Current Publications

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

Front Plant Sci. 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.

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 Rep. 2021 Oct 26

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.

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.

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

Davis JS, Pearcy MJ, Yew JY, Moyle LC

Evol Lett. 2021 Jul 17

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.

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 Dis. 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).

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 Dis. 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.

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

Bosch TCG, McFall-Ngai M

Curr Top Dev Biol. 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.

Drosophila suzukii avoidance of microbes in oviposition choice

Sato A, Tanaka KM, Yew JY, Takahashi A

R Soc Open Sci. 2021 Jan 20

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.

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

McFall-Ngai M, Bosch TCG

Epub. 2020 Nov 24

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.

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 Biol. 2020 Nov 03

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

Proc Natl Acad Sci USA . 2020 Nov 03

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.

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 Sept 01

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 Negl Trop Dis. 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.

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

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.

Natural Product Discovery by Direct Analysis in Real Time Mass Spectrometry

Yew JY

Mass Spectrom (Tokyo). 2020 Jan 11

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.

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

Mol Microbiol. 2019 Oct 11

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 7

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.

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 Test Anal. 2019 Jun 11

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.

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

Front Physiol. 2019 May 1

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.

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

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

J Vis Exp. 2019 Apr 6

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.