Axenic and gnotobiotic insect technologies in research on host-microbiota interactions.

Insects are one of the most important animal life forms on earth. Symbiotic microbes are closely related to the growth and development of the host insects and can affect pathogen transmission. For decades, various axenic insect-rearing systems have been developed, allowing further manipulation of symbiotic microbiota composition. Here we review the historical development of axenic rearing systems and the latest progress in using axenic and gnotobiotic approaches to study insect-microbe interactions. We also discuss the challenges of these emerging technologies, possible solutions to address these challenges, and future research directions that can contribute to a more comprehensive understanding of insect-microbe interactions.

Microbiome variation correlates with the insecticide susceptibility in different geographic strains of a significant agricultural pest, Nilaparvata lugens.

Microbiome-mediated insecticide resistance is an emerging phenomenon found in insect pests. However, microbiome composition can vary by host genotype and environmental factors, but how these variations may be associated with insecticide resistance phenotype remains unclear. In this study, we compared different field and laboratory strains of the brown planthopper Nilaparvata lugens in their microbiome composition, transcriptome, and insecticide resistance profiles to identify possible patterns of correlation. Our analysis reveals that the abundances of core bacterial symbionts are significantly correlated with the expression of several host detoxifying genes (especially NlCYP6ER1, a key gene previously shown involved in insecticides resistance). The expression levels of these detoxifying genes correlated with N. lugens insecticide susceptibility. Furthermore, we have identified several environmental abiotic factors, including temperature, precipitation, latitude, and longitude, as potential predictors of symbiont abundances associated with expression of key detoxifying genes, and correlated with insecticide susceptibility levels of N. lugens. These findings provide new insights into how microbiome-environment-host interactions may influence insecticide susceptibility, which will be helpful in guiding targeted microbial-based strategies for insecticide resistance management in the field.

Decline in symbiont-dependent host detoxification metabolism contributes to increased insecticide susceptibility of insects under high temperature.

The interactions between insects and their bacterial symbionts are shaped by a variety of abiotic factors, including temperature. As global temperatures continue to break high records, a great deal of uncertainty surrounds how agriculturally important insect pests and their symbionts may be affected by elevated temperatures, and its implications for future pest management. In this study, we examine the role of bacterial symbionts in the brown planthopper Nilaparvata lugens response to insecticide (imidacloprid) under different temperature scenarios. Our results reveal that the bacterial symbionts orchestrate host detoxification metabolism via the CncC pathway to promote host insecticide resistance, whereby the symbiont-inducible CncC pathway acts as a signaling conduit between exogenous abiotic stimuli and host metabolism. However, this insect-bacterial partnership function is vulnerable to high temperature, which causes a significant decline in host-bacterial content. In particular, we have identified the temperature-sensitive Wolbachia as a candidate player in N. lugens detoxification metabolism. Wolbachia-dependent insecticide resistance was confirmed through a series of insecticide assays and experiments comparing Wolbachia-free and Wolbachia-infected N. lugens and also Drosophila melanogaster. Together, our research reveals elevated temperatures negatively impact insect-bacterial symbiosis, triggering adverse consequences on host response to insecticide (imidacloprid) and potentially other xenobiotics.

Hybrid Nanopore-Illumina Genome Assembly of a Drosophila suzukii Gut Bacterial Symbiont, Gluconobacter cerinus FLW-1.

Gluconobacter is a genus of acetic acid bacteria (AAB) whose members have been shown to function as insect symbionts. Here, we report the complete genome sequence of Gluconobacter cerinus, isolated from field-collected Drosophila suzukii using a hybrid assembly approach. The data provide essential insights into the metabolic functions of the symbiont to the host.

Prevalence of Field-Collected House Flies and Stable Flies With Bacteria Displaying Cefotaxime and Multidrug Resistance.

Antibiotic use in livestock accounts for 80% of total antibiotic use in the United States and has been described as the driver for resistance evolution and spread. As clinical infections with multidrug-resistant pathogens are rapidly rising, there remains a missing link between agricultural antibiotic use and its impact on human health. In this study, two species of filth flies from a livestock operation were collected over the course of 11 mo: house flies Musca domestica (L.) (Diptera: Muscidae), representing a generalist feeder, and stable flies Stomoxys calcitrans (L.) (Diptera: Muscidae), representing a specialist (blood) feeder. The prevalence of flies carrying cefotaxime-resistant (CTX-R) bacteria in whole bodies and dissected guts were assayed by culturing on antibiotic-selective media, with distinct colonies identified by Sanger sequencing. Of the 149 flies processed, including 81 house flies and 68 stable flies, 18 isolates of 12 unique bacterial species resistant to high-level cefotaxime were recovered. These isolates also showed resistance to multiple classes of antibiotics. The CTX-R isolates were predominantly recovered from female flies, which bore at least two resistant bacterial species. The majority of resistant bacteria were isolated from the guts encompassing both enteric pathogens and commensals, sharing no overlap between the two fly species. Together, we conclude that house flies and stable flies in the field could harbor multidrug-resistant bacteria. The fly gut may serve as a reservoir for the acquisition and dissemination of resistance genes.

Draft Genome Sequence of an Environmental Vibrio cholerae Strain, 2012Env-25, Obtained Using Nanopore Sequencing Technology.

Vibrio cholerae is a halophilic Gram-negative bacterial species and the etiological agent of cholera. Here, we report the draft genome sequence of an environmental V. cholerae strain, 2012Env-25, obtained using Oxford Nanopore Technologies (ONT) to provide insights into the ecology, evolution, and pathogenic potential of this bacterium.

Activation of Vibrio cholerae quorum sensing promotes survival of an arthropod host.

Vibrio cholerae colonizes the human terminal ileum to cause cholera, and the arthropod intestine and exoskeleton to persist in the aquatic environment. Attachment to these surfaces is regulated by the bacterial quorum-sensing signal transduction cascade, which allows bacteria to assess the density of microbial neighbours. Intestinal colonization with V. cholerae results in expenditure of host lipid stores in the model arthropod Drosophila melanogaster. Here we report that activation of quorum sensing in the Drosophila intestine retards this process by repressing V. cholerae succinate uptake. Increased host access to intestinal succinate mitigates infection-induced lipid wasting to extend survival of V. cholerae-infected flies. Therefore, quorum sensing promotes a more favourable interaction between V. cholerae and an arthropod host by reducing the nutritional burden of intestinal colonization.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions.

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Sucralose Promotes Food Intake through NPY and a Neuronal Fasting Response.

Non-nutritive sweeteners like sucralose are consumed by billions of people. While animal and human studies have demonstrated a link between synthetic sweetener consumption and metabolic dysregulation, the mechanisms responsible remain unknown. Here we use a diet supplemented with sucralose to investigate the long-term effects of sweet/energy imbalance. In flies, chronic sweet/energy imbalance promoted hyperactivity, insomnia, glucose intolerance, enhanced sweet taste perception, and a sustained increase in food and calories consumed, effects that are reversed upon sucralose removal. Mechanistically, this response was mapped to the ancient insulin, catecholamine, and NPF/NPY systems and the energy sensor AMPK, which together comprise a novel neuronal starvation response pathway. Interestingly, chronic sweet/energy imbalance promoted increased food intake in mammals as well, and this also occurs through an NPY-dependent mechanism. Together, our data show that chronic consumption of a sweet/energy imbalanced diet triggers a conserved neuronal fasting response and increases the motivation to eat.

The interplay between intestinal bacteria and host metabolism in health and disease: lessons from Drosophila melanogaster.

All higher organisms negotiate a truce with their commensal microbes and battle pathogenic microbes on a daily basis. Much attention has been given to the role of the innate immune system in controlling intestinal microbes and to the strategies used by intestinal microbes to overcome the host immune response. However, it is becoming increasingly clear that the metabolisms of intestinal microbes and their hosts are linked and that this interaction is equally important for host health and well-being. For instance, an individual's array of commensal microbes can influence their predisposition to chronic metabolic diseases such as diabetes and obesity. A better understanding of host-microbe metabolic interactions is important in defining the molecular bases of these disorders and could potentially lead to new therapeutic avenues. Key advances in this area have been made using Drosophila melanogaster. Here, we review studies that have explored the impact of both commensal and pathogenic intestinal microbes on Drosophila carbohydrate and lipid metabolism. These studies have helped to elucidate the metabolites produced by intestinal microbes, the intestinal receptors that sense these metabolites, and the signaling pathways through which these metabolites manipulate host metabolism. Furthermore, they suggest that targeting microbial metabolism could represent an effective therapeutic strategy for human metabolic diseases and intestinal infection.

Behavioral Microbiomics: A Multi-Dimensional Approach to Microbial Influence on Behavior.

The role of microbes as a part of animal systems has historically been an under-appreciated aspect of animal life histories. Recently, evidence has emerged that microbes have wide-ranging influences on animal behavior. Elucidating the complex relationships between host-microbe interactions and behavior requires an expanded ecological perspective, involving the host, the microbiome and the environment; which, in combination, is termed the holobiont. We begin by seeking insights from the literature on host-parasite interactions, then expand to consider networks of interactions between members of the microbial community. A central aspect of the environment is host nutrition. We describe how interactions between the nutrient environment, the metabolic and behavioral responses of the host and the microbiome can be studied using an integrative framework called nutritional geometry, which integrates and maps multiple aspects of the host and microbial response in multidimensional nutrient intake spaces.

The Host as the Driver of the Microbiota in the Gut and External Environment of Drosophila melanogaster.

Most associations between animals and their gut microbiota are dynamic, involving sustained transfer of food-associated microbial cells into the gut and shedding of microorganisms into the external environment with feces, but the interacting effects of host and microbial factors on the composition of the internal and external microbial communities are poorly understood. This study on laboratory cultures of the fruit fly Drosophila melanogaster reared in continuous contact with their food revealed time-dependent changes of the microbial communities in the food that were strongly influenced by the presence and abundance of Drosophila. When germfree Drosophila eggs were aseptically added to nonsterile food, the microbiota in the food and flies converged to a composition dramatically different from that in fly-free food, showing that Drosophila has microbiota-independent effects on the food microbiota. The microbiota in both the flies that developed from unmanipulated eggs (bearing microorganisms) and the associated food was dominated by the bacteria most abundant on the eggs, demonstrating effective vertical transmission via surface contamination of eggs. Food coinoculated with a four-species defined bacterial community of Acetobacter and Lactobacillus species revealed the progressive elimination of Lactobacillus from the food bearing few or no Drosophila, indicating the presence of antagonistic interactions between Acetobacter and Lactobacillus. Drosophila at high densities ameliorated the Acetobacter/Lactobacillus antagonism, enabling Lactobacillus to persist. This study with Drosophila demonstrates how animals can have major, coordinated effects on the composition of microbial communities in the gut and immediate environment.

Host genetic determinants of microbiota-dependent nutrition revealed by genome-wide analysis of Drosophila melanogaster.

Animals bear communities of gut microorganisms with substantial effects on animal nutrition, but the host genetic basis of these effects is unknown. Here we use Drosophila to demonstrate substantial among-genotype variation in the effects of eliminating the gut microbiota on five host nutritional indices (weight, protein, lipid, glucose and glycogen contents); this includes variation in both the magnitude and direction of microbiota-dependent effects. Genome-wide association studies to identify the genetic basis of the microbiota-dependent variation reveal polymorphisms in largely non-overlapping sets of genes associated with variation in the nutritional traits, including strong representation of conserved genes functioning in signalling. Key genes identified by the GWA study are validated by loss-of-function mutations that altered microbiota-dependent nutritional effects. We conclude that the microbiota interacts with the animal at multiple points in the signalling and regulatory networks that determine animal nutrition. These interactions with the microbiota are probably conserved across animals, including humans.

In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors.

Symbiosis is often characterized by co-evolutionary changes in the genomes of the partners involved. An understanding of these changes can provide insight into the nature of the relationship, including the mechanisms that initiate and maintain an association between organisms. In this study we examined the genome sequences of bacteria isolated from the Drosophila melanogaster gut with the objective of identifying genes that are important for function in the host. We compared microbiota isolates with con-specific or closely related bacterial species isolated from non-fly environments. First the phenotype of germ-free Drosophila (axenic flies) was compared to that of flies colonized with specific bacteria (gnotobiotic flies) as a measure of symbiotic function. Non-fly isolates were functionally distinct from bacteria isolated from flies, conferring slower development and an altered nutrient profile in the host, traits known to be microbiota-dependent. Comparative genomic methods were next employed to identify putative symbiosis factors: genes found in bacteria that restore microbiota-dependent traits to gnotobiotic flies, but absent from those that do not. Factors identified include riboflavin synthesis and stress resistance. We also used a phylogenomic approach to identify protein coding genes for which fly-isolate sequences were more similar to each other than to other sequences, reasoning that these genes may have a shared function unique to the fly environment. This method identified genes in Acetobacter species that cluster in two distinct genomic loci: one predicted to be involved in oxidative stress detoxification and another encoding an efflux pump. In summary, we leveraged genomic and in vivo functional comparisons to identify candidate traits that distinguish symbiotic bacteria. These candidates can serve as the basis for further work investigating the genetic requirements of bacteria for function and persistence in the Drosophila gut.

Gut microbiota dictates the metabolic response of Drosophila to diet.

Animal nutrition is profoundly influenced by the gut microbiota, but knowledge of the scope and core mechanisms of the underlying animal-microbiota interactions is fragmentary. To investigate the nutritional traits shaped by the gut microbiota of Drosophila, we determined the microbiota-dependent response of multiple metabolic and performance indices to systematically varied diet composition. Diet-dependent differences between Drosophila bearing its unmanipulated microbiota (conventional flies) and experimentally deprived of its microbiota (axenic flies) revealed evidence for: microbial sparing of dietary B vitamins, especially riboflavin, on low-yeast diets; microbial promotion of protein nutrition, particularly in females; and microbiota-mediated suppression of lipid/carbohydrate storage, especially on high sugar diets. The microbiota also sets the relationship between energy storage and body mass, indicative of microbial modulation of the host signaling networks that coordinate metabolism with body size. This analysis identifies the multiple impacts of the microbiota on the metabolism of Drosophila, and demonstrates that the significance of these different interactions varies with diet composition and host sex.

The bacterial communities in plant phloem-sap-feeding insects.

The resident microbiota of animals represents an important contribution to the global microbial diversity, but it is poorly known in many animals. This study investigated the bacterial diversity in plant phloem-sap-feeding whiteflies, aphids and psyllids by pyrosequencing bacterial 16S rRNA gene amplicons. After correction for sequencing error, just 3-7 bacterial operational taxonomic units were recovered from each insect sample sequenced to sufficient depth for saturation of rarefaction curves. Most samples were dominated by primary and secondary symbionts, which are localized to insect cells or the body cavity, indicative of a dearth of bacterial colonists of the gut lumen. Diversity indices of the bacterial communities (Shannon's index: 0.40-1.46, Simpson's index: 0.15-0.74) did not differ significantly between laboratory and field samples of the phloem-feeding insects, but were significantly lower than in drosophilid flies quantified by the same methods. Both the low bacterial content of the phloem sap diet and biological processes in the insect may contribute to the apparently low bacterial diversity in these phloem-feeding insects.

The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis.

The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans.

Microbe-dependent and nonspecific effects of procedures to eliminate the resident microbiota from Drosophila melanogaster.

Comparisons of animals bearing and lacking microorganisms can offer valuable insight into the interactions between animal hosts and their resident microbiota. Most hosts are naturally infected, and therefore, these comparisons require specific procedures (e.g., antibiotic treatment or physical exclusion of microorganisms) to disrupt the microbiota, but the potential for confounding nonspecific effects of the procedure on the traits of the host exists. Microbe-dependent and nonspecific effects can be discriminated by using multiple procedures: microbe-dependent effects are evident in hosts made microbe free by different procedures, but nonspecific effects are unique to individual procedures. As a demonstration, two procedures, oral administration of chlortetracycline (50 µg ml(-1) diet) and microbiota removal by egg dechorionation, were applied to Drosophila melanogaster in a 2-by-2 factorial design. Microorganisms were undetectable in flies from dechorionated eggs and reduced by >99% in chlortetracycline-treated flies. Drosophila flies subjected to both protocols displayed an extended preadult development time, suggesting that the microbiota promotes the development rate. Female chlortetracycline-treated flies, whether from untreated or dechorionated eggs, displayed reduced protein content and egg fecundity, which could be attributed to the nonspecific effect of the antibiotic. We recommend that procedures used to disrupt the microbiota of animals should be selected, following systematic analysis of alternative mechanistically distinct procedures, on the basis of two criteria: those that achieve the greatest reduction (ideally, elimination) of the microbiota and those that achieve minimal nonspecific effects.

Impact of the resident microbiota on the nutritional phenotype of Drosophila melanogaster.

BACKGROUND: Animals are chronically infected by benign and beneficial microorganisms that generally promote animal health through their effects on the nutrition, immune function and other physiological systems of the host. Insight into the host-microbial interactions can be obtained by comparing the traits of animals experimentally deprived of their microbiota and untreated animals. Drosophila melanogaster is an experimentally tractable system to study host-microbial interactions. METHODOLOGY/PRINCIPAL FINDINGS: The nutritional significance of the microbiota was investigated in D. melanogaster bearing unmanipulated microbiota, demonstrated by 454 sequencing of 16S rRNA amplicons to be dominated by the α-proteobacterium Acetobacter, and experimentally deprived of the microbiota by egg dechorionation (conventional and axenic flies, respectively). In axenic flies, larval development rate was depressed with no effect on adult size relative to conventional flies, indicating that the microbiota promotes larval growth rates. Female fecundity did not differ significantly between conventional and axenic flies, but axenic flies had significantly reduced metabolic rate and altered carbohydrate allocation, including elevated glucose levels. CONCLUSIONS/SIGNIFICANCE: We have shown that elimination of the resident microbiota extends larval development and perturbs energy homeostasis and carbohydrate allocation patterns of of D. melanogaster. Our results indicate that the resident microbiota promotes host nutrition and interacts with the regulation of host metabolism.