A note on sampling chironomids for RNA-based studies of natural populations that retains critical morphological vouchers

Abstract

<p>The rapid uptake of transcriptomic approaches in freshwater ecology has seen a wealth of data produced concerning the ways in which organisms interact with their environment on a molecular level. Typically, such studies focus either at the community level and so don’t require species identifications, or on laboratory strains of known species identity or natural populations of large, easily identifiable taxa. For chironomids, impediments still exist for applying these technologies to natural populations because they are small-bodied and often require time-consuming secondary sorting of stream material and morphological voucher preparation to confirm species diagnosis. These procedures limit the ability to maintain RNA quantity and quality in such organisms because RNA degrades rapidly and gene expression can be altered rapidly in organisms; thereby limiting the inclusion of such taxa in transcriptomic studies. Here, we demonstrate that these limitations can be overcome and outline an optimised protocol for collecting, sorting and preserving chironomid larvae that enables retention of both morphological vouchers and RNA for subsequent transcriptomics purposes. By ensuring that sorting and voucher preparation are completed within <4 hours after collection and that samples are kept cold at all times, we successfully retained both RNA and morphological vouchers from all specimens. Although not prescriptive in specific methodology, we anticipate that this paper will assist in promoting transcriptomic investigations of the sublethal impact on chironomid gene expression of changes to aquatic environments.</p>

A transcriptome‐based analytical workflow for identifying loci for species diagnosis: a case study with Bactrocera fruit flies (Diptera: Tephritidae)

“…Cultures were kept in separate rooms for each species, and adult males and females were collected between 3 and 5 days after emergence, killed by freezing and transferred immediately to RNA later for transport to the Molecular Genetics Research Facility (MGRF), QUT, Brisbane, Australia. One individual of each sex per species was pooled in a single replicated RNA extraction, conducted using a modified Trizol/Trisure RNA isolation as described in Krosch and Bryant (). RNA quality and quantity were assessed on an Agilent 2100 Bioanalyser (Agilent Technologies, USA) and an RNA 6000 Nano kit for total RNA.…”

Differential gene expression of Australian Cricotopus draysoni (Diptera: Chironomidae) populations reveals seasonal association in detoxification gene regulation

“…Here, we utilise RNAseq data for C. draysoni obtained in a previous comparative transcriptomic study to explore patterns of differential expression within-species 40 (BioProject PRJNA350713, Transcriptome Shotgun Assembly Accession GFNI00000000, Short Read Archive Accessions in Table 1 ). Details of specimen collection, preservation, processing, RNA extraction, sequencing and read filtering are described in Krosch & Bryant (2015) 58 and Krosch (2017) 40 . Cleaned reads were assembled with Trinity Version 2014–04–13pl package 59 .…”