Gut Bacteriome Analysis of Anastrepha fraterculus sp. 1 During the Early Steps of Laboratory Colonization

Abstract

Microbial communities associated to insect species are involved in essential biological functions such as host nutrition, reproduction and survivability. Main factors have been described as modulators of gut bacterial community, such as diet, habit, developmental stage and taxonomy of the host. The present work focuses on the complex changes that gut microbial communities go through when wild insects are introduced to artificial rearing conditions. Specifically, we analyzed the effect of the laboratory colonization on the richness and diversity of the gut bacteriome hosted by the fruit fly pest Anastrepha fraterculus sp. 1. Bacterial profiles were studied by amplicon sequencing of the 16S rRNA V3-V4 hypervariable region in gut samples of males and females, in teneral (1-day-old, unfed) and post-teneral (15-day-old, fed) flies. A total of 3,147,665 sequence reads were obtained and 32 bacterial operational taxonomic units (OTUs) were identified. Proteobacteria was the most abundant phylum (93.3% of the total reads) and, Wolbachia and Enterobacter were the most represented taxa at the genus level (29.9% and 27.7%, respectively, of the total read counts). Wild and laboratory flies showed highly significant differences in the relative abundances of bacteria. The analysis of the core bacteriome showed the presence of five OTUs in all samples grouped by origin, while nine and five OTUs were exclusively detected in laboratory and wild flies, respectively. Irrespective of fly origin or sex, a dominant presence of Wolbachia was observed in teneral flies, whereas Enterobacter was highly abundant in post-teneral individuals. We evidenced significant differences in bacterial richness and diversity among generations under laboratory colonization (F0, F1, F3 and F6) and compared to laboratory and wild flies, displaying also differential patterns between teneral and post-teneral flies. Laboratory and wild A. fraterculus sp. 1 harbor different gut bacterial communities. Laboratory colonization has an important effect on the microbiota, most likely associated to the combined effects of insect physiology and environmental conditions (e.g., diet and colony management).

Analysis of the Gut Bacterial Community of Wild Larvae of Anastrepha fraterculus sp. 1: Effect of Host Fruit, Environment, and Prominent Stable Associations of the Genera Wolbachia, Tatumella, and Enterobacter

“… Müller (2013) found that the larval gut bacteriome of a Brazilian wild population of A. fraterculus was dominated by Actinobacteria. In this case, the author targeted a different region of the 16S rRNA gene, which could partially explain the differences; however, the fact that the morphotype of A. fraterculus studied is not indicated by Müller (2013) together with environmental variation, such as diet, might contribute to the differences observed with our study as well as with previous results ( Augustinos et al, 2019 ; Salgueiro et al, 2020 ). For other Anastrepha species, a study carried out based on 454 pyrosequencing analysis, reported that the gut of larvae was dominated by Escherichia in A. striata, A. ludens and A. obliqua , and by Raoultella in A. serpentina ( Ventura et al, 2018 ).…”

“…The most abundant and diverse phylum was Proteobacteria, in agreement with results previously reported for A. fraterculus sp. 1 adults ( Augustinos et al, 2019 ; Juárez et al, 2019 ; Salgueiro et al, 2020 ). Müller (2013) found that the larval gut bacteriome of a Brazilian wild population of A. fraterculus was dominated by Actinobacteria.…”

“…Wild, third instar larvae of A. fraterculus sp. 1 were surface sterilized, and subsequently dissected in a laminar flow hood, following procedures described in Salgueiro et al (2020) for adult flies. Sterilization was carried by rinsing the larvae in a sequence of sterilized distilled water (sdw), sodium hypochlorite 0.05%, ethanol 70%, and sterile PBS 1X, for 1 min each.…”

“…For DNA extraction, guts from five larvae were pooled per sample to reduce inter-individual variability. DNA extraction was performed following Baruffi et al (1995) with modifications related to the size of the sample, as it was reported previously ( Salgueiro et al, 2020 ). The quantity and quality of the extracted DNA was measured in each sample by means of a NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, NC, United States).…”

“…The gut bacterial community of A. fraterculus sp. 1 has only been studied in the adult stage ( Augustinos et al, 2019 ; Conte et al, 2019 ; Devescovi et al, 2019 ; Juárez et al, 2019 ; Salgueiro et al, 2020 ). The intimate association between fruit and fruit fly larvae envisions that fruit should influence their gut bacteria.…”