Fruit host-dependent fungal communities in the microbiome of wild Queensland fruit fly larvae.
ABSTRACT: 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: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:<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 various fungal communities that adhere to apple fruit are influenced by agricultural practices. However, the effects of fruit bagging-based management practice on the fungal microbiota are still unknown, and little is known about the fungal communities of bagged apple fruit. We conducted a study using apple fruit grown in a conventionally managed orchard where pesticide use is an indispensable practice. Fungal communities were collected from the calyx-end and peel tissues of bagged and unbagged fruit and characterized using barcode-type next-generation sequencing. Fruit bagging had a stronger effect on fungal richness, abundance, and diversity of the fungal microbiota in comparison to non-bagging. In addition, bagging also impacted the compositional variation of the fungal communities inhabiting each fruit part. We observed that fruit bagging had a tendency to maintain ecological equilibrium since Ascomycota and Basidiomycota were more distributed in bagged fruit than in unbagged fruit. These fungal communities consist of beneficial fungi rather than potentially harmful fungi. Approximately 50 dominant taxa were detected in bagged fruit, for example, beneficial genera such as <i>Articulospora</i>, <i>Bullera, Cryptococcus</i>, <i>Dioszegia</i>, <i>Erythrobasidium</i>, and <i>Sporobolomyces</i>, as well as pathogenic genera such as <i>Aureobasidium</i> and <i>Taphrina</i>. These results suggested that fruit bagging could significantly increase fungal richness and promote healthy fungal communities, especially the harmless fungal communities, which might be helpful for protecting fruit from the effects of pathogens. This study provides a foundation for understanding the impacts of bagging-based practice on the associated fungal microbiota.
Project description:Plants and fungi often produce toxic secondary metabolites that limit their consumption, but herbivores and fungivores that evolve resistance gain access to these resources and can also gain protection against nonresistant predators and parasites. Given that Drosophila melanogaster fruit fly larvae consume yeasts growing on rotting fruit and have evolved resistance to fermentation products, we decided to test whether alcohol protects flies from one of their common natural parasites, endoparasitoid wasps. Here, we show that exposure to ethanol reduces wasp oviposition into fruit fly larvae. Furthermore, if infected, ethanol consumption by fruit fly larvae causes increased death of wasp larvae growing in the hemocoel and increased fly survival without need of the stereotypical antiwasp immune response. This multifaceted protection afforded to fly larvae by ethanol is significantly more effective against a generalist wasp than a wasp that specializes on D. melanogaster. Finally, fly larvae seek out ethanol-containing food when infected, indicating that they use alcohol as an antiwasp medicine. Although the high resistance of D. melanogaster may make it uniquely suited to exploit curative properties of alcohol, it is possible that alcohol consumption may have similar protective effects in other organisms.
Project description:UNLABELLED:Chronic nonhealing wounds have been heralded as a silent epidemic, causing significant morbidity and mortality especially in elderly, diabetic, and obese populations. Polymicrobial biofilms in the wound bed are hypothesized to disrupt the highly coordinated and sequential events of cutaneous healing. Both culture-dependent and -independent studies of the chronic-wound microbiome have almost exclusively focused on bacteria, omitting what we hypothesize are important fungal contributions to impaired healing and the development of complications. Here we show for the first time that fungal communities (the mycobiome) in chronic wounds are predictive of healing time, associated with poor outcomes, and form mixed fungal-bacterial biofilms. We longitudinally profiled 100, nonhealing diabetic-foot ulcers with high-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus, estimating that up to 80% of wounds contain fungi, whereas cultures performed in parallel captured only 5% of colonized wounds. The "mycobiome" was highly heterogeneous over time and between subjects. Fungal diversity increased with antibiotic administration and onset of a clinical complication. The proportions of the phylum Ascomycota were significantly greater (P = 0.015) at the beginning of the study in wounds that took >8 weeks to heal. Wound necrosis was distinctly associated with pathogenic fungal species, while taxa identified as allergenic filamentous fungi were associated with low levels of systemic inflammation. Directed culturing of wounds stably colonized by pathogens revealed that interkingdom biofilms formed between yeasts and coisolated bacteria. Combined, our analyses provide enhanced resolution of the mycobiome during impaired wound healing, its role in chronic disease, and impact on clinical outcomes. IMPORTANCE:Wounds are an underappreciated but serious complication for a diverse spectrum of diseases. High-risk groups, such as persons with diabetes, have a 25% lifetime risk of developing a wound that can become chronic. The majority of microbiome research related to chronic wounds is focused on bacteria, but the association of fungi with clinical outcomes remains to be elucidated. Here we describe the dynamic fungal communities in 100 diabetic patients with foot ulcers. We found that communities are unstable over time, but at the first clinical presentation, the relative proportions of different phyla predict healing times. Pathogenic fungi not identified by culture reside in necrotic wounds and are associated with a poor prognosis. In wounds stably colonized by fungi, we identified yeasts capable of forming biofilms in concert with bacteria. Our findings illuminate the associations of the fungal mycobiome with wound prognosis and healing.
Project description:The olive fruit fly (OFF), Bactrocera oleae is the most devastating pest affecting olive fruit worldwide. Previous investigations have addressed the fungal microbiome associated with olive drupes or B. oleae, but the impact of the insect on fungal communities of olive fruit remains undescribed. In the present work, the fungal microbiome of olive drupes, infested and non-infested by the OFF, was investigated in four different localities and cultivars. Olive fruit fly infestations caused a general reduction of the fungal diversity, a higher quantity of the total DNA and an increase in taxa that remained unidentified or had unknown roles. The infestations led to imbalanced fungal communities with the growth of taxa that are usually outcompeted. While it was difficult to establish a cause-effect link between fly infestation and specific fungi, it is clear that the fly alters the natural microbial balance, especially the low abundant taxa. On the other hand, the most abundant ones, were not significantly influenced by the insect. In fact, despite the slight variation between the sampling locations, Aureobasidium, Cladosporium, and Alternaria, were the dominant genera, suggesting the existence of a typical olive fungal microbiome.
Project description: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:The fruit fly, Drosophila melanogaster, is preferentially found on fermenting fruits. The yeasts that dominate the microbial communities of these substrates are the primary food source for developing D. melanogaster larvae, and adult flies manifest a strong olfactory system-mediated attraction for the volatile compounds produced by these yeasts during fermentation. Although most work on this interaction has focused on the standard laboratory yeast Saccharomyces cerevisiae, a wide variety of other yeasts naturally ferment fallen fruit. Here we address the open question of whether D. melanogaster preferentially associates with distinct yeasts in different, closely-related environments. We characterized the spatial and temporal dynamics of Drosophila-associated fungi in Northern California wineries that use organic grapes and natural fermentation using high-throughput, short-amplicon sequencing. We found that there is nonrandom structure in the fungal communities that are vectored by flies both between and within vineyards. Within wineries, the fungal communities associated with flies in cellars, fermentation tanks, and pomace piles are distinguished by varying abundances of a small number of yeast species. To investigate the origins of this structure, we assayed Drosophila attraction to, oviposition on, larval development in, and longevity when consuming the yeasts that distinguish vineyard microhabitats from each other. We found that wild fly lines did not respond differentially to the yeast species that distinguish winery habitats in habitat specific manner. Instead, this subset of yeast shares traits that make them attractive to and ensure their close association with Drosophila.
Project description:Invasive fungal infections are an increasingly important cause of human morbidity and mortality. We generated a next-generation sequencing (NGS)-based method designed to detect a wide range of fungi and applied it to analysis of the fungal microbiome (mycobiome) of the lung during fungal infection. Internal transcribed spacer 1 (ITS1) amplicon sequencing and a custom analysis pipeline detected 96% of species from three mock communities comprised of potential fungal lung pathogens with good recapitulation of the expected species distributions (Pearson correlation coefficients r = 0.63, p = 0.004; r = 0.71, p < 0.001; r = 0.62, p = 0.002). We used this pipeline to analyze mycobiomes of bronchoalveolar lavage (BAL) specimens classified as culture-negative (n = 50) or culture-positive (n = 39) for Blastomyces dermatitidis/gilchristii, the causative agent of North America blastomycosis. Detected in 91.4% of the culture-positive samples, Blastomyces dominated (>50% relative abundance) the mycobiome in 68.6% of these culture-positive samples but was absent in culture-negative samples. To overcome any bias in relative abundance due to between-sample variation in fungal biomass, an abundance-weighting calculation was used to normalize the data by accounting for sample-specific PCR cycle number and PCR product concentration data utilized during sample preparation. After normalization, there was a statistically significant greater overall abundance of ITS1 amplicon in the Blastomyces-culture-positive samples versus culture-negative samples. Moreover, the normalization revealed a greater biomass of yeast and environmental fungi in several Blastomyces-culture-positive samples than in the culture-negative samples. Successful detection of Coccidioides, Scedosporium, Phaeoacremonium, and Aspergillus in 6 additional culture-positive BALs by ITS1 amplicon sequencing demonstrates the ability of this method to detect a broad range of fungi from clinical specimens, suggesting that it may be a potentially useful adjunct to traditional fungal microbiological testing for the diagnosis of respiratory mycoses.
Project description:Esca is a disease complex belonging to the grapevine trunk diseases cluster. It comprises five syndromes, three main fungal pathogenic agents and several symptoms, both internal (i.e., affecting woody tissue) and external (e.g., affecting leaves and bunches). The etiology and epidemiology of this disease complex remain, in part, unclear. Some of the points that are still under discussion concern the sudden rise in disease incidence, the simultaneous presence of multiple wood pathogens in affected grapevines, the causal agents and the discontinuity in time of leaf symptoms manifestation. The standard approach to the study of esca has been mostly through culture-dependent studies, yet, leaving many questions unanswered. In this study, we used Illumina® next-generation amplicon sequencing to investigate the mycobiome of grapevines wood in a vineyard with history of esca. We characterized the wood mycobiome composition, investigated the spatial dynamics of the fungal communities in different areas of the stem and in canes, and assessed the putative link between mycobiome and leaf symptoms. An unprecedented diversity of fungi is presented (289 taxa), including five genera reported for the first time in association with grapevines wood (Debaryomyces, Trematosphaeria, Biatriospora, Lopadostoma, and Malassezia) and numerous hitherto unreported species. Esca-associated fungi Phaeomoniella chlamydospora and Fomitiporia sp. dominate the fungal community, and numerous other fungi associated with wood syndromes are also encountered (e.g., Eutypa spp., Inonotus hispidus). The spatial analysis revealed differences in diversity, evenness and taxa abundances, the unique presence of certain fungi in specific areas of the plants, and tissue specificity. Lastly, the mycobiome composition of the woody tissue in proximity to leaves manifesting 'tiger stripes' symptoms of esca, as well as in leaf-symptomatic canes, was highly similar to that of plants not exhibiting any leaf symptomatology. This observation supports the current understanding that leaf symptoms are not directly linked with the fungal communities in the wood. This work builds to the understanding of the microbial ecology of the grapevines wood, offering insights and a critical view on the current knowledge of the etiology of esca.