a higher number of children between 6 to 17 years of age. Interestingly, we observed that an increasing number of children <5 years of age modulated both indoor and outdoor relative abundance, with a 52% increase indoors and 30% decrease outdoors. The low perception of mosquito and disease risk highlights engagement needs for vector-borne disease prevention in this region. The identified risk factors can help guide public health officials in their efforts to reduce human and vector contact.

Comparative genomics reveals dynamic genome evolution in host specialist ectomycorrhizal fungi

Lofgren LA, Nguyen NH, et al.

New Phytologist. 2021 Feb 06

While there has been significant progress characterizing the ‘symbiotic tool kit’ of ectomycorrhizal (ECM) fungi, how host specificity may be encoded into ECM fungal genomes remains poorly understood. We conducted a comparative genomic analysis of ECM fungal host specialists and generalists, focusing on the specialist genus Suillus. Global analyses of genome dynamics across 46 species were assessed, along with targeted analyses of three classes of molecules previously identified as important determinants of host specificity: small secreted proteins (SSPs), secondary metabolites (SMs), and G-protein coupled receptors (GPCRs). Relative to other ECM fungi, including other host specialists, Suillus had highly dynamic genomes including numerous rapidly evolving gene families and many domain expansions and contractions. Targeted analyses supported a role for SMs but not SSPs or GPCRs in Suillus host specificity. Phylogenomic-based ancestral state reconstruction identified Larix as the ancestral host of Suillus, with multiple independent switches between white and red pine hosts. These results suggest that like other defining characteristics of the ECM lifestyle, host specificity is a dynamic process at the genome level. In the case of Suillus, both SMs and pathways involved in the deactivation of reactive oxygen species appear to be strongly associated with enhanced host specificity.

Animal development in the microbial world: Re-thinking the conceptual framework

Bosch TCG, McFall-Ngai M

Current Topics in Developmental Biology. 2021 Jan 29

Animals have evolved within the framework of the microbes and are constantly exposed to diverse microbiota. This dominance of the microbial world is forcing all fields of biology to question some of their most basic premises, with developmental biology being no exception. While animals under laboratory conditions can develop and live without microbes, they are far from normal, and would not survive under natural conditions, where their fitness would be strongly compromised. Since much of the undescribed biodiversity on Earth is microbial, any consideration of animal development in the absence of the recognition of microbes will be incomplete. Here, we show that animal development may never have been autonomous, rather it requires transient or persistent interactions with the microbial world. We propose that to formulate a comprehensive understanding of embryogenesis and post-embryonic development, we must recognize that symbiotic microbes provide important developmental signals and contribute in significant ways to phenotype production. This offers limitless opportunities for the field of developmental biology to expand.

A method for characterizing dissolved DNA and its application to the North Pacific Subtropical Gyre

Linney MD, Schvarcz CR, Steward GF, DeLong EF, Karl DM

Limnology and Oceanography: Methods. 2021 Jan 27

Use of MICRO/BEMF Core Facility

Dissolved DNA (D-DNA) is a ubiquitous component of dissolved organic matter in aquatic systems. It is operationally defined as the DNA that passes a membrane filter and thus includes pools of truly dissolved “free” DNA (F-DNA), virion encapsidated DNA, DNA within membrane vesicles, and possibly other bound forms, each with different sources and lability. We investigated whether filtration (< 0.1 μm), concentration by tangential flow ultrafiltration (> 30 kDa), and fractionation in an equilibrium buoyant density gradient could be used to discriminate the mass contributions of the different pools of filterable DNA in seawater. Spike-in experiments with a known range of DNA standards (75–20,000 bp) indicated that this method results in high recoveries of F-DNA (68–86%) with minimal degradation. Application of the fractionation method to seawater samples collected from the oligotrophic North Pacific Ocean followed by analysis of fractions (epifluorescence and electron microscopy, DNase digestion) suggested that the low-density fractions (1.30–1.35 g mL−1) were dominated by vesicle-like particles, mid-density fractions (1.45–1.55 g mL−1) by virus-like particles, and high-density fractions (1.60–1.70 g mL−1) by F-DNA. The estimated concentration of DNA that is either F-DNA, in viruses, or in vesicles was 0.13, 0.14, and 0.08 μg L−1, respectively in the euphotic zone and 0.09, 0.04, and 0.03 μg L−1, respectively in the mesopelagic zone. The approach described should be useful for more detailed investigations of the abundance, dynamics, and sources of DNA in the distinct pools that comprise filterable DNA in aquatic environments.

Drosophila suzukii avoidance of microbes in oviposition choice

Sato A, Tanaka KM, Yew JY, Takahashi A

The Royal Society Open Science. 2021 Jan 20

Use of MGAL Core Facility

While the majority of Drosophila species lays eggs onto fermented fruits, females of Drosophila suzukii pierce the skin and lay eggs into ripening fruits using their serrated ovipositors. The changes of oviposition site preference must have accompanied this niche exploitation. In this study, we established an oviposition assay to investigate the effects of commensal microbes deposited by conspecific and heterospecific individuals and showed that the presence of microbes on the oviposition substrate enhances egg laying of Drosophila melanogaster and Drosophila biarmipes, but discourages that of D. suzukii. This result suggests that a drastic change has taken place in the lineage leading to D. suzukii in how females respond to chemical cues produced by microbes. We also found that hardness of the substrate, resembling that of either ripening or damaged and fermenting fruits, affects the response to microbial growth, indicating that mechanosensory stimuli interact with chemosensory-guided decisions to select or avoid oviposition sites.

FungalTraits: a user-friendly traits database of fungi and fungus-like stramenopiles

Põlme S, Abarenkov K, Nilsson RH, Lindahl BD, Clemmensen KE, Kauserud H, Nguyen N, et al.

Fungal Diversity. 2021 Jan 19

The cryptic lifestyle of most fungi necessitates molecular identification of the guild in environmental studies. Over the past decades, rapid development and affordability of molecular tools have tremendously improved insights of the fungal diversity in all ecosystems and habitats. Yet, in spite of the progress of molecular methods, knowledge about functional properties of the fungal taxa is vague and interpretation of environmental studies in an ecologically meaningful manner remains challenging. In order to facilitate functional assignments and ecological interpretation of environmental studies we introduce a user friendly traits and character database FungalTraits operating at genus and species hypothesis levels. Combining the information from previous efforts such as FUNGuild and FunFun together with involvement of expert knowledge, we reannotated 10,210 and 151 fungal and Stramenopila genera, respectively. This resulted in a stand-alone spreadsheet dataset covering 17 lifestyle related traits of fungal and Stramenopila genera, designed for rapid functional assignments of environmental studies. In order to assign the trait states to fungal species hypotheses, the scientific community of experts manually categorised and assigned available trait information to 697,413 fungal ITS sequences. On the basis of those sequences we were able to summarise trait and host information into 92,623 fungal species hypotheses at 1% dissimilarity threshold.

Host snail species exhibit differential Angiostrongylus cantonensis prevalence and infection intensity across an environmental gradient

Rollins RL, Cowie RH, Echaluse MV, Medeiros MCI

Acta Tropica. 2021 Jan 07

Diverse snail species serve as intermediate hosts of the parasitic nematode Angiostrongylus cantonensis, the etiological agent of human neuroangiostrongyliasis. However, levels of A. cantonensis infection prevalence and intensity vary dramatically among these host species. Factors contributing to this variation are largely unknown. Environmental factors, such as precipitation and temperature, have been correlated with overall A. cantonensis infection levels in a locale, but the influence of environment on infection in individual snail species has not been addressed. We identified levels of A. cantonensis prevalence and intensity in 16 species of snails collected from 29 sites along an environmental gradient on the island of Oahu, Hawaii. The relationship between infection levels of individual species and their environment was evaluated using AIC model selection of Generalized Linear Mixed Models incorporating precipitation, temperature, and vegetation cover at each collection site. Our results indicate that different mechanisms drive parasite prevalence and intensity in the intermediate hosts. Overall, snails from rainy, cool, green sites had higher infection levels than snails from dry, hot sites with less green vegetation. Intensity increased at the same rate along the environmental gradient in all species, though at different levels, while the relation between prevalence and environmental variables depended on species. These results have implications for zoonotic transmission, as human infection is a function of infection in the intermediate hosts, ingestion of which is the main pathway of transmission. The probability of human infection is greater in locations with higher rainfall, lower temperature and more vegetation cover because of higher infection prevalence in the gastropod hosts, but this depends on the host species. Moreover, severity of neuroangiostrongyliasis symptoms is likely to be greater in locations with higher rainfall, lower temperature, and more vegetation because of the higher numbers of infectious larvae (infection intensity) in all infected snail species. This study highlights the variation of infection prevalence and intensity in individual gastropod species, the individualistic nature of interactions between host species and their environment, and the implications for human neuroangiostrongyliasis in different environments.

Topological Twists in Nature

Simien JM, Haglund E

Trends in Biochemical Sciences. 2021 Jan 05

The first entangled protein was observed about 30 years ago, resulting in an increased interest for uncovering the biological functions and biophysical properties of these complex topologies. Recently, the Pierced Lasso Topology (PLT) was discovered in which a covalent bond forms an intramolecular loop, leaving one or both termini free to pierce the loop. This topology is related to knots and other entanglements. PLTs exist in many well-researched systems where the PLTs have previously been unnoticed. PLTs represents 18% of all disulfide containing proteins across all kingdoms of life. In this review, we investigate the biological implications of this specific topology in which the PLT-forming disulfide may act as a molecular switch for protein function and consequently human health.

Collaborative research to inform adaptive comanagement: a framework for the Heʻeia National Estuarine Research Reserve

Winter KB, Rii YM, Reppun FAWL, DeLaforgue Hintzen K, Alegado RA, Bowen BW, Bremer LL, Coffman M, Deenik JL, Donahue MJ, Falinski KA, Frank K, et al.

Ecology and Society. 2020

Globally, an increasing recognition of the importance of ecosystem-based management (EBM), Indigenous resource management (IRM), and Indigenous-led research and management is emerging; yet, case studies within scholarly literature illustrating comprehensive application of these theories and philosophies are scarce. We present the collaborative management model for the Heʻeia National Estuarine Research Reserve (NERR) as a contemporary Indigenous Community and Conserved Area (ICCA) that has synergistically operationalized these principles, as well as one that approaches research as a reciprocal collaboration with the Indigenous people and local community (IPLC) of place. The Heʻeia NERR was designated in 2017 through a process led by IPLC members in Hawaiʻi. This research framework is aimed at informing EBM within social-ecological systems. It, therefore, serves as an example of a program designed to demonstrate and provide practical solutions for adaptive resource management. The framework of the Heʻeia NERR embraces the values, perspectives, and IRM strategies that have been foundational for the people of the Pacific to thrive sustainably in the context of limited resources for millennia. As a program, the Heʻeia NERR aims to build bridges between coexisting worldviews as a means of informing policy in the realms of conservation and sustainability. We do this by weaving together conventional and Indigenous science to collaboratively develop research and collaboratively produce new knowledge. We examine these issues through the lens of holistic ecosystem services that consider both the reciprocal benefits that humans provide to nature as well as the full range of existential benefits that humans gain from nature. Research collaborations between the Heʻeia NERR and its partners (University of Hawaiʻi, state and federal agencies, and Indigenous-led NGOs operating in the community) are grounded in Indigenous and local knowledge (ILK) with applications that will guide a future of enhanced ecosystem services in a changing world.

Ecomimicry in Indigenous resource management: optimizing ecosystem services to achieve resource abundance, with examples from Hawaiʻi

Winter KB, Lincoln NK, Berkes F, Alegado RA, Kurashima N, Frank KL, et al.

Ecology and Society. 2020

Here, we expand on the term “ecomimicry” to be an umbrella concept for an approach to adaptive ecosystem-based management of social-ecological systems that simultaneously optimizes multiple ecosystem services for the benefit of people and place. In this context, we define ecomimicry as a strategy for developing and managing cultural landscapes, built upon a deep understanding of the structure and function of ecosystems, that harnesses ecosystem processes for the purpose of balancing and sustaining key ecosystem services, rather than maximizing one service (e.g., food production) to the detriment of others. Ecomimicry arises through novel, place-based innovations or is adopted from elsewhere and adapted to local conditions. Similarly, precontact Hawaiian social-ecological systems integrated a variety of ecomimicry schema to engender a complex system of adaptive resource management that enhanced biocultural diversity and supported resilient food systems, ultimately sustaining a thriving human population. In addition to presenting a synopsis of how ecomimicry was employed in the design and management of Hawaiian social-ecological systems, we identify and characterize specific ecomimicry applications. Within this context, we explore a revival of ecomimicry for biological conservation, biocultural restoration, resilience, and food security. We conclude with a discussion of how revitalizing such an approach in the restoration of social-ecological systems may address issues of conservation and sustainability in the Anthropocene.

The Arabidopsis Protein Disulfide Isomerase Subfamily M Isoform, PDI9, Localizes to the Endoplasmic Reticulum and Influences Pollen Viability and Proper Formation of the Pollen Exine During Heat Stress

Feldeverd E, et al.

Frontiers in Plant Science. 2020 Dec 29

Use of MICRO/BEMF Core Facility

Plants adapt to heat via thermotolerance pathways in which the activation of protein folding chaperones is essential. In eukaryotes, protein disulfide isomerases (PDIs) facilitate the folding of nascent and misfolded proteins in the secretory pathway by catalyzing the formation and isomerization of disulfide bonds and serving as molecular chaperones. In Arabidopsis, several members of the PDI family are upregulated in response to chemical inducers of the unfolded protein response (UPR), including both members of the non-classical PDI-M subfamily, PDI9 and PDI10. Unlike classical PDIs, which have two catalytic thioredoxin (TRX) domains separated by two non-catalytic TRX-fold domains, PDI-M isoforms are orthologs of mammalian P5/PDIA6 and possess two tandem catalytic domains. Here, PDI9 accumulation was found to be upregulated in pollen in response to heat stress. Histochemical staining of plants harboring the PDI9 and PDI10 promoters fused to the gusA gene indicated they were actively expressed in the anthers of flowers, specifically in the pollen and tapetum. Immunoelectron microscopy revealed that PDI9 localized to the endoplasmic reticulum in root and pollen cells. transfer DNA (T-DNA) insertional mutations in the PDI9 gene disrupted pollen viability and development in plants exposed to heat stress. In particular, the pollen grains of pdi9 mutants exhibited disruptions in the reticulated pattern of the exine and an increased adhesion of pollen grains. Pollen in the pdi10 single mutant did not display similar heat-associated defects, but pdi9 pdi10 double mutants (DMs) completely lost exine reticulation. Interestingly, overexpression of PDI9 partially led to heat-associated defects in the exine. We conclude that PDI9 plays an important role in pollen thermotolerance and exine biogenesis. Its role fits the mechanistic theory of proteostasis in which an ideal balance of PDI isoforms is required in the endoplasmic reticulum (ER) for normal exine formation in plants subjected to heat stress.

Animal development in the microbial world: The power of experimental model systems

McFall-Ngai M, Bosch TCG

Epub. 2020 Nov 24

Use of MICRO/BEMF Core Facility

The development of powerful model systems has been a critical strategy for understanding the mechanisms underlying the progression of an animal through its ontogeny. Here we provide two examples that allow deep and mechanistic insight into the development of specific animal systems. Species of the cnidarian genus Hydra have provided excellent models for studying host-microbe interactions and how metaorganisms function in vivo. Studies of the Hawaiian bobtail squid Euprymna scolopes and its luminous bacterial partner Vibrio fischeri have been used for over 30 years to understand the impact of a broad array of levels, from ecology to genomics, on the development and persistence of symbiosis. These examples provide an integrated perspective of how developmental processes work and evolve within the context of a microbial world, a new view that opens vast horizons for developmental biology research. The Hydra and the squid systems also lend an example of how profound insights can be discovered by taking advantage of the "experiments" that evolution had done in shaping conserved developmental processes.

Toward a revision of the bamboo corals: Part 1, species in the Muricellisidinae (Octocorallia: Isididae)

Watling L

Zootaxa. 2020 Nov 19

Use of MICRO/BEMF Core Facility

The subfamily Muricellisidinae was erected by Kükenthal in 1915 and placed in the family Isididae in order to accommodate an unusual species collected in Sagami Bay, Japan. In 1931, Thomson and Dean added a second species collected in Indonesia during the Siboga Expedition. The holotypes of both species have been re-examined. Muricellisis echinata was found to be an anthothelid living on the axis of a keratoisid bamboo coral and M. cervicornis is a melithaeid. Both species are redescribed.

Cytology reveals diverse cell morphotypes and cellin-cell interactions in normal collector sea urchins Tripneustes gratilla

Work TM, Millard E, Mariani DB, Weatherby TM, Rameyer RA, Dagenais J, Breeden R, Beale AM

Diseases of Aquatic Organisms. 2020 Nov 19

Use of MICRO/BEMF Core Facility

Echinoderms such as sea urchins are important in marine ecosystems, particularly as grazers, and unhealthy sea urchins can have important ecological implications. For instance, unexplained mortalities of Diadema antillarum in the Caribbean were followed by algal overgrowth and subsequent collapse of coral reef ecosystems. Unfortunately, few tools exist to evaluate echinoderm health, making management of mortalities or other health issues problematic. Hematology is often used to assess health in many animal groups, including invertebrates, but is seldom applied to echinoderms. We used a standard gravitometric technique to concentrate fixed coelomocytes from the collector sea urchin Tripneustes gratilla onto microscope slides, permitting staining and enumeration. Using Romanowsky stain and electron microscopy to visualize cell details, we found that urchin cells could be partitioned into different morphotypes. Specifically, we enumerated phagocytes, phagocytes with perinuclear cytoplasmic dots, vibratile cells, colorless spherule cells, red spherule cells, and red spherule cells with pink granules. We also saw cell-in-cell interactions characterized by phagocytes apparently phagocytizing mainly the motile cells including red spherule cells, colorless spherule cells, and vibratile cells disproportionate to underlying populations of circulating cells. Cell-in-cell interactions were seen in 71% of sea urchins, but comprised <1% of circulating cells. Finally, about 40% of sea urchins had circulating phagocytes that were apparently phagocytizing spicules. The coelomic fluid collection and slide preparation methods described here are simple, field portable, and might be a useful complementary tool for assessing health of other marine invertebrates, revealing heretofore unknown physiological phenomena in this animal group.

The noncoding small RNA SsrA is released by Vibrio fischeri and modulates critical host responses

Moriano-Gutierrez S, Bongrand C, Essock-Burns T, Wu L, McFall-Ngai M, Ruby EG

PLoS Biology. 2020 Nov 03

Use of MICRO/BEMF Core Facility

The regulatory noncoding small RNAs (sRNAs) of bacteria are key elements influencing gene expression; however, there has been little evidence that beneficial bacteria use these molecules to communicate with their animal hosts. We report here that the bacterial sRNA SsrA plays an essential role in the light-organ symbiosis between Vibrio fischeri and the squid Euprymna scolopes. The symbionts load SsrA into outer membrane vesicles, which are transported specifically into the epithelial cells surrounding the symbiont population in the light organ. Although an SsrA-deletion mutant (ΔssrA) colonized the host to a normal level after 24 h, it produced only 2/10 the luminescence per bacterium, and its persistence began to decline by 48 h. The host's response to colonization by the ΔssrA strain was also abnormal: the epithelial cells underwent premature swelling, and host robustness was reduced. Most notably, when colonized by the ΔssrA strain, the light organ differentially up-regulated 10 genes, including several encoding heightened immune-function or antimicrobial activities. This study reveals the potential for a bacterial symbiont's sRNAs not only to control its own activities but also to trigger critical responses promoting homeostasis in its host. In the absence of this communication, there are dramatic fitness consequences for both partners.

The cytokine MIF controls daily rhythms of symbiont nutrition in an animal–bacterial association

Koch EJ, Bongrand C, Bennett BD, Lawhorn S, Moriano-Gutierrez S, Pende M, Vadiwala K, Dodt H, Raible F, Goldman W, Ruby EG, McFall-Ngai M

PNAS. 2020 Nov 03

Use of MICRO/BEMF Core Facility

The recent recognition that many symbioses exhibit daily rhythms has encouraged research into the partner dialogue that drives these biological oscillations. Here we characterized the pivotal role of the versatile cytokine macrophage migration inhibitory factor (MIF) in regulating a metabolic rhythm in the model light-organ symbiosis between Euprymna scolopes and Vibrio fischeri. As the juvenile host matures, it develops complex daily rhythms characterized by profound changes in the association, from gene expression to behavior. One such rhythm is a diurnal shift in symbiont metabolism triggered by the periodic provision of a specific nutrient by the mature host: each night the symbionts catabolize chitin released from hemocytes (phagocytic immune cells) that traffic into the light-organ crypts, where the population of V. fischeri cells resides. Nocturnal migration of these macrophage-like cells, together with identification of an E. scolopes MIF (EsMIF) in the light-organ transcriptome, led us to ask whether EsMIF might be the gatekeeper controlling the periodic movement of the hemocytes. Western blots, ELISAs, and confocal immunocytochemistry showed EsMIF was at highest abundance in the light organ. Its concentration there was lowest at night, when hemocytes entered the crypts. EsMIF inhibited migration of isolated hemocytes, whereas exported bacterial products, including peptidoglycan derivatives and secreted chitin catabolites, induced migration. These results provide evidence that the nocturnal decrease in EsMIF concentration permits the hemocytes to be drawn into the crypts, delivering chitin. This nutritional function for a cytokine offers the basis for the diurnal rhythms underlying a dynamic symbiotic conversation.

Vibration attraction response is a plastic trait in blind Mexican tetra (Astyanax mexicanus), variable within subpopulations inhabiting the same cave

Espinasa L, Heintz C, Rétaux S, Yoshizawa M, Agnès F, Ornelas-Garcia P, Balogh-Robinson R

Journal of Fish Biology. 2020 Oct 30

Animals evolve their sensory systems and foraging behaviours to adapt and colonize new and challenging habitats such as the dark cave environment. Vibration attraction behaviour (VAB) gives fish the ability to locate the source of a water disturbance in the darkness. VAB evolved in the blind Mexican cave tetra, Astyanax mexicanus. VAB is triggered in cavefish by vibration stimuli peaking at 35 Hz, which is within the main spectrum of water fluctuations produced by many prey crustaceans and insects. VAB has a genetic component and is correlated to an increased number of head mechanosensory neuromasts in the eye orbital region when compared to surface fish. Previous competitive prey capture assays have supported the advantage of VAB for foraging in the dark. Despite its putative adaptive function, VAB has been described as absent in some Astyanax cave populations (Tinaja and Molino) but present in others (Pachón, Piedras, Toro and Sabinos). Here we have tested the occurrence of VAB in the field and in multiple cave populations using a vibrating device in natural pools. Our results confirmed the presence of VAB in caves such as Pachón, Toro and Sabinos but showed that VAB is also present in the Tinaja and Molino cave populations, previously reported as VAB-negative in laboratory experiments. Thus, VAB is available throughout the range of hypogean A. mexicanus. However, and most notably, within a given cave the levels of VAB were highly variable among different pools. Fish at one pool may express no VAB, while fish at another nearby pool of the same cave may actively show VAB. While a variety of environmental conditions may foster this diversity, we found that individuals inhabiting pools with a high abundance of organic matter have reduced expression of VAB. In contrast, in pools with little organic debris where fish probably depend more on hunting than on scavenging, VAB is enhanced. Our results suggest that expression of VAB is a plastic trait whose variability can depend on local conditions. Such plasticity may be required within and among caves where high environmental variability between pools results in a diverse availability of food.

Taxonomy and Phylogenetic Research on Ralstonia solanacearum Species Complex: A Complex Pathogen with Extraordinary Economic Consequences

Paudel S, Dobhal S, Alvarez AM, Arif M

Pathogens. 2020 Oct 25

The bacterial wilt pathogen, first known as Bacillus solanacearum, has undergone numerous taxonomic changes since its first description in 1896. The history and significance of this pathogen is covered in this review with an emphasis on the advances in technology that were used to support each reclassification that finally led to the current separation of Ralstonia solanacearum into three genomic species. Frequent name changes occurred as methodology transitioned from phenotypic, biochemical, and molecular studies, to genomics and functional genomics. The diversity, wide host range, and geographical distribution of the bacterial wilt pathogen resulted in its division into three species as genomic analyses elucidated phylogenetic relationships among strains. Current advances in phylogenetics and functional genomics now open new avenues for research into epidemiology and control of the devastating bacterial wilt disease.

Pharmaceutically Acceptable Carboxylic Acid-Terminated Polymers Show Activity and Selectivity against HSV-1 and HSV-2 and Synergy with Antiviral Drugs

Yadavalli T, et al.

ACS Infectious Diseases. 2020 Oct 20

Use of MICRO/BEMF Core Facility

Polyanionic macromolecules including carboxylate-terminated polymers (polycarboxylates) are capable of inhibiting sexually transmitted viruses such as human immunodeficiency virus (HIV) and herpes simplex virus (HSV). Cellulose acetate phthalate (CAP), a pharmaceutically acceptable pH-sensitive polycarboxylate polymer, showed promising prophylactic activity against HIV and HSV, but the instability of CAP in an aqueous environment prevented its clinical development. Interestingly, several pharmaceutically acceptable polycarboxylates have features similar to CAP with an aqueous stability significantly higher than that of CAP. However, their activity against sexually transmitted viruses remains unexplored. Here, we evaluate the activity of various polycarboxylates such as polyvinyl acetate phthalate (PVAP), various grades of hydroxypropyl methylcellulose phthalate (HPMCP-50, HPMCP-55, and HPMCP-55S), and various grades of methacrylic acid copolymers (Eudragit L100-55, Eudragit L100, Eudragit S100, and Kollicoat MAE 100P) against HSV. We, for the first time, demonstrate that PVAP, HPMCP-55S, and Eudragit S100 have activity and selectivity against HSV-1 and HSV-2. Further, we report that polycarboxylates can be easily transformed into nanoparticles (NPs) and in the nanoparticulate form, they show similar or enhanced activity against HSV. Finally, using PVAP NPs, as a model, we demonstrate using in vitro HSV therapy studies that polycarboxylate NPs are capable of synergizing with antiviral drugs such as acyclovir (ACV), tenofovir, and tenofovir disoproxil fumarate. Thus, pharmaceutically acceptable carboxylic acid-terminated polymers and their NPs have the potential to be developed into topical formulations for the prevention and treatment of HSV infection.

Genomic divergence between Dickeya zeae strain EC2 isolated from rice and previously identified strains, suggests a different rice foot rot strain

Zhang J, Arif M, Shen H, Hu J, Sun D, Pu X, Yang Q, Lin B

PLoS One. 2020 Oct 20

Rice foot rot caused by Dickeya zeae is an important bacterial disease of rice worldwide. In this study, we identified a new strain EC2 from rice in Guangdong province, China. This strain differed from the previously identified strain from rice in its biochemical characteristics, pathogenicity, and genomic constituents. To explore genomic discrepancies between EC2 and previously identified strains from rice, a complete genome sequence of EC2 was obtained and used for comparative genomic analyses. The complete genome sequence of EC2 is 4,575,125 bp in length. EC2 was phylogenetically closest to previously identified Dickeya strains from rice, but not within their subgroup. In terms of secretion systems, genomic comparisons revealed that EC2 harbored only type I (T1SS), typeⅡ (T2SS), and type VI (T6SS) secretion systems. The flagella cluster of this strain possessed specific genomic characteristics like other D. zeae strains from Guangdong and from rice; within this locus, the genetic diversity among strains from rice was much lower than that of within strains from non-rice hosts. Unlike other strains from rice, EC2 lost the zeamine cluster, but retained the clustered regularly interspaced short palindromic repeats-1 (CRISPR-1) array. Compared to the other D. zeae strains containing both exopolysaccharide (EPS) and capsular polysaccharide (CPS) clusters, EC2 harbored only the CPS cluster, while the other strains from rice carried only the EPS cluster. Furthermore, we found strain MS1 from banana, carrying both EPS and CPS clusters, produced significantly more EPS than the strains from rice, and exhibited different biofilm-associated phenotypes. Comparative genomics analyses suggest EC2 likely evolved through a pathway different from the other D. zeae strains from rice, producing a new type of rice foot rot pathogen. These findings emphasize the emergence of a new type of D. zeae strain causing rice foot rot, an essential step in the early prevention of this rice bacterial disease.

Exploring the Use of High-Resolution Melting Analysis and Helicase-Dependent Amplification for Discrimination of Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species and Trialeurodes vaporariorum

Andreason SA, Arif M, Brown JK, Ochoa-Corona F, Wayadande A

Journal of Economic Entomology. 2020 Oct 16

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera; Aleyrodidae), and greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), are highly problematic plant pests and virus vectors with worldwide distributions. Identification of whitefly species is typically accomplished by observation of distinct morphological characters; however, because of morphological inconsistency and indistinguishability, the discrimination of B. tabaci species variants is dependent on molecular techniques based on genetic differences. New assays were designed for the detection of B. tabaci A, B, and Q mitotype groups, and T. vaporariorum. Specific primer sets were designed for amplification of the mitochondrial cytochrome c oxidase I gene of the four targets to perform in end-point PCR, real-time PCR coupled to high-resolution melting analysis (HRM), and the isothermal helicase-dependent amplification (HDA). Primer specificities were validated using end-point PCR, then tested in HRM and HDA. Bemisia tabaci A, B, and Q mitotypes, and T. vaporariorum-targeted primer sets discriminately amplified specimens of different populations within their target whitefly group. These tests provide three novel discrimination assays for the high-consequence, exotic B. tabaci B and Q groups, along with the native B. tabaci A group and T. vaporariorum.

Fungal heavy metal adaptation through single nucleotide polymorphisms and copy-number variation

Bazzicalupo AL, Ruytinx J, Ke YH, Coninx L, Colpaert JV, Nguyen NH, Vilgalys R, Branco S

Molecular Ecology. 2020 Sep 15

Human-altered environments can shape the evolution of organisms. Fungi are no exception, although little is known about how they withstand anthropogenic pollution. Here, we document adaptation in the mycorrhizal fungus Suillus luteus driven by soil heavy metal contamination. Genome scans across individuals from recently polluted and nearby unpolluted soils in Belgium revealed low divergence across isolates and no evidence of population structure based on soil type. However, we detected single nucleotide polymorphism divergence and gene copy-number variation, with different genetic combinations potentially conferring the ability to persist in contaminated soils. Variants were shared across the population but found to be under selection in isolates exposed to pollution and located across the genome, including in genes involved in metal exclusion, storage, immobilization and reactive oxygen species detoxification. Together, our results point to S. luteus undergoing the initial steps of adaptive divergence and contribute to understanding the processes underlying local adaptation under strong environmental selection.

HbtR, a Heterofunctional Homolog of the Virulence Regulator TcpP, Facilitates the Transition between Symbiotic and Planktonic Lifestyles in Vibrio fischeri

Bennett BD, Essock-Burns T, Ruby EG

mBio. 2020 Sep 01

Use of MICRO/BEMF Core Facility

The bioluminescent bacterium Vibrio fischeri forms a mutually beneficial symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, in which the bacteria, housed inside a specialized light organ, produce light used by the squid in its nocturnal activities. Upon hatching, E. scolopes juveniles acquire V. fischeri from the seawater through a complex process that requires, among other factors, chemotaxis by the bacteria along a gradient of N-acetylated sugars into the crypts of the light organ, the niche in which the bacteria reside. Once inside the light organ, V. fischeri transitions into a symbiotic, sessile state in which the quorum-signaling regulator LitR induces luminescence. In this work we show that expression of litR and luminescence are repressed by a homolog of the Vibrio cholerae virulence factor TcpP, which we have named HbtR. Further, we demonstrate that LitR represses genes involved in motility and chemotaxis into the light organ and activates genes required for exopolysaccharide production.

The Aedes albopictus (Diptera: Culicidae) microbiome varies spatially and with Ascogregarine infection

Seabourn P, Spafford H, Yoneishi N, Medeiros M

PLoS Neglected Tropical Diseases. 2020 Aug 19

The mosquito microbiome alters the physiological traits of medically important mosquitoes, which can scale to impact how mosquito populations sustain disease transmission. The mosquito microbiome varies significantly within individual mosquitoes and among populations, however the ecological and environmental factors that contribute to this variation are poorly understood. To further understand the factors that influence variation and diversity of the mosquito microbiome, we conducted a survey of the bacterial microbiome in the medically important mosquito, Aedes albopictus, on the high Pacific island of Maui, Hawai‘i. We detected three bacterial Phyla and twelve bacterial families: Proteobacteria, Acitinobacteria, and Firmicutes; and Anaplasmataceae, Acetobacteraceae, Enterobacteriaceae, Burkholderiaceae, Xanthobacteraceae, Pseudomonadaceae, Streptomycetaceae, Staphylococcaceae, Xanthomonadaceae, Beijerinckiaceae, Rhizobiaceae, and Sphingomonadaceae. The Ae. albopictus bacterial microbiota varied among geographic locations, but temperature and rainfall were uncorrelated with this spatial variation. Infection status with an ampicomplexan pathosymbiont Ascogregarina taiwanensis was significantly associated with the composition of the Ae. albopictus bacteriome. The bacteriomes of mosquitoes with an A. taiwanensis infection were more likely to include several bacterial symbionts, including the most abundant lineage of Wolbachia sp. Other symbionts like Asaia sp. and several Enterobacteriaceae lineages were less prevalent in A. taiwanensis-infected mosquitoes. This highlights the possibility that inter- and intra-domain interactions may structure the Ae. albopictus microbiome.

First Report of Pectobacterium brasiliense Causing Soft Rot on Brassica oleracea var. sabellica in Hawaii, United States

Boluk G, Arizala D, Ocenar J, Mokwele J, Silva J, Dobhal S, Uyeda J, Alvarez AM, Arif M

Plant Disease. 2020 Aug 18

Kale (Brassica oleracea var. sabellica L.), family Brassicaceae, is an economically important vegetable crop and an excellent source of natural antioxidants, consumed in many countries worldwide (Ligor et al. 2013). In June 2019, 11 kale plants (variety: Starbor), showing symptoms of wilting and soft rot on the stem, were collected from a field on Oahu, Hawaii. An estimated 30 to 40% of total plants were infected. Small pieces of stems were taken (5 × 5 mm), surface sterilized with 0.6% sodium hypochlorite for 1 min, and rinsed three times with sterilized water. Later, the tissues were macerated in a sterile 1.5-ml centrifuge tube containing 100 μl of sterile water; macerated tissues (∼10 μl) were streaked onto crystal violet pectate (CVP) agar medium and incubated at 26 to 28°C for 48 to 78 h (Dobhal et al. 2020; Helias et al. 2012). Pectolytic bacteria that created pits on CVP were restreaked on nutrient agar plates containing tetrazolium chloride to obtain pure single colonies.

Genomic DNA from 11 bacterial isolates was extracted using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer’s instructions. The dnaA, gapA, and gyrB housekeeping genes were used for identity determination and phylogenetic analysis. For amplification, the dnaA primer set PCR conditions and components, as described by Dobhal et al. (2020), were used for all three primer sets (dnaA gene primers from Dobhal et al. 2020; gapA gene: gapA-F1, 5′-ATATGACTATCAAAGTAGGTATCAACGG-3′, and gapA-R2, 5′-CGTACCAGGAAACCAGTTTCAC-3′; and gyrB gene: gyrB-F1, 5′-ATGTCGAATTCTTATGACTCCTCA-3′, and gyrB-R1, 5′-TGGCTTTCTTGCTGTAGCCTTC-3′). The amplification reaction contained 10 μl of GoTaq Green Master Mix (Promega), 1 μl (5 mM) of each forward and reverse primer, 1 μl of template DNA, and 7 μl of distilled water. The PCR conditions, PCR product cleanup, and sequencing protocols were followed as described by Dobhal et al. (2020). Using the error-free consensus sequences, BLASTn results showed 100% query coverage and ∼98% identity with Pectobacterium brasiliense sequences. All the sequences were submitted to the NCBI GenBank database under the following accession numbers: for dnaA gene, MN544620 to MN544630 (all 11 strains); gapA gene, MN732539 and MN732540 (PL131, PL141); and gyrB gene, MN732542 and MN732543 (PL131, PL141). Pathogenicity assays were performed with three strains on kale plants grown in plastic pots under the temperature-controlled greenhouse. Bacterial inoculum (200 µl of 3.4 × 108 CFU/ml) was inoculated into three consecutive nodes using sterile pipet tips. Controls were inoculated with sterile water; the experiment was performed in three replicates. Typical disease symptoms, wilting across the plant and soft rot on the stem, were observed after 24 h, whereas no symptoms were observed on control plants. To confirm successful P. brasiliense infection, strains were reisolated from inoculated plants and confirmed as P. brasiliense through sequencing. In the phylogenetic tree, the kale strains were grouped with seven other P. brasiliense strains retrieved from the NCBI GenBank database. Pectobacterium and Dickeya species, causing soft rot, have been reported in Hawaii on potato but not on kale (Arizala et al. 2020; Boluk and Arif 2019). Only in Brazil, P. brasiliense has been reported to cause soft rot on kale (Queiroz et al. 2017). So far, this is the first report of P. brasiliense causing bacterial soft rot on kale in Hawaii and the United States.

Does fungal competitive ability explain host specificity or rarity in ectomycorrhizal symbioses?

Kennedy PG, Gagne J, Perez-Pazos E, Lofgren LA, Nguyen NH

PLoS One. 2020 Aug 18

Two common ecological assumptions are that host generalist and rare species are poorer competitors relative to host specialist and more abundant counterparts. While these assumptions have received considerable study in both plant and animals, how they apply to ectomycorrhizal fungi remains largely unknown. To investigate how interspecific competition may influence the anomalous host associations of the rare ectomycorrhizal generalist fungus, Suillus subaureus, we conducted a seedling bioassay. Pinus strobus seedlings were inoculated in single- or two-species treatments of three Suillus species: S. subaureus, S. americanus, and S. spraguei. After 4 and 8 months of growth, seedlings were harvested and scored for mycorrhizal colonization as well as dry biomass. At both time points, we found a clear competitive hierarchy among the three ectomycorrhizal fungal species: S. americanus > S. subaureus > S. spraguei, with the competitive inferior, S. spraguei, having significantly delayed colonization relative to S. americanus and S. subaureus. In the singlespecies treatments, we found no significant differences in the dry biomasses of P. strobus seedlings colonized by each Suillus species, suggesting none was a more effective plant symbiont. Taken together, these results indicate that the rarity and anomalous host associations exhibited by S. subaureus in natural settings are not driven by inherently poor competitive ability or host growth promotion, but that the timing of colonization is a key factor determining the outcome of ectomycorrhizal fungal competitive interactions.

LapG mediates biofilm dispersal in Vibrio fischeri by controlling maintenance of the VCBS-containing adhesin LapV

Christensen DG, Marsden AE, Hodge-Hanson K, Essock-Burns T, Visick KL

Molecular Microbiology. 2020 Aug 03

Use of MICRO/BEMF Core Facility

Efficient symbiotic colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation on the surface of the squid’s light organ. Subsequently, the bacteria disperse from the biofilm via an unknown mechanism and enter through pores to reach interior colonization sites. Here, we identify a homolog of Pseudomonas fluorescens LapG as a dispersal factor that promotes cleavage of a biofilm-promoting adhesin, LapV. Overproduction of LapG inhibited biofilm formation and, unlike the wild-type parent, a ΔlapG mutant formed biofilms in vitro. Although V. fischeri encodes two putative large adhesins, LapI (near lapG on chromosome II) and LapV (on chromosome I), only the latter contributed to biofilm formation. Consistent with the Pseudomonas Lap system model, our data support a role for the predicted c-di-GMP-binding protein LapD in inhibiting LapG-dependent dispersal. Furthermore, we identified a phosphodiesterase, PdeV, whose loss promotes biofilm formation similar to that of the ΔlapG mutant and dependent on both LapD and LapV. Finally, we found a minor defect for a ΔlapD mutant in initiating squid colonization, indicating a role for the Lap system in a relevant environmental niche. Together, these data reveal new factors and provide important insights into biofilm dispersal by V. fischeri.

First Report of Pectobacterium brasiliense Causing Bacterial Soft Rot and Blackleg Diseases of Potato in Hawaii

Arizala D, Dobhal S, Paudel S, Boluk G, Silva J, Ahmad AA, Uyeda J, Sugano J, Alvarez AM, Arif M

Plant Disease. 2020 Jul 22

Pectobacterium brasiliense, first described in Brazil (Duarte et al. 2004), and recently elevated to the species level (Portier et al. 2019), has emerged as one of the most destructive bacterial species in the heterogenous Pectobacterium genus, which causes numerous diseases including blackleg of potato (Arizala and Arif 2019; Li et al. 2019). In January 2019, 67 potato plants showing characteristic symptoms of blackleg, stem rot, and leaf necrosis were observed in a field on Oahu, Hawaii. Affected tissues (light brown, macerated, and water-soaked stem sections) were cut into 1-cm pieces, followed by surface sterilization using 0.6% sodium hypochlorite for 30 s and then three successive rinses with distilled water. The tissues were ground and streaked on plates containing crystal violet pectate medium. Plates were incubated for 48 h at 26 ± 2°C; colonies producing pits were restreaked and purified on dextrose peptone agar.

Later, the bacterial DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). The Pec.dnaA-F1 and Pec.dnaA-R1 primer set (Dobhal et al. 2020) was used to amplify the dnaA gene, and PCR products were treated using the ExoSAP-IT kit (Affymetrix) and sent for sequencing at the GENEWIZ facility using both forward and reverse primers. Both strands were aligned and manually corrected for errors; the consensus sequence of each bacterial strain was analyzed with the NCBI BLASTn tool. Eleven strains isolated from different infected plants were confirmed as P. brasiliense with 100% query cover and 99 to 100% identity. After performing a phylogenetic analysis using the dnaA sequences representing different Pectobacterium sp., all 11 strains grouped within the clade of P. brasiliense. The dnaA consensus sequences were submitted to the GenBank database under the accession numbers MN428420 to MN428430 for the strains PL68, PL69, PL76, PL78, PL79, PL80, PL81, PL82, PL107, PL108, and PL109. To fulfill Koch’s postulates, a pathogenicity test was performed by injecting 100 µl of a bacterial suspension of strains PL108 (3.6 × 108 CFU/ml) and PL109 (3.8 × 108 CFU/ml) into the stem of healthy potato seedlings (3 weeks old); control plants were inoculated with 100 µl of distilled water. The experiment was performed in three replicates in a temperature-controlled greenhouse. Four days postinoculation, all inoculated seedlings (except the controls) showed typical symptoms of stem darkening, maceration, and watery ooze that resembled those manifested by the naturally infected plants on the field. Bacteria were reisolated from symptomatic tissues, and the dnaA region was resequenced. Sequence analysis confirmed the identity of these strains as P. brasiliense. This is the first report of P. brasiliense on potato in Hawaii. This along with first reports of other pectolytic soft rot bacteria associated with potato in Hawaii (Arizala et al. 2020; Boluk and Arif 2019) indicates a need for conducting further state-wide surveys of these vegetable crops.

Species Identity and Size are Associated with Rat Lungworm Infection in Gastropods

Medeiros MCI, Rollins RL, Echaluse MV, Cowie RH

Ecohealth. 2020 Jul 16

Angiostrongylus cantonensis, the rat lungworm, is an emerging zoonotic pathogen that cycles between definitive rat and intermediate gastropod hosts. Zoonotic infection occurs when humans intentionally or accidentally consume infectious larvae in a gastropod host, and may manifest as neuroangiostrongyliasis, characterized by eosinophilic meningitis, severe neurological impairment, and even death. Thus, the risk of A. cantonensis zoonoses may be related to the distribution of A. cantonensis larvae across gastropod hosts. We screened 16 gastropod species from 14 communities on the island of O'ahu, Hawai'i, USA, to characterize the distribution of A. cantonensis among species and across host size. Prevalence (proportion of the population infected) and infection intensity (density of worms in host tissue) varied among gastropod species. Prevalence also varied with gastropod host size, but this relationship differed among host species. Most host species showed a positive increase in the probability of infection with host size, suggesting that within species relatively larger hosts had higher prevalence. The density of worms in an infected snail was unrelated to host size. These results suggest that variation in A. cantonensis infection is associated with demographic structure and composition of gastropod communities, which could underlie heterogeneity in the risk of human angiostrongyliasis across landscapes.

Cholesterol crystals and atherosclerosis

Baumer Y, Mehta NN, Dey AK, Powell-Wiley TM, Boisvert WA

European Heart Journal. 2020 Jun 21

Use of MICRO/BEMF Core Facility

The use of scanning electron microscopy and transmission electron microscopy to elucidate the role of cholesterol crystals in atherosclerotic plaque rupture is discussed. Atherosclerosis is a chronic inflammatory process accompanied by dyslipidaemia and other cellular derangements.1 As the quest for the development of innovative future therapies is ongoing, the ability to examine atherosclerotic vessels and the plaques within at an ultrastructural level has become requisite in order to further understand the underlying aetiological processes. In particular, electron microscopy techniques developed over the last several decades, together with classic histological imaging, have been of tremendous importance to understand plaque composition. Both scanning and transmission electron microscopy (SEM and TEM, respectively) have been used to illustrate the timeline of atherosclerosis.2–4 Advances made in these techniques have allowed for a deeper insight into the pathology of atherogenesis.

Genome-Informed Recombinase Polymerase Amplification Assay Coupled with a Lateral Flow Device for In-Field Detection of Dickeya Species

Boluk, G, Dobhal S, Crockford AB, Melzer M, Alvarez AM, Arif M

Plant Disease. 2020 Jun 12

Dickeya spp. cause blackleg and soft rot diseases of potato and several other plant species worldwide, resulting in high economic losses. Rapid detection and identification of the pathogen is essential for facilitating efficient disease management. Our aim in this research was to develop a rapid and field-deployable recombinase polymerase amplification (RPA) assay coupled with a lateral flow device (LFD) that will accurately detect Dickeya spp. in infected plant tissues without the need for DNA isolation. A unique genomic region (mglA/mglC genes) conserved among Dickeya spp. was used to design highly specific robust primers and probes for an RPA assay. Assay specificity was validated with 34 representative strains from all Dickeya spp. and 24 strains from other genera and species; no false positives or negatives were detected. An RPA assay targeting the internal transcribed spacer region of the host genome was included to enhance the reliability and accuracy of the Dickeya assay. The detection limit of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effects were observed when 1 µl of host sap, macerated in Tris-EDTA buffer, was added to each reaction in the sensitivity tests. The developed RPA assay is rapid, highly accurate, sensitive, and fully field deployable. It has numerous applications in routine diagnostics, surveillance, biosecurity, and disease management.

Interactions of Symbiotic Partners Drive the Development of a Complex Biogeography in the Squid-Vibrio Symbiosis

Essock-Burns T, Bongrand C, Goldman WE, Ruby EG, McFall-Ngai MJ

mBio. 2020 May 26

Use of MICRO/BEMF Core Facility

Microbes live in complex microniches within host tissues, but how symbiotic partners communicate to create such niches during development remains largely unexplored. Using confocal microscopy and symbiont genetics, we characterized the shaping of host microenvironments during light organ colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri. During embryogenesis, three pairs of invaginations form sequentially on the organ’s surface, producing pores that lead to interior compressed tubules at different stages of development. After hatching, these areas expand, allowing V. fischeri cells to enter and migrate 120 m through three anatomically distinct regions before reaching blind-ended crypt spaces. A dynamic gatekeeper, or bottleneck, connects these crypts with the migration path. Once V. fischeri cells have entered the crypts, the bottlenecks narrow, and colonization by the symbiont population becomes spatially restricted. The actual timing of constriction and restriction varies with crypt maturity and with different V. fischeri strains. Subsequently, starting with the first dawn following colonization, the bottleneck controls a lifelong cycle of dawn-triggered expulsions of most of the symbionts into the environment and a subsequent regrowth in the crypts. Unlike other developmental phenotypes, bottleneck constriction is not induced by known microbe-associated molecular patterns (MAMPs) or by V. fischeri-produced bioluminescence, but it does require metabolically active symbionts. Further, while symbionts in the most mature crypts have a higher proportion of live cells and a greater likelihood of expulsion at dawn, they have a lower resistance to antibiotics. The overall dynamics of these distinct microenvironments reflect the complexity of the host-symbiont dialogue.

An abundant new genus and species of fan worms (Polychaeta: Sabellidae) from Hawaii

Magalhães WF, Bailey-Brock JH, Tovar-Hernández MA

Zootaxa. 2020 Apr 08

Use of MICRO/BEMF Core Facility

A new genus and species of Hawaiian sabellid polychaete, Euchonoides moeone n. gen. et n. sp. is described. This new species has consistently been one of the most abundant polychaetes collected in Mamala Bay, Hawaii, reaching densities of 141,046 ind. m-2 and representing up to 30.6% of all polychaetes collected in that region. The species has a small body (~2 mm length) with a reduced number of abdominal chaetigers (8-10), and is distinguished by the presence of the following features: 1) three pairs of radioles; 2) radiolar skeleton with two longitudinal rows of cells from radiole base to third proximal pair of pinnules, remainder of each radiole skeleton with single rows of cells; 3) pinnules unpaired, alternating (snowflake arrangement); 4) thoracic uncini acicular with a large tooth above the main fang followed by a series of small ones; 5) thoracic chaetiger 3 enlarged; 6) a wide belt on third abdominal chaetiger; and 7) a pre-pygidial depression composed of three chaetigers, with lateral wings, among a combination of several others features. Histological sections have shown that the abdominal belt seems to be a clitellum-like structure where oogenesis takes place. The new genus is compared with other plesiomorphic genera sharing similar morphological features. Patterns of abundance of the new species are presented for the past 27 years in Mamala Bay.

Study of degradation and spatial performance of low Pt-loaded proton exchange membrane fuel cells under exposure to sulfur dioxide in an oxidant stream

Reshetenko T, Laue V, Krewer U, Artyushkova K

Journal of Power Sources. 2020 Mar 26

Use of MICRO/BEMF Core Facility

Sulfur dioxide is a common air pollutant that has an adverse impact on proton exchange membrane fuel cells (PEMFCs). The present study reports the spatial performance and degradation of low-Pt PEMFCs exposed to trace concentrations of SO2 in a cathode feed stream. PEMFCs exposed to 2 ppm SO2 resulted in a performance loss of 240 and 345 mV for constant current holds of 0.2 and 0.8 A cm−2. However, the SO2 contamination of low-Pt fuel cells at 1.0 A cm−2 led to a drastic cell potential drop below 0.1 V from the initial 0.605 V and demonstrated that the cathode exposure to SO2 should be limited to concentrations significantly below 2 ppm for high power operating conditions. The observed voltage and spatial current behavior was attributed to the reduction of SO2 on Pt to zero-valent sulfur at potentials below 0.6 V. PEMFCs recovered performance only after potential cycling, insuring the oxidation of the formed S0 species at potentials higher than 0.8 V. A comparison of the electrochemical parameters before and after PEMFC poisoning revealed a substantial decrease in electrochemical surface area of up to 45.2% and a final performance loss of 60–100 mV.

Light organ photosensitivity in deep-sea shrimp may suggest a novel role in counterillumination

Bracken-Grissom HD, DeLeo DM, Porter ML, et al.

Scientific Reports. 2020 Mar 11

Use of MICRO/BEMF Core Facility

Extraocular photoreception, the ability to detect and respond to light outside of the eye, has not been previously described in deep-sea invertebrates. Here, we investigate photosensitivity in the bioluminescent light organs (photophores) of deep-sea shrimp, an autogenic system in which the organism possesses the substrates and enzymes to produce light. Through the integration of transcriptomics, in situ hybridization and immunohistochemistry we find evidence for the expression of opsins and phototransduction genes known to play a role in light detection in most animals. Subsequent shipboard light exposure experiments showed ultrastructural changes in the photophore similar to those seen in crustacean eyes, providing further evidence that photophores are light sensitive. In many deep-sea species, it has long been documented that photophores emit light to aid in counterillumination - a dynamic form of camouflage that requires adjusting the organ's light intensity to "hide" their silhouettes from predators below. However, it remains a mystery how animals fine-tune their photophore luminescence to match the intensity of downwelling light. Photophore photosensitivity allows us to reconsider the organ's role in counterillumination - not only in light emission but also light detection and regulation.

Comparative Genomic Analysis Confirms Five Genetic Populations of the Select Agent, Rathayibacter toxicus

Yasuhara-Bell J, Arif M, Busot GY, Mann R, Rodoni B, Stack JP

Microorganisms. 2020 Mar 05

Rathayibacter toxicus is a Gram-positive, nematode-vectored bacterium that infects several grass species in the family Poaceae. Unique in its genus, R. toxicus has the smallest genome, possesses a complete CRISPR-Cas system, a vancomycin-resistance cassette, produces tunicamycin, a corynetoxin responsible for livestock deaths in Australia, and is designated a Select Agent in the United States. In-depth, genome-wide analyses performed in this study support the previously designated five genetic populations, with a core genome comprising approximately 80% of the genome for all populations. Results varied as a function of the type of analysis and when using different bioinformatics tools for the same analysis; e.g., some programs failed to identify specific genomic regions that were actually present. The software variance highlights the need to verify bioinformatics results by additional methods; e.g., PCR, mapping genes to genomes, use of multiple algorithms). These analyses suggest the following relationships among populations: RT-IV ↔ RT-I ↔ RT-II ↔ RT-III ↔ RT-V, with RT-IV and RT-V being the most unrelated. This is the most comprehensive analysis of R. toxicus that included populations RT-I and RT-V. Future studies require underrepresented populations and more recent isolates from varied hosts and geographic locations.

Landscape and demographic determinants of Culex infection with West Nile virus during the 2012 epidemic in Dallas County, TX

Poh KC, Medeiros MCI, Hamer GL

Spatial and Spatio-temporal Epidemiology. 2020 Feb 12

In 2012, the United States experienced one of the largest outbreaks of West Nile virus (WNV)-associated deaths, with the majority occurring in Dallas County (Co.), Texas (TX) and surrounding areas. In this study, logistic mixed models were used to identify associations between the landscape, human population, and WNV-infected Culex quinquefasciatus mosquitoes during the 2012 WNV epidemic in Dallas Co. We found increased probabilities for WNV-positive mosquitoes in north and central Dallas Co. The most significant predictors of the presence of WNV in Cx. quinquefasciatus pools were increased urbanization (based on an index composed of greater population density, lower normalized difference vegetation index, higher coverage of urban land types, and more impervious surfaces), older human populations, and lower elevation. These relationships between the landscape, sociodemographics, and risk of enzootic transmission identified regions of Dallas Co., TX with highest risk of spillover to human disease during the 2012 WNV epidemic.

Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real-time qPCR assay to detect the genus Dickeya and Dickeya dianthicola

Dobhal S, Boluk G, Babler B, Stulberg MJ, Rascoe J, Nakhla MK, Chapman TA, Crockford AB, Melzer MJ, Alvarez AM, Arif M

Journal of Applied Microbiology. 2020 Feb 12

Dickeya species are high consequence plant pathogenic bacteria; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, Dickeya dianthicola.

Dual roles of the retinal pigment epithelium and lens in cavefish eye degeneration

Ma L, Ng M, van der Weele CM, Yoshizawa M, Jeffery WR

The Journal of Experimental Zoology Part B: Molecular and Developmental Evolution. 2020 Jan 12

Astyanax mexicanus consists of two forms, a sighted surface dwelling form (surface fish) and a blind cave-dwelling form (cavefish). Embryonic eyes are initially formed in cavefish but they are subsequently arrested in growth and degenerate during larval development. Previous lens transplantation studies have shown that the lens plays a central role in cavefish eye loss. However, several lines of evidence suggest that additional factors, such as the retinal pigment epithelium (RPE), which is morphologically altered in cavefish, could also be involved in the eye regression process. To explore the role of the RPE in cavefish eye degeneration, we generated an albino eyed (AE) strain by artificial selection for hybrid individuals with large eyes and a depigmented RPE. The AE strain exhibited an RPE lacking pigment granules and showed reduced expression of the RPE specific enzyme retinol isomerase, allowing eye development to be studied by lens ablation in an RPE background resembling cavefish. We found that lens ablation in the AE strain had stronger negative effects on eye growth than in surface fish, suggesting that an intact RPE is required for normal eye development. We also found that the AE strain develops a cartilaginous sclera lacking boney ossicles, a trait similar to cavefish. Extrapolation of the results to cavefish suggests that the RPE and lens have dual roles in eye degeneration, and that deficiencies in the RPE may be associated with evolutionary changes in scleral ossification.

Natural Product Discovery by Direct Analysis in Real Time Mass Spectrometry

Yew JY

Mass Spectrometry (Tokyo). 2020 Jan 11

Use of MGAL Core Facility

Direct analysis in real time mass spectrometry (DART MS) is one of the first ambient ionization methods to be introduced and commercialized. Analysis by DART MS requires minimal sample preparation, produces nearly instantaneous results, and provides detection over a broad range of compounds. These advantageous features are particularly well-suited for the inherent complexity of natural product analysis. This review highlights recent applications of DART MS for species identification by chemotaxonomy, chemical profiling, genetic screening, and chemical spatial analysis from plants, insects, microbes, and metabolites from living systems.

Expression of extraocular opsin genes and light-dependent basal activity of blind cavefish

Simon N, Fujita S, Porter M, Yoshizawa M

PeerJ. 2019 Dec 17

Animals living in well-lit environments utilize optical stimuli for detecting visual information, regulating the homeostatic pacemaker, and controlling patterns of body pigmentation. In contrast, many subterranean animal species without optical stimuli have evolved regressed binocular eyes and body pigmentation. Interestingly, some fossorial and cave-dwelling animals with regressed eyes still respond to light. These light-dependent responses may be simply evolutionary residuals or they may be adaptive, where negative phototaxis provides avoidance of predator-rich surface environments. However, the relationship between these non-ocular light responses and the underlying light-sensing Opsin proteins has not been fully elucidated.

Poisoning effects of sulfur dioxide in an air stream on spatial proton exchange membrane fuel cell performance

Reshetenko T, Laue V, Krewer U, Artyushkova K

Journal of Power Sources. 2019 Oct 31

Use of MICRO/BEMF Core Facility

The paper examines spatial and temporal proton exchange membrane fuel cell (PEMFC) performance under exposure to SO2 in an air stream at low and high operating currents. Cathode poisoning by 2 ppm SO2 resulted in cell voltage losses of 60 and 295 mV for overall cell currents of 0.2 and 1.0 A cm−2, respectively. The voltage drop was accompanied by localized current redistributions, which depend on the operating current hold. The observed spatial PEMFC behavior is attributed to different electrochemical reactions of adsorbed SO2 occurring at high and low cathode potentials. SO2 is mainly oxidized at potentials corresponding to 0.2 A cm−2, while reduction to zero-valent sulfur takes place at 1.0 A cm−2, as confirmed by XPS. Full self-recovery was not observed for either operating current hold, and only potential cycling recovered the performances. Moreover, comparison of the initial and after-poisoning electrochemical PEMFC parameters revealed that SO2 exposure led to a decrease in Pt electrochemical surface area due to growth of the Pt particle size and caused final performance losses of 20–30 and 25–50 mV for low and high current operations. A physics-based mathematical model was successfully developed and applied for analysis of SO2 effects on PEMFC performance.

Description of Echthrogaleus spinulus n. sp. (Copepoda: Pandaridae) parasitic on a torpedo ray from the central Pacific Ocean utilising a morphological and molecular approach

Morales-Serna FN, Crow GL, Montes MM, González MT

Systematic Parasitology. 2019 Oct 25

Use of MICRO/BEMF Core Facility

A new species of parasitic copepod, Echthrogaleus spinulus n. sp. (Pandaridae), is described from the torpedo ray Tetronarce tokionis (Tanaka) (Torpedinidae) captured in pelagic Hawaiian waters. The new species has pediger 4 bearing large dorsal plates with denticles on posterior margin, genital complex with posterolateral lobes widely curved medially and overlapping, leg 4 exopod incompletely 3-segmented, and the largest body size (maximum length 16 mm from anterior rim of frontal plates to tip of caudal rami, excluding setae). This morphology does not match any of the seven valid species of Echthrogaleus Steenstrup & Lütken, 1861. Analysis of 28S rDNA sequences separated the new material from the Central Pacific from samples of E. coleoptratus in the Atlantic and Eastern Pacific Oceans. However, due to the lack of DNA sequences in the databases, the new 28S rDNA sequence cannot used to confirm the species identity. The unique morphological characteristics of the Central Pacific female copepods combined with 28S rDNA sequencing was used as a basis to validate the new species.

Ambient pH Alters the Protein Content of Outer Membrane Vesicles, Driving Host Development in a Beneficial Symbiosis

Lynch JB, Schwartzman JA, Bennett BD, McAnulty SJ, Knop M, Nyholm SV, Ruby EG

Journal of Bacteriology. 2019 Oct 15

Use of MICRO/BEMF Core Facility

Outer membrane vesicles (OMVs) are continuously produced by Gram-negative bacteria and are increasingly recognized as ubiquitous mediators of bacterial physiology. In particular, OMVs are powerful effectors in interorganismal interactions, driven largely by their molecular contents. These impacts have been studied extensively in bacterial pathogenesis but have not been well documented within the context of mutualism. Here, we examined the proteomic composition of OMVs from the marine bacterium Vibrio fischeri, which forms a specific mutualism with the Hawaiian bobtail squid, Euprymna scolopes. We found that V. fischeri upregulates transcription of its major outer membrane protein, OmpU, during growth at an acidic pH, which V. fischeri experiences when it transitions from its environmental reservoir to host tissues. We used comparative genomics and DNA pulldown analyses to search for regulators of ompU and found that differential expression of ompU is governed by the OmpR, H-NS, and ToxR proteins. This transcriptional control combines with nutritional conditions to govern OmpU levels in OMVs. Under a host-encountered acidic pH, V. fischeri OMVs become more potent stimulators of symbiotic host development in an OmpU-dependent manner. Finally, we found that symbiotic development could be stimulated by OMVs containing a homolog of OmpU from the pathogenic species Vibrio cholerae, connecting the role of a well-described virulence factor with a mutualistic element. This work explores the symbiotic effects of OMV variation, identifies regulatory machinery shared between pathogenic and mutualistic bacteria, and provides evidence of the role that OMVs play in animal-bacterium mutualism.

Acidic pH promotes lipopolysaccharide modification and alters colonization in a bacteria-animal mutualism

Schwartzman JA, Lynch JB, Flores Ramos S, Zhou L, Apicella MA, Yew JY, Ruby EG

Molecular Microbiology. 2019 Oct 11

Use of MGAL Core Facility

Environmental pH can be an important cue for symbiotic bacteria as they colonize their eukaryotic hosts. Using the model mutualism between the marine bacterium Vibrio fischeri and the Hawaiian bobtail squid, we characterized the bacterial transcriptional response to acidic pH experienced during the shift from planktonic to host-associated lifestyles. We found several genes involved in outer membrane structure were differentially expressed based on pH, indicating alterations in membrane physiology as V. fischeri initiates its symbiotic program. Exposure to host-like pH increased the resistance of V. fischeri to the cationic antimicrobial peptide polymixin B, which resembles antibacterial molecules that are produced by the squid to select V. fischeri from the ocean microbiota. Using a forward genetic screen, we identified a homolog of eptA, a predicted phosphoethanolamine transferase, as critical for antimicrobial defense. We used MALDI-MS to verify eptA as an ethanolamine transferase for the lipid-A portion of V. fischeri lipopolysaccharide. We then used a DNA pulldown approach to discover that eptA transcription is activated by the global regulator H-NS. Finally, we revealed that eptA promotes successful squid colonization by V. fischeri, supporting its potential role in initiation of this highly specific symbiosis.

Carbon-carbon double bond position elucidation in fatty acids using ozone-coupled direct analysis in real time mass spectrometry

Cetraro N, Cody RB, Yew JY

Analyst. 2019 Oct 07

Use of MGAL Core Facility

The carbon-carbon double bond positions of unsaturated fatty acids can have markedly different effects on biological function and also serve as biomarkers of disease pathology, dietary history, and species identity. As such, there is great interest in developing methods for the facile determination of double bond position for natural product chemistry, the pharmaceutical industry, and forensics. We paired ozonolysis with direct analysis in real time mass spectrometry (DART MS) to cleave and rapidly identify carbon-carbon double bond position in fatty acids, fatty alcohols, wax esters, and crude fatty acid extracts. In addition, ozone exposure time and DART ion source temperature were investigated to identify optimal conditions. Our results reveal that brief, offline exposure to ozone-generated aldehyde and carboxylate products that are indicative of carbon-carbon double bond position. The relative abundance of diagnostic fragments quantitatively reflects the ratios of isobaric fatty acid positional isomers in a mixture with a correlation coefficient of 0.99. Lastly, the unsaturation profile generated from unfractionated, fatty acid extracts can be used to differentiate insect species and populations. The ability to rapidly elucidate lipid double bond position by combining ozonolysis with DART MS will be useful for lipid structural elucidation, assessing isobaric purity, and potentially distinguishing between animals fed on different diets or belonging to different ecological populations.

Wide-Bandgap Cu(In,Ga)S2 Photocathodes Integrated on Transparent Conductive F:SnO2 Substrates for Chalcopyrite-Based Water Splitting Tandem Devices

Gaillard N, Prasher D, Chong M, Deangelis A, Horsley K, Ishii HA, Bradley JP, Varley J, Ogitsu T

ACS Applied Energy Materials. 2019 Jul 25

Use of MICRO/BEMF Core Facility

Whole-chalcopyrite-based tandem devices for photoelectrochemical (PEC) water splitting have emerged as a promising route for obtaining ∼20% solar-to-hydrogen efficiencies. Here we pursue this approach by demonstrating integration of the top cell wide-bandgap (EG) chalcopyrite onto a transparent conductor, which is a critical step in the realization of tandem devices. We report specifically on our efforts to synthesize photoactive Cu(In,Ga)S2 thin films on transparent conductive F:SnO2 (FTO), while preserving the optoelectronic properties of the FTO substrate and preventing the formation of a resistive SnSx interfacial layer. We demonstrate that such attributes can be achieved via close-space sulfurization (CSS) of lower EG Cu(In,Ga)Se2 precursors, coevaporated on FTO at low temperature. Depending on Cu(In,Ga)Se2 precursors’ Ga and In content, the resulting Cu(In,Ga)S2 solar absorbers have EG energies spanning from 2.05 to 2.45 eV. The CSS process, which includes a low-temperature annealing in sulfur vapor followed by a high-temperature crystallization under inert atmosphere, allowed for up to 95% Se substitution with S in the chalcopyrite lattice, tuning both EG and band edge positions that impact PEC performance. Photoelectrochemical measurements performed under AM1.5G illumination in 0.5 M H2SO4 on the 2.05 eV CuInGaS2 photocathode revealed a saturation photocurrent density (JSAT) of −5.25 mA/cm2, a value corresponding to 38% of the absorber’s optical limit. We further concluded that such low JSAT originates from subpar optical absorption of Cu(In,Ga)S2 absorbers. Future improvements of the CSS process are expected to improve material quality toward our end goal of achieving whole-chalcopyrite tandem PEC devices.

Ultramorphological analysis of plaque advancement and cholesterol crystal formation in Ldlr knockout mouse atherosclerosis

Baumer Y, McCurdy S, Jin X, Weatherby TM, Dey AK, Mehta NN, Yap JK, Kruth HS, Boisvert WA

Atherosclerosis. 2019 Jun 12

Use of MICRO/BEMF Core Facility

The low-density lipoprotein receptor-deficient (Ldlr-/-) mouse has been utilized by cardiovascular researchers for more than two decades to study atherosclerosis. However, there has not yet been a systematic effort to document the ultrastructural changes that accompany the progression of atherosclerotic plaque in this model.

Areca alkaloids measured from buccal cells using DART-MS serve as accurate biomarkers for areca nut chewing

Franke AA, Biggs L, Yew JY, Lai JF

Drug Testing and Analysis. 2019 Jun 11

Use of MGAL Core Facility

Areca nut (AN) chewing is carcinogenic and biomarkers reflecting it are urgently needed to determine the effectiveness of emergent cessation programs. Buccal cells (BCs) may serve as an ideal matrix to measure such biomarkers; however, their utility for this purpose is unknown. Direct analysis in real time-mass spectrometry (DART-MS) is a sensitive technique that analyzes materials in the open air and requires minimal/no sample preparation. We utilized DART-MS to analyze BCs to test the usefulness of this method in measuring areca alkaloids as biomarkers for AN chewing.

On two cryptogenic maldanids (Annelida) from coral rubble habitats in Hawaii

Magalhães WF, Bailey-Brock JH

Marine Biology Research. 2019 May 29

Use of MICRO/BEMF Core Facility

Dissolution of coral rubble collected in the vicinities of a tropical ocean outfall yielded two maldanid species, one of them not previously recorded to Hawaii. The putative species Boguea cf. enigmatica was originally described from North Carolina in coral sand. The Hawaiian material fits very well the original and subsequent descriptions but it is the first record in the Pacific Ocean and molecular data would be necessary to confirm this wide distributional range. This species broods its young and it would also limit its areal distribution unless it has been introduced. Axiothella quadrimaculata is also described from coral rubbles of Hawaii being previously recorded from Australia, New Zealand and South Africa.

Cultivation and Characterization of Viruses Infecting Eukaryotic Phytoplankton from the Tropical North Pacific Ocean.

Schvarcz CR

ScholarSpace - Thesis. 2019 May 28

Use of MICRO/BEMF Core Facility

Death by viral infection rivals predation as a source of mortality for all types of microscopic plankton in the ocean, including phytoplankton that are the foundation of the marine food web. This has profound consequences for plankton ecology and nutrient cycling in the sea. Viruses tend to be quite specific in the cells they infect, so the known extraordinary diversity among the marine phytoplankton implies that there is a similar high diversity of viruses in the sea. Our knowledge of viral diversity in the ocean has dramatically improved in recent years using metagenomic techniques (random sequencing of genome fragments from mixed communities). However, basic information about the viruses being detected, such as which organisms they infect and the details of their infection cycle cannot be reliably determined from sequence data alone. Having more model virus-host systems in culture that can be experimentally manipulated and studied in the lab would provide valuable new insights into the functional roles of the viruses in the marine food web. In this dissertation, cultivation-based techniques were used to characterize novel virus-host systems for eukaryotic phytoplankton from the tropical North Pacific Ocean, thereby identifying new virus-host linkages and establishing model systems for further study. Over 300 phytoplankton strains were cultivated and used in the isolation of over 60 virus strains. Described herein is a summary of these isolation efforts, including preliminary characterizations for 19 virus isolates using electron microscopy and genome sequencing. This is followed by more in-depth analyses of the genome and virion proteome of the giant virus Tetraselmis virus 1 (TetV-1), which infects the cosmopolitan green alga Tetraselmis. This work establishes new virus-host linkages and highlights previously uncharacterized viral diversity, including the first protist-infecting isolates from viral families previously only known to infect plants or animals. Furthermore, the genomic analyses have revealed a high number of viral encoded metabolic genes not previously seen in viruses, and the proteomic analyses have identified novel virion-associated enzymes. Marine viruses continue to represent an enormous amount of unknown or uncharacterized taxonomic and metabolic diversity, and this work demonstrates the utility of cultivation-based approaches in illuminating some of these mysteries.

Pleiotropic Effects of ebony and tan on Pigmentation and Cuticular Hydrocarbon Composition in Drosophila melanogaster

Massey JH, Akiyama N, Bien T, Dreisewerd K, Wittkopp PJ, Yew JY, Takahashi A

Frontiers in Physiology. 2019 May 01

Use of MGAL Core Facility

Pleiotropic genes are genes that affect more than one trait. For example, many genes required for pigmentation in the fruit fly Drosophila melanogaster also affect traits such as circadian rhythms, vision, and mating behavior. Here, we present evidence that two pigmentation genes, ebony and tan, which encode enzymes catalyzing reciprocal reactions in the melanin biosynthesis pathway, also affect cuticular hydrocarbon (CHC) composition in D. melanogaster females. More specifically, we report that ebony loss-of-function mutants have a CHC profile that is biased toward long (>25C) chain CHCs, whereas tan loss-of-function mutants have a CHC profile that is biased toward short (<25C) chain CHCs. Moreover, pharmacological inhibition of dopamine synthesis, a key step in the melanin synthesis pathway, reversed the changes in CHC composition seen in ebony mutants, making the CHC profiles similar to those seen in tan mutants. These observations suggest that genetic variation affecting ebony and/or tan activity might cause correlated changes in pigmentation and CHC composition in natural populations. We tested this possibility using the Drosophila Genetic Reference Panel (DGRP) and found that CHC composition covaried with pigmentation as well as levels of ebony and tan expression in newly eclosed adults in a manner consistent with the ebony and tan mutant phenotypes. These data suggest that the pleiotropic effects of ebony and tan might contribute to covariation of pigmentation and CHC profiles in Drosophila.

Detection of very long-chain hydrocarbons by laser mass spectrometry reveals novel species-, sex-, and age-dependent differences in the cuticular profiles of three Nasonia species

Bien T, Gadau J, Schnapp A, Yew JY, Sievert C, Dreisewerd K

Analytical and Bioanalytical Chemistry. 2019 Apr 08

Use of MGAL Core Facility

Long-chain cuticular hydrocarbons (CHC) are key components of chemical communication in many insects. The parasitoid jewel wasps from the genus Nasonia use their CHC profile as sex pheromone and for species recognition. The standard analytical tool to analyze CHC is gas chromatography coupled with mass spectrometric detection (GC/MS). This method reliably identifies short- to long-chain alkanes and alkenes, but CHC with more than 40 carbon atoms are usually not detected. Here, we applied two laser mass spectrometry (MS) techniques, namely direct laser desorption/ionization (d)LDI and silver-assisted (Ag-)LDI MS, respectively, to analyze CHC profiles of N. vitripennis, N. giraulti, and N. longicornis directly from the cuticle or extracts. Furthermore, we applied direct analysis in real-time (DART) MS as another orthogonal technique for extracts. The three methods corroborated previous results based on GC/MS, i.e., the production of CHC with carbon numbers between C25 and C40. However, we discovered a novel series of long-chain CHC ranging from C41 to C51/C52. Additionally, several previously unreported singly and doubly unsaturated alkenes in the C31-C39 range were found. Use of principal component analysis (PCA) revealed that the composition of the newly discovered CHC varies significantly between species, sex, and age of the animals. Our study adds to the growing literature on the presence of very long-chain CHC in insects and hints at putative roles in insect communication. Graphical abstract.

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Worsham M, Fernandes VFL, Settle A, Balaan C, Lactaoen K, Tuttle LJ, Iwashita M, Yoshizawa M

JoVE. 2019 Apr 06

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Opheliidae (Annelida: Polychaeta) from the western Pacific islands, including five new species

Magalhães WF, Rizzo AE, Bailey-Brock JH

Zootaxa. 2019 Feb 12

Use of MICRO/BEMF Core Facility

Intertidal, shallow subtidal and deep-water opheliids from the Hawaiian Islands, Guam in the Mariana Islands and Tutuila in American Samoa were examined and nine species are described, five of which are new. Armandia garretti sp. nov. and A. circumpapillata sp. nov. are new species proposed for previous records of A. intermedia Fauvel, 1902 from Hawaii. Armandia cf. paraintermedia Parapar Moreira, 2015 is recorded from intertidal muddy bottoms of Tutuila and A. amakusaesis Saito, Tamaki Imajima, 2000 from subtidal regions in Guam. Polyophthalmus mauliola sp. nov. is described from Hawaiian material and a comparative table with relevant morphological features of eight species of this genus is presented. Ophelina cf. chaetifera (Hartman, 1965) and Ophelina abranchiata Stop-Bowitz, 1948 are new records to Hawaii from up to 507 m depth, Ophelina kohni sp. nov. is described from shallow waters of Guam and Ophelina manana sp. nov. is described from Mamala Bay, Oahu.

Surveillance of Aedes aegypti indoors and outdoors using Autocidal Gravid Ovitraps in South Texas during local transmission of Zika virus, 2016 to 2018

Martin E, Medeiros MCI, Carbajal E, Valdez E, Juarez JG, Garcia-Luna S, Salazar A, Qualls WA, Hinojosa S, Borucki MK, Manley HA, Badillo-Vargas IE, Frank M, Hamer GL

Acta Tropica. 2019 Feb 11

The yellow fever mosquito, Aedes aegypti, has facilitated the re-emergence of dengue virus (DENV) and emergence of chikungunya virus (CHIKV) and Zika virus (ZIKV) in the Americas and the Caribbean. The recent transmission of these arboviruses in the continental United States has been limited, to date, to South Florida and South Texas despite Ae. aegypti occurring over a much larger geographical region within the country. The main goal of our study was to provide the first long term longitudinal study of Ae. aegypti and enhance the knowledge about the indoor and outdoor relative abundance of Ae. aegypti as a proxy for mosquito-human contact in South Texas, a region of the United States that is at high risk for mosquito-borne virus transmission. Here, the relative abundance of indoors and outdoors mosquitoes of households in eight different communities was described. Surveillance was done weekly from September 2016 to April 2018 using the CDC Autocidal Gravid Ovitraps in low- and middle-income communities. A total of 69 houses were included in this survey among which 36 were in the low-income communities (n = 11 for Donna, n = 15 for Progresso, n = 5 for Mesquite, n = 5 for Chapa) and 33 in middle-income communities (n = 9 for La Feria, n = 8 for Weslaco, n = 11 for McAllen, and n = 5 for Rio Rico). Overall, Ae. aegypti was the dominant species (59.2% of collections, n = 7255) followed by Culex spp. mosquitoes (27.3% of collections, n = 3350). Furthermore, we demonstrated for Ae. aegypti that 1) outdoor relative abundance was higher compared to indoor relative abundance, 2) low-income communities were associated with an increase in mosquito relative abundance indoors when compared to middle-income communities, 3) no difference was observed in the number of mosquitoes collected outdoors between low-income and middle-income communities, and 4) warmer months were positively correlated with outdoor relative abundance whereas no seasonality was observed in the relative abundance of mosquitoes indoors. Additionally, Ae. aegypti mosquitoes collected in South Texas were tested using a specific ZIKV/CHIKV multiplex real-time PCR assay, however, none of the mosquitoes tested positive. Our data highlights the occurrence of mosquitoes indoors in the continental United States and that adults are collected nearly every week of the calendar year. These mosquito data, obtained concurrently with local ZIKV transmission of 10 locally acquired cases in nearby communities, represent a baseline for future studies in the Lower Rio Grande Valley (LRGV) including vector control interventions relying on the oviposition behavior to reduce mosquito populations and pathogen transmission.

Piko A, Piko O, Piko I: Those That Came Before, Those That are Here Now, and Those That Will Come After

Ginsberg AD, Chieza N, Frank K, Rands A, Vilutis J

JoDS. 2019 Jan 29

What if the fastest road to the future has to do with slowing down and looking at—and tasting—where we’ve already been? Indigenous peoples who live in symbiosis with the natural systems that surround them have developed a unique and deep-rooted awareness of the connection between people, place, and practice. This awareness influences the shape and function of their societies, which in turn can reveal other ways of knowledge-making, offering alternatives to the anthropocentric view endemic in science today. In this conversation, Ahilapalapa and Kiana explore other cultural models and approaches to ecological understandings that expand biodesign, sending it in new, sustainable directions.

Culicoides species community composition and infection status with parasites in an urban environment of east central Texas, USA

Martin E, Chu E, Shults P, Golnar A, Swanson DA, Benn J, Kim D, Schneider P, Pena S, Culver C, Medeiros MCI, Hamer SA, Hamer GL

Parasites & Vectors. 2019 Jan 16

Despite their importance as vectors of zoonotic parasites that can impact human and animal health, Culicoides species distribution across different habitat types is largely unknown. Here we document the community composition of Culicoides found in an urban environment including developed and natural sites in east central Texas, a region of high vector diversity due to subtropical climates, and report their infection status with haemoparasites.

A new species of Paradoneis Hartman, 1965 (Annelida: Paraonidae) from Hawaii with notes on its reproductive strategy

Magalhães WF, Bailey-Brock JH, Barroso R

Marine Biodiversity. 2018 Dec 03

Use of MICRO/BEMF Core Facility

A new and abundant species of Paradoneis Hartman, 1965 is described from the vicinities of a sewage outfall in Mamala Bay, Oahu, Hawaii. This species belongs to a group of at least six Paradoneis species that have three prebranchiate segments, modified notopodial lyrate chaetae from chaetiger 3, and lack additional types of modified chaetae. Paradoneis kamaehu sp. nov. is unique among its congeners by the presence of 25–48 pairs of branchiae, reddish-brown body color, peristomium distinct dorsally, and presence of up to two lyrate notopodial chaetae. Epitokes and individuals with regenerating anterior ends are reported and the reproductive strategy of this species is discussed. The capacity of regeneration of the anterior end is reported for the first time in paraonids.

The role of gene flow in rapid and repeated evolution of cave-related traits in Mexican tetra, Astyanax mexicanus

Herman A, Brandvain Y, Weagley J, Jeffery WR, Keene AC, Kono TJY, Bilandžija H, Borowsky R, Espinasa L, O'Quin K, Ornelas-García CP, Yoshizawa M, Carlson B, Maldonado E, Gross JB, Cartwright RA, Rohner N, Warren WC, McGaugh SE

Molecular Ecology. 2018 Nov 27

Understanding the molecular basis of repeatedly evolved phenotypes can yield key insights into the evolutionary process. Quantifying gene flow between populations is especially important in interpreting mechanisms of repeated phenotypic evolution, and genomic analyses have revealed that admixture occurs more frequently between diverging lineages than previously thought. In this study, we resequenced 47 whole genomes of the Mexican tetra from three cave populations, two surface populations and outgroup samples. We confirmed that cave populations are polyphyletic and two Astyanax mexicanus lineages are present in our data set. The two lineages likely diverged much more recently than previous mitochondrial estimates of 5-7 mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between ~161 and 191 k generations ago. The favoured demographic model for most population pairs accounts for divergence with secondary contact and heterogeneous gene flow across the genome, and we rigorously identified gene flow among all lineages sampled. Therefore, the evolution of cave-related traits occurred more rapidly than previously thought, and trogolomorphic traits are maintained despite gene flow with surface populations. The recency of these estimated divergence events suggests that selection may drive the evolution of cave-derived traits, as opposed to disuse and drift. Finally, we show that a key trogolomorphic phenotype QTL is enriched for genomic regions with low divergence between caves, suggesting that regions important for cave phenotypes may be transferred between caves via gene flow. Our study shows that gene flow must be considered in studies of independent, repeated trait evolution.

Achieving a multi-strain symbiosis: strain behavior and infection dynamics

Bongrand C, Ruby EG

The ISME Journal. 2018 Oct 23

Use of MICRO/BEMF Core Facility

Strain diversity, while now recognized as a key driver underlying partner dynamics in symbioses, is usually difficult to experimentally manipulate and image in hosts with complex microbiota. To address this problem, we have used the luminous marine bacterium Vibrio fischeri, which establishes a symbiosis within the crypts of the nascent light organ of the squid Euprymna scolopes. Competition assays in newly hatched juvenile squid have shown that symbiotic V. fischeri are either niche-sharing “S strains”, which share the light organ when co-inoculated with other S strains, or niche-dominant “D strains”, which are typically found alone in the light organ after a co-colonization. To understand this D strain advantage, we determined the minimum time that different V. fischeri strains needed to initiate colonization and used confocal microscopy to localize the symbionts along their infection track. Further, we determined whether symbiont-induced host morphogenic events also occurred earlier during a D strain colonization. We conclude that D strains colonized more quickly than S strains. Nevertheless, light-organ populations in field-caught adult squid often contain both D and S strains. We determined experimentally that this symbiont population heterogeneity might be achieved in nature by a serial encounter of different strains in the environment.

Evolution of the developmental plasticity and a coupling between left mechanosensory neuromasts and an adaptive foraging behavior

Fernandes VFL, Macaspac C, Lu L, Yoshizawa M

Developmental Biology. 2018 Sep 15

Many animal species exhibit laterality in sensation and behavioral responses, namely, the preference for using either the left or right side of the sensory system. For example, some fish use their left eye when observing social stimuli, whereas they use their right eye to observe novel objects. However, it is largely unknown whether such laterality in sensory-behavior coupling evolves during rapid adaptation processes. Here, in the Mexican tetra, Astyanax mexicanus, we investigate the laterality in the relationship between an evolved adaptive behavior, vibration attraction behavior (VAB), and its main sensors, mechanosensory neuromasts. A. mexicanus has a surface-dwelling form and cave-dwelling forms (cavefish), whereby a surface fish ancestor colonized the new environment of a cave, eventually evolving cave-type morphologies such as increased numbers of neuromasts at the cranium. These neuromasts are known to regulate VAB, and it is known that, in teleosts, the budding (increasing) process of neuromasts is accompanied with dermal bone formation. This bone formation is largely regulated by endothelin signaling. To assess the evolutionary relationship between bone formation, neuromast budding, and VAB, we treated 1-3 month old juvenile fish with endothelin receptor antagonists. This treatment significantly increased cranial neuromasts in both surface and cavefish, and the effect was significantly more pronounced in cavefish. Antagonist treatment also increased the size of dermal bones in cavefish, but neuromast enhancement was observed earlier than dermal bone formation, suggesting that endothelin signaling may independently regulate neuromast development and bone formation. In addition, although we did not detect a major change in VAB level under this antagonist treatment, cavefish did show a positive correlation of VAB with the number of neuromasts on their left side but not their right. This laterality in correlation was observed when VAB emerged during cavefish development, but it was not seen in surface fish under any conditions tested, suggesting this laterality emerged through an evolutionary process. Above all, cavefish showed higher developmental plasticity in neuromast number and bone formation, and they showed an asymmetric correlation between the number of left-right neuromasts and VAB.

Maternal genetic effects in Astyanax cavefish development

Ma L, Strickler AG, Parkhurst A, Yoshizawa M, Shi J, Jeffery WR

Developmental Biology. 2018 Sep 15

The role of maternal factors in the evolution of development is poorly understood. Here we describe the use of reciprocal hybridization between the surface dwelling (surface fish, SF) and cave dwelling (cavefish, CF) morphs of the teleost Astyanax mexicanus to investigate the roles of maternal genetic effects in cavefish development. Reciprocal hybridization, a procedure in which F1 hybrids are generated by fertilizing SF eggs with CF sperm (SF X CF hybrids) and CF eggs with SF sperm (CF X SF hybrids), revealed that the CF degenerative eye phenotype showed maternal genetic effects. The eyes of CF X SF hybrids resembled the degenerate eyes of CF in showing ventral reduction of the retina and corresponding displacement of the lens within the optic cup, a smaller lens and eyeball, more lens apoptosis, a smaller cartilaginous sclera, and lens-specific gene expression characteristics compared to SF X CF hybrids, which showed eye and lens gene expression phenotypes resembling SF. In contrast, reciprocal hybridization failed to support roles for maternal genetic effects in the CF regressive pigmentation phenotype or in CF constructive changes related to enhanced jaw development. Maternal transcripts encoded by the pou2f1b, runx2b, and axin1 genes, which are involved in determining ventral embryonic fates, were increased in unfertilized CF eggs. In contrast, maternal mRNAs encoded by the ß-catenin and syntabulin genes, which control dorsal embryonic fates, showed similar expression levels in unfertilized SF and CF eggs. Furthermore, maternal transcripts of a sonic hedgehog gene were detected in SF and CF eggs and early cleaving embryos. This study reveals that CF eye degeneration is controlled by changes in maternal factors produced during oogenesis and introduces A. mexicanus as a model system for studying the role of maternal changes in the evolution of development.

Four new species of Magelona (Annelida: Magelonidae) from Easter Island, Guam and Hawaii

MagalhÃes WF, Bailey-Brock J, Watling L

Zootaxa. 2018 Aug 09

Use of MICRO/BEMF Core Facility

Several collections of magelonids from Easter Island, Guam in the Mariana Islands and Hawaii were examined. Five magelonids are fully described and illustrated: Magelona anuheone sp. nov. (Easter Island), M. cf. symmetrica Mortimer Mackie, 2006 (Guam), M. alexandrae sp. nov. (Hawaii), M. cinthyae sp. nov. (Hawaii) and M. paulolanai sp. nov. (Hawaii and Guam). Magelona anuheone sp. nov. has a distinctly longer than wide prostomium with inflated and well-developed prostomial horns, similar to M. montera from the Indian Ocean. Magelona alexandrae sp. nov. belongs to Magelona species with crenulated upper margin of thoracic postchaetal lamellae and also well-developed prostomial horns. Magelona cinthyae sp. nov. is unique among its congeners in that several collected adult individuals presented two or more eyespots in the prostomium, a feature only previously recorded to pelagic larval stages. Magelona paulolanai sp. nov. belongs to a group of species with rudimentary prostomial horns, simple capillaries on chaetiger 9 and tridentate abdominal hooks. All species are illustrated and compared to their morphologically similar congeners.

Gene expression across tissues, sex, and life stages in the sea urchin Tripneustes gratilla [Toxopneustidae, Odontophora, Camarodonta]

Láruson AJ, Coppard SE, Pespeni MH, Reed FA

Marine Genomics. 2018 Jul 29

The pan-tropical sea urchin Tripneustes gratilla is an ecologically and economically important shallow water algal grazer. The aquaculture of T. gratilla has spurred growing interest in the population biology of the species, and by extension the generation of more molecular resources. To this purpose, de novo transcriptomes of T. gratilla were generated for two adults, a male and a female, as well as for a cohort of approximately 1000 plutei larvae. Gene expression profiles of three adult tissue samples were quantified and compared. These samples were of gonadal tissue, the neural ring, and pooled tube feet and pedicellariae. Levels of shared and different gene expression between sexes, as well as across functional categories of interest, including the immune system, toxins, genes involved in fertilization, and sensory genes are highlighted. Differences in expression of isoforms between the sexes and Sex determining Region Y-related High Mobility Group box groups is observed. Additionally an expansion of the tumor suppressor DMBT1 is observed in T. gratilla when compared to the annotated genome of the sea urchin Strongylocentrotus purpuratus. The draft transcriptome of T. gratilla is presented here in order to facilitate more genomic level analysis of emerging model sea urchin systems.

Neural Crest Transplantation Reveals Key Roles in the Evolution of Cavefish Development

Yoshizawa M, Hixon E, Jeffery WR

Integrative and Comparative Biology. 2018 May 02

Evolutionary changes in Astyanax mexicanus cavefish with respect to conspecific surface fish, including the regression of eyes, loss of pigmentation, and modification of the cranial skeleton, involve derivatives of the neural crest. However, the role of neural crest cells in cavefish evolution and development is poorly understood. One of the reasons is that experimental methods for neural crest analysis are not well developed in the Astyanax system. Here we describe neural crest transplantation between Astyanax surface fish and cavefish embryos. We found differences in the migration of cranial neural crest cells transplanted from the surface fish anterior hindbrain to the same region of surface fish or cavefish hosts. Cranial neural crest cells migrated extensively throughout the head, and to a lesser extent the trunk, in surface fish hosts but their migration was mostly restricted to the anterior and dorsal head regions in cavefish hosts. Cranial neural crest cells derived from the surface fish transplants invaded the degenerating eyes of cavefish hosts, resulting in increased eye size and suggesting that cavefish neural crest cells are defective in forming optic derivatives. We found that melanophores were formed in albino cavefish from grafts of surface fish trunk neural crest cells, showing that the cavefish tissue environment is conducive for pigment cell development, and implicating intrinsic changes in cavefish neural crest cells in loss of body pigmentation. It is concluded that changes in neural crest cells play key roles in the evolution of cavefish development.

A new Calliovarica species (Seguenzioidea: Chilodontidae) from the Eocene of Oregon, USA: Persistence of a relict Mesozoic gastropod group in a unique forearc tectonic setting

Hickman CS

PaleoBios. 2018 Apr 18

Use of MICRO/BEMF Core Facility

A new, enigmatic chilodontid gastropod with distinctive periodic varices is described as Calliovarica oregonensis. It is based on ten specimens from slope deposits of the early late Eocene Nestucca Formation on the coastal Cascadia margin of present day Oregon, U.S.A. It is the last appearance of a Mesozoic group of epifaunal basal gastropods with periodic varices. It is the third species in a Cenozoic genus previously known only from the early Eocene Lodo Formation in California and the late Paleocene to early Eocene Red Bluff Tuff in New Zealand. The type species, C. eocensis, is refigured to clarify the nature of the axial varices as well as a terminal thickening and flaring of the apertural lip immediately following deposition of the final varix. Detailed preservation of microstructure in the nacreous layers of crushed and disintegrating shell fragments demonstrates the value of collecting material typically left behind in the field. Calliovarica oregonensis n. sp. is part of a poorly understood molluscan fauna that lived during an unusual paleoclimatic interval immediately prior to global cooling and extinctions in the late Eocene. It also thrived in an unusual volcano-sedimentary interval at the onset of subduction at the Cascadia margin. The tectonic setting provides a unique snapshot of a depositional environment receiving periodic influxes of ash from the young volcanic arc to the east and periodic basalt intrusions from the underlying asthenosphere into the forerarc over a hotspot or through slab window emplacement. Links between biofacies and lithofacies demonstrate the ability of paleontology and geology to provide reciprocal illumination, especially in dynamic settings with no modern counterparts. The link between local persistence of relict Mesozoic taxa and localized tectonic events merits further integrative investigation.

Synergy among Microbiota and Their Hosts: Leveraging the Hawaiian Archipelago and Local Collaborative Networks To Address Pressing Questions in Microbiome Research

Hynson N, Frank KL, Alegado RA, Amend AS, Arif M, Bennett GM, Jani AJ, Medeiros MCI, Mileyko Y, Nelson CE, Nguyen NH, Nigro OD, Prisic S, Shin S, Takagi D, Wilson ST, Yew JY

mSystems. 2018 Mar 13

Despite increasing acknowledgment that microorganisms underpin the healthy functioning of basically all multicellular life, few cross-disciplinary teams address the diversity and function of microbiota across organisms and ecosystems. Our newly formed consortium of junior faculty spanning fields such as ecology and geoscience to mathematics and molecular biology from the University of Hawai‘i at Mānoa aims to fill this gap. We are united in our mutual interest in advancing a new paradigm for biology that incorporates our modern understanding of the importance of microorganisms. As our first concerted research effort, we will assess the diversity and function of microbes across an entire watershed on the island of Oahu, Hawai‘i. Due to its high ecological diversity across tractable areas of land and sea, Hawai‘i provides a model system for the study of complex microbial communities and the processes they mediate. Owing to our diverse expertise, we will leverage this study system to advance the field of biology.

Rheinheimera salexigens sp. nov., isolated from a fishing hook, and emended description of the genus Rheinheimera

Hayashi K, Busse H, Golke J, Anderson J, Wan X, Hou S, Chain PSG, Prescott RD, Donachie SP

International Journal of Systematic and Evolutionary Microbiology. 2018 Jan 01

Use of MICRO/BEMF Core Facility

A Gram-negative, rod-shaped bacterium, designated KH87T, was isolated from a fishing hook that had been baited and suspended in seawater off O'ahu, Hawai'i. Based on a comparison of 1524 nt of the 16S rRNA gene sequence of strain KH87T, its nearest neighbours were the GammaproteobacteriaRheinheimera nanhaiensis E407-8T (96.2 % identity), Rheinheimera chironomi K19414T (96.0 %), Rheinheimera pacifica KMM 1406T (95.8 %), Rheinheimera muenzenbergensis E49T (95.7 %), Alishewanella solinquinati KMK6T (94.9 %) and Arsukibacterium ikkense GCM72T (94.6 %). Cells of KH87T were motile by a single polar flagellum, strictly aerobic, and catalase- and oxidase-positive. Growth occurred between 4 and 39 °C, and in a circumneutral pH range. Major fatty acids in whole cells of strain KH87T were cis-9-hexadecenoic acid, hexadecanoic acid and cis-11-octadecenoic acid. The quinone system contained mostly menaquinone MK-7, and a minor amount of ubiquinone Q-8. The polar lipid profile contained the major lipids phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, an unidentified aminolipid, and a lipid not containing phosphate, an amino group or a sugar moiety. Putrescine was the major polyamine. Physiological, biochemical and genomic data, including obligate halophily, absence of amylolytic activity, a quinone system dominated by MK-7 and DNA G+C content (42.0 mol%) distinguished KH87T from extant Rheinheimera species; strain KH87T was also distinguished by a multi-locus sequence analysis of aligned and concatenated 16S rRNA, gyrB, rpoB and rpoD gene sequences. Based on phenotypic and genotypic differences, the species Rheinheimera salexigens sp. nov. is proposed to accommodate KH87T as the type strain (=ATCC BAA-2715T=CIP 111115T). An emended description of the genus Rheinheimera is also proposed.