Identification of Putative Nuclear Receptors and Steroidogenic Enzymes in Murray-Darling Rainbowfish (Melanotaenia fluviatilis) Using RNA-Seq and De Novo Transcriptome Assembly

Peter A Bain,Anupama Kumar,Alexie Papanicolaou,John A Craft

doi:10.1371/journal.pone.0142636

Peter A Bain, Anupama Kumar + Show 2 more

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https://doi.org/10.1371/journal.pone.0142636

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Abstract

Murray-Darling rainbowfish (Melanotaenia fluviatilis [Castelnau, 1878]; Atheriniformes: Melanotaeniidae) is a small-bodied teleost currently under development in Australasia as a test species for aquatic toxicological studies. To date, efforts towards the development of molecular biomarkers of contaminant exposure have been hindered by the lack of available sequence data. To address this, we sequenced messenger RNA from brain, liver and gonads of mature male and female fish and generated a high-quality draft transcriptome using a de novo assembly approach. 149,742 clusters of putative transcripts were obtained, encompassing 43,841 non-redundant protein-coding regions. Deduced amino acid sequences were annotated by functional inference based on similarity with sequences from manually curated protein sequence databases. The draft assembly contained protein-coding regions homologous to 95.7% of the complete cohort of predicted proteins from the taxonomically related species, Oryzias latipes (Japanese medaka). The mean length of rainbowfish protein-coding sequences relative to their medaka homologues was 92.1%, indicating that despite the limited number of tissues sampled a large proportion of the total expected number of protein-coding genes was captured in the study. Because of our interest in the effects of environmental contaminants on endocrine pathways, we manually curated subsets of coding regions for putative nuclear receptors and steroidogenic enzymes in the rainbowfish transcriptome, revealing 61 candidate nuclear receptors encompassing all known subfamilies, and 41 putative steroidogenic enzymes representing all major steroidogenic enzymes occurring in teleosts. The transcriptome presented here will be a valuable resource for researchers interested in biomarker development, protein structure and function, and contaminant-response genomics in Murray-Darling rainbowfish.

Highlights

The Murray-Darling rainbowfish (Melanotaenia fluviatilis [Castelnau, 1878]; Atheriniformes: Melanotaeniidae) is a small-bodied freshwater fish endemic to the Murray-Darling basin, a region that produces approximately half of the Australia’s total irrigated agricultural output [1]
The major focus to date has been on estrogenic endocrine disrupting chemicals (EDCs), which have been shown to induce the expression of female-specific biomarkers in male fish and result in intersex condition in some species [20,21,22,23,24]
Redundancy in the complete set of open reading frames (ORFs) was reduced by clustering sequences based on similarity of the deduced protein sequences, resulting in 43,841 non-redundant ORFs corresponding to a deduced amino acid sequence identity of 95%

Summary

Introduction

The Murray-Darling rainbowfish (Melanotaenia fluviatilis [Castelnau, 1878]; Atheriniformes: Melanotaeniidae) is a small-bodied freshwater fish endemic to the Murray-Darling basin, a region that produces approximately half of the Australia’s total irrigated agricultural output [1]. In the case of xenoestrogens, inter-species differences sensitivity can be predicted using in vitro reporter assays [25] and are least partly explained by sequence diversity in the ligand binding domain of estrogen receptor α (ERα) [26], suggesting that characterising the phylogenetics and in vitro sensitivity of key receptors can provide insight into the potential susceptibility of a given fish species to the effects of EDCs. Since the environmental persistence of endocrine-active contaminants such as pharmaceuticals varies with temperature, sunlight and water chemistry [27] estimation of potential risks is likely to be improved through the use of endemic species as test organisms and by taking local environmental conditions into consideration during experimental design. While some progress has been made with regard to the effects of EDCs on endemic species in the southern hemisphere, a recent report highlighted the need for further research in native freshwater fish species in Australia [28]

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