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

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.

Environmental microbes promote phenotypic plasticity in reproduction and sleep behaviour.

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.

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

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.

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

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.

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.

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.

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.

Steroid Hormone Signaling Is Essential for Pheromone Production and Oenocyte Survival.

Many of the lipids found on the cuticles of insects function as pheromones and communicate information about age, sex, and reproductive status. In Drosophila, the composition of the information-rich lipid profile is dynamic and changes over the lifetime of an individual. However, the molecular basis of this change is not well understood. To identify genes that control cuticular lipid production in Drosophila, we performed a RNA interference screen and used Direct Analysis in Real Time and gas chromatography mass spectrometry to quantify changes in the chemical profiles. Twelve putative genes were identified whereby transcriptional silencing led to significant differences in cuticular lipid production. Amongst them, we characterized a gene which we name spidey, and which encodes a putative steroid dehydrogenase that has sex- and age-dependent effects on viability, pheromone production, and oenocyte survival. Transcriptional silencing or overexpression of spidey during embryonic development results in pupal lethality and significant changes in levels of the ecdysone metabolite 20-hydroxyecdysonic acid and 20-hydroxyecdysone. In contrast, inhibiting gene expression only during adulthood resulted in a striking loss of oenocyte cells and a concomitant reduction of cuticular hydrocarbons, desiccation resistance, and lifespan. Oenocyte loss and cuticular lipid levels were partially rescued by 20-hydroxyecdysone supplementation. Taken together, these results identify a novel regulator of pheromone synthesis and reveal that ecdysteroid signaling is essential for the maintenance of cuticular lipids and oenocytes throughout adulthood.

Pheromone evolution and sexual behavior in Drosophila are shaped by male sensory exploitation of other males.

Animals exhibit a spectacular array of traits to attract mates. Understanding the evolutionary origins of sexual features and preferences is a fundamental problem in evolutionary biology, and the mechanisms remain highly controversial. In some species, females choose mates based on direct benefits conferred by the male to the female and her offspring. Thus, female preferences are thought to originate and coevolve with male traits. In contrast, sensory exploitation occurs when expression of a male trait takes advantage of preexisting sensory biases in females. Here, we document in Drosophila a previously unidentified example of sensory exploitation of males by other males through the use of the sex pheromone CH503. We use mass spectrometry, high-performance liquid chromatography, and behavioral analysis to demonstrate that an antiaphrodisiac produced by males of the melanogaster subgroup also is effective in distant Drosophila relatives that do not express the pheromone. We further show that species that produce the pheromone have become less sensitive to the compound, illustrating that sensory adaptation occurs after sensory exploitation. Our findings provide a mechanism for the origin of a sex pheromone and show that sensory exploitation changes male sexual behavior over evolutionary time.

Sequential Collision- and Ozone-Induced Dissociation Enables Assignment of Relative Acyl Chain Position in Triacylglycerols.

Unambiguous identification of isomeric lipids by mass spectrometry represents a significant analytical challenge in contemporary lipidomics. Herein, the combination of collision-induced dissociation (CID) with ozone-induced dissociation (OzID) on an ion-trap mass spectrometer is applied to the identification of triacylglycerol (TG) isomers that vary only by the substitution pattern of fatty acyl (FA) chains esterified to the glycerol backbone. Isolated product ions attributed to loss of a single FA arising from CID of [TG + Na](+) ions react rapidly with ozone within the ion trap. The resulting CID/OzID spectra exhibit abundant ions that unequivocally reveal the relative position of FAs along the backbone. Isomeric TGs containing two or three different FA substituents are readily differentiated by diagnostic ions present in their CID/OzID spectra. Compatibility of this method with chromatographic separations enables the characterization of unusual TGs containing multiple short-chain FAs present in Drosophila.

Insulin signaling mediates sexual attractiveness in Drosophila.

Sexually attractive characteristics are often thought to reflect an individual's condition or reproductive potential, but the underlying molecular mechanisms through which they do so are generally unknown. Insulin/insulin-like growth factor signaling (IIS) is known to modulate aging, reproduction, and stress resistance in several species and to contribute to variability of these traits in natural populations. Here we show that IIS determines sexual attractiveness in Drosophila through transcriptional regulation of genes involved in the production of cuticular hydrocarbons (CHC), many of which function as pheromones. Using traditional gas chromatography/mass spectrometry (GC/MS) together with newly introduced laser desorption/ionization orthogonal time-of-flight mass spectrometry (LDI-MS) we establish that CHC profiles are significantly affected by genetic manipulations that target IIS. Manipulations that reduce IIS also reduce attractiveness, while females with increased IIS are significantly more attractive than wild-type animals. IIS effects on attractiveness are mediated by changes in CHC profiles. Insulin signaling influences CHC through pathways that are likely independent of dFOXO and that may involve the nutrient-sensing Target of Rapamycin (TOR) pathway. These results suggest that the activity of conserved molecular regulators of longevity and reproductive output may manifest in different species as external characteristics that are perceived as honest indicators of fitness potential.

Analysis of Drosophila lipids by matrix-assisted laser desorption/ionization mass spectrometric imaging.

Drosophila melanogaster is a major model organism for numerous lipid-related diseases. While comprehensive lipidomic profiles have been generated for D. melanogaster, little information is available on the localization of individual lipid classes and species. Here, we show the use of matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) to profile lipids in D. melanogaster tissue sections. The preparation of intact cryosections from whole insects presents a challenge due to the brittle hydrophobic cuticle surrounding the body and heterogeneous tissue types beneath the cuticle. However, the introduction of a novel sucrose infiltration step and gelatin as an embedding media greatly improved the quality of tissue sections. We generated MS image profiles of six major lipid classes: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and triacylglycerides. In addition, signals corresponding to two male-specific sex pheromones were detected in the ejaculatory bulb, a specialized site of pheromone production. MSI performed with 35 µm lateral resolution provided high sensitivity detection of at least 92 different lipid species, based on exact mass. In contrast, MSI with 10 µm lateral resolution enabled the detection of 36 lipid species but allowed lipid profiling of individual organs. The ability to localize lipid classes in intact sections from whole Drosophila provides a powerful tool for characterizing the effects of diet, age, stress, and environment on lipid production and distribution.

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

Unlike many species of Drosophila flies that colonize decaying fruits, Drosophila suzukii lay eggs in ripening fruits. The oviposition and feeding site preferences for bacterial growth were quantified in multiple strains of D. suzukii and its closely related species, D. subpulchrella and D. biarmipes . A continuous degree of preference for oviposition sites with Acetobacter growth both within and across species suggested that the separation in resource usage is notable but not complete among these species. The lack of interspecific differences in feeding site preference for Acetobacter -containing media implied that the oviposition site preferences evolved independently from the feeding site preference.

Long-sperm precedence and other cryptic female choices in Drosophila melanogaster.

Females that mate multiply make postmating choices about which sperm fertilize their eggs (cryptic female choice); however, the male characteristics they use to make such choices remain unclear. In this study, we sought to understand female sperm use patterns by evaluating whether Drosophila melanogaster females adjust sperm use (second male paternity) in response to four main factors: male genotype, male courtship effort, male pheromone alteration, and male postmating reproductive morphology. Our experiment was replicated across four different D. melanogaster lines, in a full factorial design, including a pheromone manipulation in which second males were perfumed to resemble heterospecific (D. yakuba) males. We found that females prefer longer sperm-regardless of mating order-in almost all contexts; this observed pattern of 'long-sperm precedence' is consistent with female postcopulatory choice of high-fitness male traits. Nonetheless, we also found that this general preference can be plastically altered by females in response to effects including perfuming treatment; this differential female sperm use is between otherwise identical males, and therefore solely female-mediated. Furthermore, our finding that females exercise choice using diverse criteria suggests a possible mechanism for the maintenance of variation in sexually selected male traits.

Pheromonal and behavioral cues trigger male-to-female aggression in Drosophila.

Appropriate displays of aggression rely on the ability to recognize potential competitors. As in most species, Drosophila males fight with other males and do not attack females. In insects, sex recognition is strongly dependent on chemosensory communication, mediated by cuticular hydrocarbons acting as pheromones. While the roles of chemical and other sensory cues in stimulating male to female courtship have been well characterized in Drosophila, the signals that elicit aggression remain unclear. Here we show that when female pheromones or behavior are masculinized, males recognize females as competitors and switch from courtship to aggression. To masculinize female pheromones, a transgene carrying dsRNA for the sex determination factor transformer (traIR) was targeted to the pheromone producing cells, the oenocytes. Shortly after copulation males attacked these females, indicating that pheromonal cues can override other sensory cues. Surprisingly, masculinization of female behavior by targeting traIR to the nervous system in an otherwise normal female also was sufficient to trigger male aggression. Simultaneous masculinization of both pheromones and behavior induced a complete switch in the normal male response to a female. Control males now fought rather than copulated with these females. In a reciprocal experiment, feminization of the oenocytes and nervous system in males by expression of transformer (traF) elicited high levels of courtship and little or no aggression from control males. Finally, when confronted with flies devoid of pheromones, control males attacked male but not female opponents, suggesting that aggression is not a default behavior in the absence of pheromonal cues. Thus, our results show that masculinization of either pheromones or behavior in females is sufficient to trigger male-to-female aggression. Moreover, by manipulating both the pheromonal profile and the fighting patterns displayed by the opponent, male behavioral responses towards males and females can be completely reversed. Therefore, both pheromonal and behavioral cues are used by Drosophila males in recognizing a conspecific as a competitor.

Synthesis and bioassay of the eight analogues of the CH503 male pheromone (3-acetoxy-11,19-octacosadien-1-ol) of the Drosophila melanogaster fruit fly.

Eight analogues of (3R,11Z,19Z)-CH503 (3-acetoxy-11,19-octacosadien-1-ol), the anti-aphrodisiac pheromone of male Drosophila melanogaster, were synthesized for a bioassay. These were the enantiomers of 3-acetoxy-11,19-octacosadiyn-1-ol (1), 3-acetoxyoctacosan-1-ol (2), (Z)-3-acetoxy-11-octacosen-1-ol (3), and (Z)-3-acetoxy-19-octacosen-1-ol (4). None of them were pheromonally active, indicating that the two double bonds at C-11 and C-19 were necessary for bioactivity.

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

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.

Microbes control Drosophila germline stem cell increase and egg maturation through hormonal pathways.

Reproduction is highly dependent on environmental and physiological factors including nutrition, mating stimuli and microbes. Among these factors, microbes facilitate vital functions for host animals such as nutritional intake, metabolic regulation, and enhancing fertility under poor nutrition conditions. However, detailed molecular mechanisms by which microbes control germline maturation, leading to reproduction, remain largely unknown. In this study, we show that environmental microbes exert a beneficial effect on Drosophila oogenesis by promoting germline stem cell (GSC) proliferation and subsequent egg maturation via acceleration of ovarian cell division and suppression of apoptosis. Moreover, insulin-related signaling is not required; rather, the ecdysone pathway is necessary for microbe-induced increase of GSCs and promotion of egg maturation, while juvenile hormone contributes only to increasing GSC numbers, suggesting that hormonal pathways are activated at different stages of oogenesis. Our findings reveal that environmental microbes can enhance host reproductivity by modulating host hormone release and promoting oogenesis.

Bacterial and fungal components of the gut microbiome have distinct, sex-specific roles in Hawaiian Drosophila reproduction.

Gut microbiomes provide numerous physiological benefits for host animals. The role of bacterial members of microbiomes in host physiology is well-documented. However, much less is known about the contributions and interactions of fungal members of the microbiome even though fungi are significant components of many microbiomes, including those of humans and insects. Here, we used antibacterial and antifungal drugs to manipulate the gut microbiome of a Hawaiian picture-wing Drosophila species, D. grimshawi, and identified distinct, sex-specific roles for the bacteria and fungi in microbiome community stability and reproduction. Female oogenesis, fecundity and mating drive were significantly diminished when fungal communities were suppressed. By contrast, male fecundity was more strongly affected by bacterial but not fungal populations. For males and females, suppression of both bacteria and fungi severely reduced fecundity and altered fatty acid levels and composition, implicating the importance of interkingdom interactions on reproduction and lipid metabolism. Overall, our results reveal that bacteria and fungi have distinct, sexually-dimorphic effects on host physiology and interkingdom dynamics in the gut help to maintain microbiome community stability and enhance reproduction.

Microbiomes and metabolomes of dominant coral reef primary producers illustrate a potential role for immunolipids in marine symbioses.

The dominant benthic primary producers in coral reef ecosystems are complex holobionts with diverse microbiomes and metabolomes. In this study, we characterize the tissue metabolomes and microbiomes of corals, macroalgae, and crustose coralline algae via an intensive, replicated synoptic survey of a single coral reef system (Waimea Bay, O'ahu, Hawaii) and use these results to define associations between microbial taxa and metabolites specific to different hosts. Our results quantify and constrain the degree of host specificity of tissue metabolomes and microbiomes at both phylum and genus level. Both microbiome and metabolomes were distinct between calcifiers (corals and CCA) and erect macroalgae. Moreover, our multi-omics investigations highlight common lipid-based immune response pathways across host organisms. In addition, we observed strong covariation among several specific microbial taxa and metabolite classes, suggesting new metabolic roles of symbiosis to further explore.