Next-Generation Sequencing reveals relationship between the larval microbiome and food substrate in the polyphagous Queensland fruit fly.
ABSTRACT: Insects typically host substantial microbial communities (the 'microbiome') that can serve as a vital source of nutrients and also acts as a modulator of immune function. While recent studies have shown that diet is an important influence on the gut microbiome, very little is known about the dynamics underpinning microbial acquisition from natural food sources. Here, we addressed this gap by comparing the microbiome of larvae of the polyphagous fruit fly Bactrocera tryoni ('Queensland fruit fly') that were collected from five different fruit types (sapodilla [from two different localities], hog plum, pomegranate, green apple, and quince) from North-east to South-east Australia. Using Next-Generation Sequencing on the Illumina MiSeq platform, we addressed two questions: (1) what bacterial communities are available to B. tryoni larvae from different host fruit; and (2) how does the microbiome vary between B. tryoni larvae and its host fruit? The abundant bacterial taxa were similar for B. tryoni larvae from different fruit despite significant differences in the overall microbial community compositions. Our study suggests that the bacterial community structure of B. tryoni larvae is related less to the host fruit (diet) microbiome and more to vertical transfer of the microbiome during egg laying. Our findings also suggest that geographic location may play a quite limited role in structuring of larval microbiomes. This is the first study to use Next-Generation Sequencing to analyze the microbiome of B. tryoni larvae together with the host fruit, an approach that has enabled greatly increased resolution of relationships between the insect's microbiome and that of the surrounding host tissues.
Project description:BACKGROUND:Gut microbiota affects tephritid (Diptera: Tephritidae) fruit fly development, physiology, behavior, and thus the quality of flies mass-reared for the sterile insect technique (SIT), a target-specific, sustainable, environmentally benign form of pest management. The Queensland fruit fly, Bactrocera tryoni (Tephritidae), is a significant horticultural pest in Australia and can be managed with SIT. Little is known about the impacts that laboratory-adaptation (domestication) and mass-rearing have on the tephritid larval gut microbiome. Read lengths of previous fruit fly next-generation sequencing (NGS) studies have limited the resolution of microbiome studies, and the diversity within populations is often overlooked. In this study, we used a new near full-length (>?1300 nt) 16S rRNA gene amplicon NGS approach to characterize gut bacterial communities of individual B. tryoni larvae from two field populations (developing in peaches) and three domesticated populations (mass- or laboratory-reared on artificial diets). RESULTS:Near full-length 16S rRNA gene sequences were obtained for 56 B. tryoni larvae. OTU clustering at 99% similarity revealed that gut bacterial diversity was low and significantly lower in domesticated larvae. Bacteria commonly associated with fruit (Acetobacteraceae, Enterobacteriaceae, and Leuconostocaceae) were detected in wild larvae, but were largely absent from domesticated larvae. However, Asaia, an acetic acid bacterium not frequently detected within adult tephritid species, was detected in larvae of both wild and domesticated populations (55 out of 56 larval gut samples). Larvae from the same single peach shared a similar gut bacterial profile, whereas larvae from different peaches collected from the same tree had different gut bacterial profiles. Clustering of the Asaia near full-length sequences at 100% similarity showed that the wild flies from different locations had different Asaia strains. CONCLUSIONS:Variation in the gut bacterial communities of B. tryoni larvae depends on diet, domestication, and horizontal acquisition. Bacterial variation in wild larvae suggests that more than one bacterial species can perform the same functional role; however, Asaia could be an important gut bacterium in larvae and warrants further study. A greater understanding of the functions of the bacteria detected in larvae could lead to increased fly quality and performance as part of the SIT.
Project description:<h4>Background</h4>The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera, Tephritidae) is the most significant insect pest of Australian horticulture. Bactrocera tryoni is controlled using a range of tools including the Sterile Insect Technique (SIT). Mass-rearing and irradiation of pupae in SIT can reduce the fitness and quality of the released sterile insects. Studies have also showed reduced microbial gut diversity in domesticated versus wild tephritids.<h4>Results</h4>Transmission electron microscopy confirmed the presence of the bacterial isolates in the mid-gut of mass-reared larvae, and plate counts from individual larval guts showed increased numbers of bacteria in supplemented larvae. Several developmental and fitness parameters were tested including larval development time (egg-hatch to pupation), pupal weight, emergence, flight ability, sex-ratio, and time to adult eclosion (egg-hatch to adult eclosion). Enterobacter sp. and Asaia sp. shortened larval development time, while this was delayed by Lactobacillus sp., Leuconostoc sp. and a blend of all four bacteria. The mean time from egg hatch to adult eclosion was significantly reduced by Leuconostoc sp. and the blend for males and females, indicating that the individual bacterium and consortium affect flies differently depending on the life stage (larval or pupal). There was no impact of bacterial supplemented larvae on pupal weight, emergence, flight ability, or sex ratio.<h4>Conclusions</h4>Our findings show that bacteria fed to the larval stage of B. tryoni can impart fitness advantages, but the selection of probiotic strains (individual or a consortium) is key, as each have varying effects on the host. Bacteria added to the larval diet particularly Leuconostoc sp. and the blend have the capacity to reduce costs and increase the number of flies produced in mass-rearing facilities by reducing time to adult eclosion by 1.3 and 0.8 mean days for males, and 1.2 and 0.8 mean days for females.
Project description:Larval diets used for artificial rearing can have a significant effect on insect biology. The Queensland fruit fly (aka "Qfly"), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is one of the greatest challenges for fruit growers in Australia. The sterile insect technique (SIT) is being developed to manage outbreaks in regions that remain free of Qfly and to reduce populations in regions where this species is endemic. Factory scale rearing is essential for SIT; however, artificial larval diets are known to affect the microbiome of Qfly, which may then affect fly performance. In this study, high-throughput Illumina sequencing was used to assess the Qfly microbiome in colonies reared, for five generations from nature, on two common artificial diets (carrot and gel). At generation five (G5), the microbiome was assessed in larvae, pupae, adult males and adult females and standard fly quality control parameters were assessed together with additional performance measures of mating propensity and survival under nutritional stress. At the genus level, bacterial communities were significantly different between the colonies reared on the two larval diets. However, communities converged at Phyla to family taxonomic levels. Bacterial genera of Morganella, Citrobacter, Providencia, and Burkholderia were highly abundant in all developmental stages of Qfly reared on the gel diet, when compared to the carrot diet. Despite abundance of these genera, a greater percentage of egg hatching, heavier pupal weight and a higher percentage of fliers were found in the Qfly reared on the gel diet. Mating propensity and survival under nutritional stress was similar for adult Qfly that had been reared on the two larval diets. Overall, our findings demonstrate that the artificial larval diet strongly influences the microbiome and quality control measures of Qfly, with likely downstream effects on performance of flies released in SIT programs.
Project description:Bactrocera tryoni (Froggatt), the Queensland fruit fly (Qfly), is a highly polyphagous tephritid fly that is widespread in Eastern Australia. Qfly physiology is closely linked with its fungal associates, with particular relationship between Qfly nutrition and yeast or yeast-like fungi. Despite animal-associated fungi typically occurring in multi-species communities, Qfly studies have predominately involved the culture and characterisation of single fungal isolates. Further, only two studies have investigated the fungal communities associated with Qfly, and both have used culture-dependant techniques that overlook non-culturable fungi and hence under-represent, and provide a biased interpretation of, the overall fungal community. In order to explore a potentially hidden fungal diversity and complexity within the Qfly mycobiome, we used culture-independent, high-throughput Illumina sequencing techniques to comprehensively, and holistically characterized the fungal community of Qfly larvae and overcome the culture bias. We collected larvae from a range of fruit hosts along the east coast of Australia, and all had a mycobiome dominated by ascomycetes. The most abundant fungal taxa belonged to the genera Pichia (43%), Candida (20%), Hanseniaspora (10%), Zygosaccharomyces (11%) and Penicillium (7%). We also characterized the fungal communities of fruit hosts, and found a strong degree of overlap between larvae and fruit host communities, suggesting that these communities are intimately inter-connected. Our data suggests that larval fungal communities are acquired from surrounding fruit flesh. It is likely that the physiological benefits of Qfly exposure to fungal communities is primarily due to consumption of these fungi, not through syntrophy/symbiosis between fungi and insect 'host'.
Project description:<h4>Backround</h4>Commensal microbes can promote survival and growth of developing insects, and have important fitness implications in adulthood. Insect larvae can acquire commensal microbes through two main routes: by vertical acquisition from maternal deposition of microbes on the eggshells and by horizontal acquisition from the environment where the larvae develop. To date, however, little is known about how microbes acquired through these different routes interact to shape insect development. In the present study, we investigated how vertically and horizontally acquired microbiota influence larval foraging behaviour, development time to pupation and pupal production in the Queensland fruit fly ('Qfly'), Bactrocera tryoni.<h4>Results</h4>Both vertically and horizontally acquired microbiota were required to maximise pupal production in Qfly. Moreover, larvae exposed to both vertically and horizontally acquired microbiota pupated sooner than those exposed to no microbiota, or only to horizontally acquired microbiota. Larval foraging behaviour was also influenced by both vertically and horizontally acquired microbiota. Larvae from treatments exposed to neither vertically nor horizontally acquired microbiota spent more time overall on foraging patches than did larvae of other treatments, and most notably had greater preference for diets with extreme protein or sugar compositions.<h4>Conclusion</h4>The integrity of the microbiota early in life is important for larval foraging behaviour, development time to pupation, and pupal production in Qflies. These findings highlight the complexity of microbial relations in this species, and provide insights to the importance of exposure to microbial communities during laboratory- or mass-rearing of tephritid fruit flies.
Project description:The family Tephritidae includes some of the most notorious insect pests of agricultural and horticultural crops in tropical and sub-tropical regions. Despite the interest in the study of their gut microbiome, our present knowledge is largely based on the analysis of laboratory strains. In this study, we present a first comparative analysis of the gut microbiome profiles of field populations of ten African and Mediterranean tephritid pests. For each species, third instar larvae were sampled from different locations and host fruits and compared using 16S rRNA amplicon sequencing and a multi-factorial sampling design. We observed considerable variation in gut microbiome diversity and composition both between and within fruit fly species. A "core" microbiome, shared across all targeted species, could only be identified at most at family level (Enterobacteriaceae). At genus level only a few bacterial genera (Klebsiella, Enterobacter, and Bacillus) were present in most, but not all, samples, with high variability in their relative abundance. Higher relative abundances were found for seven bacterial genera in five of the fruit fly species considered. These were Erwinia in Bactrocera oleae, Lactococcus in B. zonata, Providencia in Ceratitis flexuosa, Klebsiella, and Rahnella in C. podocarpi and Acetobacter and Serratia in C. rosa. With the possible exception of C. capitata and B. dorsalis (the two most polyphagous species considered) we could not detect obvious relationships between fruit fly dietary breadth and microbiome diversity or abundance patterns. Similarly, our results did not suggest straightforward differences between the microbiome profiles of species belonging to Ceratitis and the closely related Bactrocera/Zeugodacus. These results provide a first comparative analysis of the gut microbiomes of field populations of multiple economically relevant tephritids and provide base line information for future studies that will further investigate the possible functional role of the observed associations.
Project description:In holometabolous insects, adult fitness depends on the quantity and quality of resource acquired at the larval stage. Diverse ecological factors can influence larval resource acquisition, but little is known about how these factors in the larval environment interact to modulate larval development and adult traits.Here, we addressed this gap by considering how key ecological factors of larval density, diet nutritional composition, and microbial growth interact to modulate pupal and adult traits in a polyphagous tephritid fruit fly, Bactrocera tryoni (aka "Queensland fruit fly").Larvae were allowed to develop at two larval densities (low and high), on diets that were protein-rich, standard, or sugar-rich and prepared with or without preservatives to inhibit or encourage microbial growth, respectively.Percentage of adult emergence and adult sex ratio were not affected by the interaction between diet composition, larval density, and preservative treatments, although low preservative content increased adult emergence in sugar-rich diets but decreased adult emergence in protein-rich and standard diets.Pupal weight, male and female adult dry weight, and female (but not male) body energetic reserves were affected by a strong three-way interaction between diet composition, larval density, and preservative treatment, whereby in general, low preservative content increased pupal weight and female lipid storage in sugar-rich diets particularly at low-larval density and differentially modulated the decrease in adult body weight caused by larval density across diets.Our findings provide insights into the ecological factors modulating larval development of a polyphagous fly species and shed light into the ecological complexity of the larval developmental environment in frugivorous insects.
Project description:Unlike for vertebrates, the impact of starvation on the gut microbiome of invertebrates is poorly studied. Deciphering shifts in metabolically active associated bacterial communities in vertebrates has led to determining the role of the associated microbiome in the sensation of hunger and discoveries of associated regulatory mechanisms. From an invertebrate perspective, such as the black soldier fly, such information could lead to enhanced processes for optimized biomass production and waste conversion. Bacteria associated with food substrates of black soldier fly are known to impact corresponding larval life-history traits (e.g., larval development); however, whether black soldier fly larval host state (i.e., starved) impacts the gut microbiome is not known. In this study, we measured microbial community structural and functional shifts due to black soldier fly larvae starvation. Data generated demonstrate such a physiological state (i.e., starvation) does in fact impact both aspects of the microbiome. At the phylum level, community diversity decreased significantly during black soldier fly larval starvation (<i>p</i> = 0.0025). Genus level DESeq2 analysis identified five genera with significantly different relative abundance (<i>q</i> < 0.05) across the 24 and 48 H post initiation of starvation: <i>Actinomyces</i>, <i>Microbacterium</i>, <i>Enterococcus</i>, <i>Sphingobacterium</i>, and <i>Leucobacter</i>. Finally, we inferred potential gene function and significantly predicted functional KEGG Orthology (KO) abundance. We demonstrated the metabolically active microbial community structure and function could be influenced by host-feeding status. Such perturbations, even when short in duration (e.g., 24 H) could stunt larval growth and waste conversion due to lacking a full complement of bacteria and associated functions.
Project description:Fruit production is negatively affected by a wide range of frugivorous insects, among them tephritid fruit flies are one of the most important. As a replacement for pesticide-based controls, enhancing natural fruit resistance through biotechnology approaches is a poorly researched but promising alternative. The use of quantitative reverse transcription PCR (RT-qPCR) is an approach to studying gene expression which has been widely used in studying plant resistance to pathogens and non-frugivorous insect herbivores, and offers a starting point for fruit fly studies. In this paper, we develop a gene selection pipe-line for known induced-defense genes in tomato fruit, <i>Solanum lycopersicum,</i> and putative detoxification genes in Queensland fruit fly, <i>Bactrocera tryoni,</i> as a basis for future RT-qPCR research. The pipeline started with a literature review on plant/herbivore and plant/pathogen molecular interactions. With respect to the fly, this was then followed by the identification of gene families known to be associated with insect resistance to toxins, and then individual genes through reference to annotated <i>B. tryoni</i> transcriptomes and gene identity matching with related species. In contrast for tomato, a much better studied species, individual defense genes could be identified directly through literature research. For <i>B. tryoni</i>, gene selection was then further refined through gene expression studies. Ultimately 28 putative detoxification genes from cytochrome P450 (P450), carboxylesterase (CarE), glutathione S-transferases (GST), and ATP binding cassette transporters (ABC) gene families were identified for <i>B. tryoni</i>, and 15 induced defense genes from receptor-like kinase (RLK), D-mannose/L-galactose, mitogen-activated protein kinase (MAPK), lipoxygenase (LOX), gamma-aminobutyric acid (GABA) pathways and polyphenol oxidase (PPO), proteinase inhibitors (PI) and resistance (R) gene families were identified from tomato fruit. The developed gene selection process for <i>B. tryoni</i> can be applied to other herbivorous and frugivorous insect pests so long as the minimum necessary genomic information, an annotated transcriptome, is available.
Project description:The majority of insect species have a clearly defined larval stage during development. Larval nutrition is crucial for individuals' growth and development, and larval foraging success often depends on both resource availability and competition for those resources. To date, however, little is known about how these factors interact to shape larval development and behaviour. Here we manipulated the density of larvae of the polyphagous fruit fly pest Bactrocera tryoni ('Queensland fruit fly'), and the diet concentration of patches in a foraging arena to address this gap. Using advanced statistical methods of machine learning and linear regression models, we showed that high larval density results in overall high larval aggregation across all diets except in extreme diet dilutions. Larval aggregation was positively associated with larval body mass across all diet concentrations except in extreme diet dilutions where this relationship was reversed. Over time, larvae in low-density arenas also tended to aggregate while those in high-density arenas tended to disperse, an effect that was observed for all diet concentrations. Furthermore, larvae in high-density arenas displayed significant avoidance of low concentration diets - a behaviour that was not observed amongst larvae in low-density arenas. Thus, aggregation can help, rather than hinder, larval growth in high-density environments, and larvae may be better able to explore available nutrition when at high-density than when at low-density.