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The olfactory cues used by various animals to detect and identify food items often include volatile organic compounds (VOCs) produced by food-associated microorganisms. Microbial VOCs have potential as lures to trap animal pests, including insect crop pests. This study investigated microorganisms whose VOCs are attractive to natural populations of the spotted wing drosophila (SWD), an invasive insect pest of ripening fruits. The microorganisms readily cultured from wild SWD and SWD-infested fruits included yeasts, especially Hanseniaspora species, and various bacteria, including Proteobacteria (especially Acetobacteraceae and Enterobacteriaceae) and Actinobacteria. Traps in a raspberry planting that were baited with cultures of Hanseniaspora uvarum, H. opuntiae and the commercial lure Scentry trapped relatively high numbers of both SWD and non-target drosophilids. The VOCs associated with these baits were dominated by ethyl acetate and, for yeasts, other esters. By contrast, Gluconobacter species (Acetobacteraceae), whose VOCs were dominated by acetic acid and acetoin and lacked detectable ethyl acetate, trapped 60-75% fewer SWD but with very high selectivity for SWD. VOCs of two other taxa tested, the yeast Pichia sp. and Curtobacterium sp. (Actinobacteria), trapped very few SWD or other insects. Our demonstration of among-microbial variation in VOCs and their attractiveness to SWD and non-pest insects under field conditions provides the basis for improved design of lures for SWD management. Further research is required to establish how different microbial VOC profiles may function as reliable cues of habitat suitability for fly feeding and oviposition, and how this variation maps onto among-insect species differences in habitat preference.

An important goal for many nutrition-based microbiome studies is to identify the metabolic function of microbes in complex microbial communities and their impact on host physiology. This research can be confounded by poorly understood effects of community composition and host diet on the metabolic traits of individual taxa. Here, we investigated these multiway interactions by constructing and analyzing metabolic models comprising every combination of five bacterial members of the Drosophila gut microbiome (from single taxa to the five-member community of Acetobacter and Lactobacillus species) under three nutrient regimes. We show that the metabolic function of Drosophila gut bacteria is dynamic, influenced by community composition, and responsive to dietary modulation. Furthermore, we show that ecological interactions such as competition and mutualism identified from the growth patterns of gut bacteria are underlain by a diversity of metabolic interactions, and show that the bacteria tend to compete for amino acids and B vitamins more frequently than for carbon sources. Our results reveal that, in addition to fermentation products such as acetate, intermediates of the tricarboxylic acid (TCA) cycle, including 2-oxoglutarate and succinate, are produced at high flux and cross-fed between bacterial taxa, suggesting important roles for TCA cycle intermediates in modulating Drosophila gut microbe interactions and the potential to influence host traits. These metabolic models provide specific predictions of the patterns of ecological and metabolic interactions among gut bacteria under different nutrient regimes, with potentially important consequences for overall community metabolic function and nutritional interactions with the host. IMPORTANCEDrosophila is an important model for microbiome research partly because of the low complexity of its mostly culturable gut microbiota. Our current understanding of how Drosophila interacts with its gut microbes and how these interactions influence host traits derives almost entirely from empirical studies that focus on individual microbial taxa or classes of metabolites. These studies have failed to capture fully the complexity of metabolic interactions that occur between host and microbe. To overcome this limitation, we reconstructed and analyzed 31 metabolic models for every combination of the five principal bacterial taxa in the gut microbiome of Drosophila. This revealed that metabolic interactions between Drosophila gut bacterial taxa are highly dynamic and influenced by cooccurring bacteria and nutrient availability. Our results generate testable hypotheses about among-microbe ecological interactions in the Drosophila gut and the diversity of metabolites available to influence host traits.

Annotated genome sequences provide valuable insight into the functional capabilities of members of microbial communities. Nevertheless, most studies on the microbiome in animal guts use metagenomic data, hampering the assignment of genes to specific microbial taxa. Here, we make use of the readily culturable bacterial communities in the gut of the fruit fly Drosophila melanogaster to obtain draft genome sequences for 96 isolates from wild flies. These include 81 new de novo assembled genomes, assigned to three orders (Enterobacterales, Lactobacillales, and Rhodospirillales) with 80% of strains identified to species level using average nucleotide identity and phylogenomic reconstruction. Based on annotations by the RAST pipeline, among-isolate variation in metabolic function partitioned strongly by bacterial order, particularly by amino acid metabolism (Rhodospirillales), fermentation, and nucleotide metabolism (Lactobacillales) and arginine, urea, and polyamine metabolism (Enterobacterales). Seven bacterial species, comprising 2–3 species in each order, were well-represented among the isolates and included ≥5 strains, permitting analysis of metabolic functions in the accessory genome (i.e., genes not present in every strain). Overall, the metabolic function in the accessory genome partitioned by bacterial order. Two species, Gluconobacter cerinus (Rhodospirillales) and Lactiplantibacillus plantarum (Lactobacillales) had large accessory genomes, and metabolic functions were dominated by amino acid metabolism (G. cerinus) and carbohydrate metabolism (La. plantarum). The patterns of variation in metabolic capabilities at multiple phylogenetic scales provide the basis for future studies of the ecological and evolutionary processes shaping the diversity of microorganisms associated with natural populations of Drosophila.

An important goal for many nutrition-based microbiome studies is to identify the metabolic function of microbes in complex microbial communities and its impact on host physiology. This research can be confounded by poorly-understood effects of community composition and host diet on the metabolic traits of individual taxa. Here, we investigated these multi-way interactions by constructing and analyzing metabolic models comprising every combination of five bacterial members of theDrosophilagut microbiome (from single taxa to the five-member community ofAcetobacterandLactobacillusspecies) under three nutrient regimes. We show that the metabolic function ofDrosophilagut bacteria is dynamic, influenced by community composition and responsive to dietary modulation. Furthermore, we show that ecological interactions such as competition and mutualism identified from the growth patterns of gut bacteria are underlain by a diversity of metabolic interactions, and show that the bacteria tend to compete for amino acids and B vitamins more frequently than for carbon sources. Our results reveal that in addition to fermentation products such as acetate, intermediates of the tricarboxylic acid (TCA) cycle including 2-oxoglutarate and succinate are produced at high flux and cross-fed between bacterial taxa suggesting important roles for TCA cycle intermediates in modulatingDrosophilagut microbe interactions and the potential to influence host traits. These metabolic models provide specific predictions of the patterns of ecological and metabolic interactions among gut bacteria under different nutrient regimes, with potentially important consequences for overall community metabolic function and nutritional interactions with the host.IMPORTANCEDrosophilais an important model for microbiome research partly because of the low complexity of its mostly culturable gut microbiota. Our current understanding of howDrosophilainteracts with its gut microbes and how these interactions influence host traits derives almost entirely from empirical studies that focus on individual microbial taxa or classes of metabolites. These studies have failed to capture fully the complexity of metabolic interactions that occur between host and microbe. To overcome this limitation, we reconstructed and analyzed 31 metabolic models for every combination of the five principal bacterial taxa in the gut microbiome ofDrosophila. This revealed that metabolic interactions between betweenDrosophilagut bacterial taxa are highly dynamic and influenced by co-occurring bacteria and nutrient availability. Our results generate testable hypothesis about among-microbe ecological interactions in theDrosophilagut and the diversity of metabolites available to influence host traits.

Most research on the impact of the gut microbiome on animal nutrition is designed to identify the effects of single microbial taxa and single metabolites of microbial origin, without considering the potentially complex network of interactions among co-occurring microorganisms. Here, we investigated how different microbial associations and their fermentation products affect host nutrition, using Drosophila melanogaster colonized with three gut microorganisms (the bacteria Acetobacter fabarum and Lactobacillus brevis, and the yeast Hanseniaspora uvarum) in all seven possible combinations. Some microbial effects on host traits could be attributed to single taxa (e.g. yeast-mediated reduction of insect development time), while other effects were sex specific and driven by among-microbe interactions (e.g. male lipid content determined by interactions between the yeast and both bacteria). Parallel analysis of nutritional indices of microbe-free flies administered different microbial fermentation products (acetic acid, acetoin, ethanol and lactic acid) revealed a single consistent effect: that the lipid content of both male and female flies is reduced by acetic acid. This effect was recapitulated in male flies colonized with both yeast and A. fabarum, but not for any microbial treatment in females or males with other microbial complements. These data suggest that the effect of microbial fermentation products on host nutritional status is strongly context dependent, with respect to both the combination of associated microorganisms and host sex. Taken together, our findings demonstrate that among-microbe interactions can play a critically important role in determining the physiological outcome of host–microbiome interactions in Drosophila and, likely, in other animal hosts.

Acetic acid is a fermentation product of many microorganisms, including some that inhabit the food and guts of Drosophila. Here, we investigated the effect of dietary acetic acid on oviposition and larval performance of Drosophila. At all concentrations tested (0.34–3.4%), acetic acid promoted egg deposition by mated females in no-choice assays; and females preferred to oviposit on diet with acetic acid relative to acetic acid-free diet. However, acetic acid depressed larval performance, particularly extending the development time of both larvae colonized with the bacterium Acetobacter pomorum and axenic (microbe-free) larvae. The larvae may incur an energetic cost associated with dissipating the high acid load on acetic acid-supplemented diets. This effect was compounded by suppressed population growth of A. pomorum on the 3.4% acetic acid diet, such that the gnotobiotic Drosophila on this diet displayed traits characteristic of axenic Drosophila, specifically reduced developmental rate and elevated lipid content. It is concluded that acetic acid is deleterious to larval Drosophila, and hypothesized that acetic acid may function as a reliable cue for females to oviposit in substrates bearing microbial communities that promote larval nutrition.

RNA interference (RNAi) plays a key role in insect defense against viruses and transposable elements, and it is being applied as an experimental tool and for insect pest control. However, RNAi efficiency is highly variable for some insects, notably the pea aphid Acyrthosiphon pisum. In this study, we used natural variation in RNAi susceptibility of pea aphids to identify genes that influence RNAi efficiency. Susceptibility to orally-delivered dsRNA against the gut aquaporin gene AQP1 (ds-AQP1) varied widely across a panel of 83 pea aphid genotypes, from zero to total mortality. Genome-wide association between aphid performance on ds-AQP1 supplemented diet and aphid genetic variants yielded 103 significantly associated single nucleotide polymorphisms (SNPs), including variants in 55 genes, at the 10-4 probability cut-off. When ds-AQP1 was co-administered with dsRNA against six candidate genes, aphid mortality was reduced for three (50%) genes: the orthologs of the Drosophila genes trachealess (CG42865), headcase (CG15532) and a gene coding a peritrophin-A domain (CG8192), indicating that these genes function to promote RNAi efficiency against AQP1 in the pea aphid. Aphid susceptibility (quantified as mortality) to ds-AQP1 was correlated with RNAi against a further gene, snakeskin with essential gut function unrelated to AQP1, for some but not all aphid genotypes tested, suggesting that the determinants of RNAi efficiency may be partly gene-specific. This study demonstrates high levels of natural variation in susceptibility to RNAi and demonstrates the value of harnessing this variation to identify genes influencing RNAi efficiency.

Background Steinernematid nematodes form obligate symbioses with bacteria from the genus Xenorhabdus. Together Steinernema nematodes and their bacterial symbionts successfully infect, kill, utilize, and exit their insect hosts. During this process the nematodes and bacteria disassociate requiring them to re-associate before emerging from the host. This interaction can be complicated when two different nematodes co-infect an insect host. Results Non-cognate nematode-bacteria pairings result in reductions for multiple measures of success, including total progeny production and virulence. Additionally, nematode infective juveniles carry fewer bacterial cells when colonized by a non-cognate symbiont. Finally, we show that Steinernema nematodes can distinguish heterospecific and some conspecific non-cognate symbionts in behavioral choice assays. Conclusions Steinernema-Xenorhabdus symbioses are tightly governed by partner recognition and fidelity. Association with non-cognates resulted in decreased fitness, virulence, and bacterial carriage of the nematode-bacterial pairings. Entomopathogenic nematodes and their bacterial symbionts are a useful, tractable, and reliable model for testing hypotheses regarding the evolution, maintenance, persistence, and fate of mutualisms. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0939-6) contains supplementary material, which is available to authorized users.

Xenorhabdus bovienii bacteria have a dual lifestyle: they are mutualistic symbionts to many species of Steinernema nematodes and are pathogens to a wide array of insects. Previous studies have shown that virulence of X. bovienii–Steinernema spp. pairs decreases when the nematodes associate with non-cognate bacterial strains. However, the virulence of the X. bovienii strains alone has not been fully investigated. In this study, we characterized the virulence of nine X. bovienii strains in Galleria mellonella and Spodoptera littoralis and performed a comparative genomic analysis to correlate observed phenotypes with strain genotypes. Two X. bovienii strains were found to be highly virulent against the tested insect hosts, while three strains displayed attenuated insect virulence. Comparative genomic analyses revealed the presence of several clusters present only in virulent strains, including a predicted type VI secretion system (T6SS). We performed intra-species-competition assays, and showed that the virulent T6SS(+) strains generally outcompeted the less virulent T6SS(−) strains. Thus, we speculate that the T6SS in X. bovienii may be another addition to the arsenal of antibacterial mechanisms expressed by these bacteria in an insect, where it could potentially play three key roles: (1) competition against the insect host microbiota; (2) protection of the insect cadaver from necrotrophic microbial competitors; and (3) outcompeting other Xenorhabdus species and/or strains when co-infections occur.

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont–free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock’s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.

Two mouse lines transgenic with the human poliovirus receptor gene (PVR), TGM-PRG-1 and TGM-PRG-3, were characterized to determine whether transgene copy number and PVR expression levels influence susceptibility to poliovirus. The mouse lines have been bred for more than 10 generations and the transgene was stably transmitted to progeny as determined by Southern blot hybridization and restriction fragment length polymorphism. The transgene copy number is 10 in the TGM-PRG-3 mouse line and one in the TGM-PRG-1 mouse line. Abundance of PVR RNA is on average three-fold higher in TGM-PRG-3 relative to TGM-PRG-1 tissues, and the abundance of the receptor molecule is three-fold higher in TGM-PRG-3 central nervous system tissues compared to TGM-PRG-1 tissues as determined by Western blot analysis. When TGM-PRG-1 and TGM-PRG-3 mice were inoculated intracranially with a neurovirulent type III poliovirus strain, they developed clinical symptoms and CNS lesions characteristic of human poliomyelitis. These results indicate that the PVR gene is expressed as a functional receptor in the CNS of both mouse lines rendering the mice susceptible to poliovirus infection. Even though the two mouse lines have different copy numbers of the transgene and different levels of PVR RNA and protein, they are similar in their susceptibility to poliovirus.

In this study, we assessed the effect of the saprobic fungus, Fusarium oxysporum (Ascomycota: Hypocreales) on the fitness of the entomopathogenic nematode Heterorhabditis sonorensis (Caborca strain). Sand column assays were considered to evaluate the effect of fungal mycelia on infective juvenile (IJ) movement and host access. Additionally, we investigated the effect of fungal spores on the nematodes’ ability to search for a host, its virulence, penetration efficiency and reproduction. Three application timings were considered to assess interactions between the fungus and the nematodes. In vitro assays were also considered to determine the effect of fungal extracts on the nematode’s symbiotic bacteria. Our observations indicate that presence and age of fungal mycelia significantly affect IJ movement in the sand columns and their ability to establish in the host. These results were also reflected in a reduced insect mortality. In particular, treatments with the 15 days old mycelia showed a significant reduction in insect mortality and penetration efficiency. Presence of fungal spores also impacted nematode virulence and reproduction. In particular, two of the application timings tested (simultaneous [EPN and fungal spores applied at the same time] and alternate I [EPN applied first, fungus applied 24 h later]) resulted in antagonistic interactions. Moreover, IJ progeny was reduced to half in the simultaneous application. In vitro assays revealed that fungal extracts at the highest concentration tested (10 mg/ml) inhibited the growth of the symbiotic bacteria. Overall, these results suggest that saprobic fungi may play an important role in regulating. EPN populations in the soil, and that they may be one of the factors that impact nematode survival in the soil and their access to insect hosts.

In southern New South Wales, Australia, grazing wheat during the vegetative and early reproductive growth stages (typically during winter) can provide a valuable contribution of high quality feed during a period of low pasture growth. This paper reports results from a series of experiments investigating the agronomic management of grazed wheats in southern NSW. The effect of sowing date and grazing on dry matter production and subsequent grain yield of a range of wheat cultivars was measured in five experiments in 2004 and 2005. In all experiments, results were compared with ungrazed spring wheat (cv. Diamondbird). Grain yield of the best winter cultivar was either the same or significantly greater than the spring cultivar in each of the five experiments. Within the winter wheat cultivars, there was significant variation in grain yield, protein content and screenings, depending on site and year with the cultivar Marombi out-yielding all others. Interestingly, this cultivar usually had the least dry matter post-grazing but the greatest dry matter by anthesis of the winter wheats. Generally, if sowing of the winter wheat was delayed, then the effects on yield were small or non-existent. The results are discussed with respect to the benefits of incorporating grazing cereals into cropping programs in the medium rainfall zone of southern Australia.

Young crossbred sheep grazed dual-purpose wheat crops in three experiments evaluating the factors affecting variability in liveweight gain. All crops had high crude protein contents [22–33% dry matter (DM)] and in vitro DM digestibilities (0.754–0.876), which would not have limited liveweight gains. Digestibilities measured in vivo in grazing animals were even higher (0.839–0.854). No significant differences were found in preference of sheep for six different wheat cultivars. The diet composition and herbage intake of the sheep were measured in all experiments using plant wax marker profiles. Wheat forage made up 0.88–0.98 of the diet in sheep supplemented with unchopped oaten hay and 0.97–0.99 of the diet in unsupplemented sheep. Total intakes in the first two experiments were ~1380 g DM/day (3.7–4.0% of liveweight). Intakes in Experiment 3 were lower (960–1180 g DM/day; 2.6–3.1% of liveweight) because drought conditions reduced the amount of forage available. There were marked liveweight gain responses (30–50%) to mineral supplements based on NaCl, CaCO3 and MgO. Evidence is presented that these were mainly due to the Na and Mg, since the consumed diet was more than adequate for Ca. Relative to the requirements of young sheep for growth, wheat forage had high K levels (6–8 times requirement) and very low Na levels (as low as 0.02–0.03 of requirement) and thus very high K : Na ratios. Since high dietary K : Na ratios can greatly reduce Mg absorption from the rumen, the response to Na may have been partly mediated through improved Mg absorption. The liveweight gain responses to Na/Mg were large and economic, so it is recommended that young sheep grazing dual-purpose wheat be routinely supplemented with a 1 : 1 mix of NaCl : MgO.