Dataset Information

Impact of Sample Preservation and Manipulation on Insect Gut Microbiome Profiling. A Test Case With Fruit Flies (Diptera, Tephritidae).

ABSTRACT: High-throughput sequencing (HTS) techniques are of great value for the investigation of microbial communities, and have been extensively used to study the gut microbiome. While most studies focus on the human gut, many others have investigated insects. However, because of the rapid spread of HTS techniques, a lot of variation exists in the protocols for sample preparation. In the present study, we investigated the impact of two widely adopted sample-processing procedures preceding library preparation, i.e., preservation of insect tissue in 70% ethanol (EtOH) and sample dissection. We used the fruit fly Ceratitis capitata (Diptera: Tephritidae) as a model organism and set up two experiments, one comparing the effects of sample manipulation and preservation across life stages and the other across fruit samples from different sources. The results of this study showed no major effects of dissection on the outcome of HTS. However, EtOH preservation did have effects on the recovered gut microbiome, the main effect being a significant reduction of the dominant genus, Providencia, in EtOH-preserved samples. Less abundant bacterial groups were also affected resulting in altered microbial profiles obtained from samples preserved in 70% EtOH. These results have important implications for the planning of future studies and when comparing studies that used different sample preparation protocols.

SUBMITTER: De Cock M

PROVIDER: S-EPMC6923184 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Impact of Sample Preservation and Manipulation on Insect Gut Microbiome Profiling. A Test Case With Fruit Flies (Diptera, Tephritidae).

De Cock Maarten M Virgilio Massimiliano M Vandamme Peter P Augustinos Antonios A Bourtzis Kostas K Willems Anne A De Meyer Marc M

Frontiers in microbiology 20191213

High-throughput sequencing (HTS) techniques are of great value for the investigation of microbial communities, and have been extensively used to study the gut microbiome. While most studies focus on the human gut, many others have investigated insects. However, because of the rapid spread of HTS techniques, a lot of variation exists in the protocols for sample preparation. In the present study, we investigated the impact of two widely adopted sample-processing procedures preceding library prepar ...[more]

PMID: 31921020

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Project description:BackgroundIn the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2.ResultsThe gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features.ConclusionsThese results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.

Project description:BackgroundFruit flies (Diptera: Tephritidae) comprise species of agricultural and economic importance. Five such fruit fly species are known to affect commercial fruit production and export in South Africa: Ceratitis capitata, Ceratitis cosyra, Ceratitis rosa, Ceratitis quilicii, and Bactrocera dorsalis. Management practices for these pests include monitoring, application of pest control products, post-harvest disinfestation measures and inspection of consignments both prior to shipment and at ports of entry. In activities relating to monitoring and inspection, accurate identification of these pests to species level is required. While morphological keys for adult stages of these fruit fly species have been well developed, morphological keys for earlier life stages remain problematic. In instances where closely related species cannot be reliably distinguished morphologically, there is a need for molecular tools to assist in identifying these five fruit fly species during surveillance practices, where sequencing-based approaches would be beneficial.ResultsTwo complete mitochondrial genomes were assembled for each fruit fly species investigated using high throughput sequencing data generated in this study. A single primer set was designed to amplify a region between tRNAile and tRNAmet. The amplicon consists of a partial segment of tRNAile, intergenic region I (tRNAile - tRNAgln), the complete sequence of tRNAgln, intergenic region II (tRNAgln - tRNAmet), and a partial segment of tRNAmet. PCR amplicons were generated for 20 specimens of each species, five of which were colony adult males, five colony larvae, and 10 wild, trap-collected specimens. Upon analysis of the amplicon, intergenic region I was identified as the most informative region, allowing for unambiguous identification of the five fruit fly species. The similarity in intergenic region II was too high between C. rosa and C. quilicii for accurate differentiation of these species.ConclusionThe identity of all five fruit flies investigated in this study can be determined through sequence analysis of the mitochondrial intergenic regions. Within the target amplicon, intergenic region I (tRNAile - tRNAgln) shows interspecific variation sufficient for species differentiation based on multiple sequence alignment. The variation in the length of intergenic region I is proposed as a potential tool for accurately identifying these five fruit flies in South Africa.

Dataset Information

Impact of Sample Preservation and Manipulation on Insect Gut Microbiome Profiling. A Test Case With Fruit Flies (Diptera, Tephritidae).

Publications

Impact of Sample Preservation and Manipulation on Insect Gut Microbiome Profiling. A Test Case With Fruit Flies (Diptera, Tephritidae).

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