MGEs as the MVPs of Partner Quality Variation in Legume-Rhizobium Symbiosis.

Despite decades of research, we are only just beginning to understand the forces maintaining variation in the nitrogen-fixing symbiosis between rhizobial bacteria and leguminous plants. In their recent work, Alexandra Weisberg and colleagues use genomics to document the breadth of mobile element diversity that carries the symbiosis genes of Bradyrhizobium in natural populations. Studying rhizobia from the perspective of their mobile genetic elements, which have their own transmission modes and fitness interests, reveals novel mechanisms for the generation and maintenance of diversity in natural populations of these ecologically and economically important mutualisms.

Transcriptomic Analysis of Steinernema Nematodes Highlights Metabolic Costs Associated to Xenorhabdus Endosymbiont Association and Rearing Conditions.

Entomopathogenic nematodes of the genus Steinernema have a mutualistic relationship with bacteria of the genus Xenorhabdus and together they form an antagonist partnership against their insect hosts. The nematodes (third-stage infective juveniles, or IJs) protect the bacteria from the external environmental stressors and vector them from one insect host to another. Xenorhabdus produce secondary metabolites and antimicrobial compounds inside the insect that protect the cadaver from soil saprobes and scavengers. The bacteria also become the nematodes' food, allowing them to grow and reproduce. Despite these benefits, it is yet unclear what the potential metabolic costs for Steinernema IJs are relative to the maintenance and vectoring of Xenorhabdus. In this study, we performed a comparative dual RNA-seq analysis of IJs of two nematode-bacteria partnerships: Steinernema carpocapsae-Xenorhabdus nematophila and Steinernema. puntauvense-Xenorhbdus bovienii. For each association, three conditions were studied: (1) IJs reared in the insect (in vivo colonized), (2) colonized IJs reared on liver-kidney agar (in vitro colonized), and (3) IJs depleted by the bacteria reared on liver-kidney agar (in vitro aposymbiotic). Our study revealed the downregulation of numerous genes involved in metabolism pathways, such as carbohydrate, amino acid, and lipid metabolism when IJs were reared in vitro, both colonized and without the symbiont. This downregulation appears to impact the longevity pathway, with the involvement of glycogen and trehalose metabolism, as well as arginine metabolism. Additionally, a differential expression of the venom protein known to be secreted by the nematodes was observed when both Steinernema species were depleted of their symbiotic partners. These results suggest Steinernema IJs may have a mechanism to adapt their virulence in absence of their symbionts.

Genome-Inferred Correspondence between Phylogeny and Metabolic Traits in the Wild Drosophila Gut Microbiome.

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.

Predicted Metabolic Function of the Gut Microbiota of Drosophila melanogaster.

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.IMPORTANCE Drosophila 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.

How gut microbiome interactions affect nutritional traits of Drosophila melanogaster.

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.

Candidate genetic determinants of intraspecific variation in pea aphid susceptibility to RNA interference.

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.

Response of Wild Spotted Wing Drosophila (Drosophila suzukii) to Microbial Volatiles.

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.

Physiological responses of insects to microbial fermentation products: Insights from the interactions between Drosophila and acetic acid.

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.

Variable virulence phenotype of Xenorhabdus bovienii (γ-Proteobacteria: Enterobacteriaceae) in the absence of their vector hosts.

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.

Fitness costs of symbiont switching using entomopathogenic nematodes as a model.

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.

Xenorhabdus bovienii CS03, the bacterial symbiont of the entomopathogenic nematode Steinernema weiseri, is a non-virulent strain against lepidopteran insects.

Xenorhabdus bacteria (γ-proteobacteria: Enterobacteriaceae) have dual lifestyles. They have a mutualistic relationship with Steinernema nematodes (Nematoda: Steinernematidae) and are pathogenic to a wide range of insects. Each Steinernema nematode associates with a specific Xenorhabdus species. However, a Xenorhabdus species can have multiple nematode hosts. For example, Xenorhabdus bovienii (Xb) colonizes at least nine Steinernema species from two different phylogenetic clades. The Steinernema-Xb partnership has been found in association with different insect hosts. Biological and molecular data on the Steinernema jollieti-Xb strain SS-2004 pair have recently been described. In particular, the Xb SS-2004 bacteria are virulent alone after direct injection into insect, making this strain a model for studying Xb virulence. In this study, we searched for Xb strains attenuated in virulence. For this purpose, we underwent infection assays with five Steinernema spp.-Xb pairs with two insects, Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). The S. weiseri-Xb CS03 pair showed attenuated virulence and lower fitness in S. littoralis in comparison to the other nematode-bacteria pairs. Furthermore, when injected alone into the hemolymph of G. mellonella or S. littoralis, the Xb CS03 bacterial strain was the only non-virulent strain. By comparison with the virulent Xb SS-2004 strain, Xb CS03 showed an increased sensitivity to the insect antimicrobial peptides, suggesting an attenuated response to the insect humoral immunity. To our current knowledge, Xb CS03 is the first non-virulent Xb strain identified. We propose this strain as a new model for studying the Xenorhabdus virulence.

In vivo and in vitro rearing of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae).

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.