Small-scale transcriptomics reveals differences among gonadal stages in Asian seabass (Lates calcarifer)

Abstract

BackgroundThe Asian seabass (Lates calcarifer) is a protandrous hermaphrodite that typically matures as a male at approximately 2–4 years of age and then changes sex in subsequent years. Although several sexual maturation stages have been described histologically for both testis and ovary, the underlying gene expression profiles remain lacking. The development of a gene expression platform is therefore necessary to improve our understanding of the gonad development of this cultured teleost species.MethodsThirty Asian seabass gonads were collected from farms in Singapore, examined histologically and staged according to their sex and gonadal maturation status. Partial coding sequences of 24 sex-related genes were cloned using degenerate primers and were sequenced. Additional 13 cDNA sequences were obtained through next-generation sequencing. A real-time qPCR was then performed using the microfluidic-based Fluidigm 48.48 Dynamic arrays.ResultsWe obtained 17 ovaries and 13 testes at various stages of sexual maturation. Of the 37 genes that were tested, 32 (86%) showed sexually dimorphic expression. These genes included sex-related genes, sox9, wt1, amh, nr5a2, dmrt1 and nr0b1, which showed testis-enhanced expression similar to other vertebrate species. Known male- and female-enhanced germ cells markers, which were established from studies in other species, similarly showed testis- and ovary-enhanced expression, respectively, in the Asian seabass. Three pro-Wnt signaling genes were also upregulated in the ovary, consistent with existing studies that suggested the role of Wnt signaling in ovarian differentiation in teleosts and mammals. The expression patterns of genes involved in steroidogenesis, retinoic acid metabolism, apoptosis and NF-κB signaling were also described. We were able to classify gonads according to sex and gonadal maturation stages, based on their small-scale transcriptomic profiles, and to uncover a wide variation in expression profiles among individuals of the same sex.ConclusionsThe analysis of a selected set of genes related to reproduction and in sufficient number of individuals using a qPCR array can elucidate new insights into the molecular mechanisms involved in Asian seabass gonad development. Given the conservation of gene expression patterns found in this study, these insights may also help us draw parallels with other teleosts.

Transcriptome Survey of a Marine Food Fish: Asian Seabass (Lates calcarifer)

“…Gene ontology terms of these differentially expressed genes included biological processes such as reproduction (GO:0000003), sexual reproduction (GO:0019953), the reproductive process (GO:0022414), the reproductive developmental process (GO:0003006), gamete generation (GO:0007276), fertilization (GO:0009566) and the reproductive cellular process (GO:0048610). Twenty-one of these transcripts were also shown to have sex-related roles or differential expression in the gonads in previous studies (Table 4) [8,[30][31][32][33][34]. This finding showed that the assembled transcriptome could be reliably used as a reference for differential expression analyses.…”

“…Subsequently, the dataset was augmented by additional sequences in the form of pair-end reads generated on the Illumina HiSeq 2000 platform (Illumina Inc., San Diego, CA, USA) to provide sequence depth in order to improve the assembly. The 454 and SOLiD sequence data (incorporated and reassembled here) has been published and released previously [8]. The assembly from the initial round of Illumina HiSeq sequencing (HiSeq Round 1, HR1) was also utilized as part of a survey of repetitive elements in Asian seabass [7].…”

“…Our publication adds to the growing number of NGS-based transcriptome studies on the Asian seabass [7][8][9], and that of other food fishes such as Atlantic salmon (Salmo salar [10]), common carp (Cyprinus carpio [11]), European seabass (Dicentrarchus labrax [12,13]), rainbow trout (Oncorhynchus mykiss [14]), turbot (Scophthalmus maximus [15]), and striped bass (Morone saxatilis [16]). …”

“…The genome of the Asian seabass is estimated to be ~700 Mb and is currently being assembled and annotated [5], while the mitochondrial genome has previously been completely sequenced [6]. Transcriptome information for the Asian seabass to date is mainly represented by ~22,000 EST sequences in GenBank along with a limited number of organ-specific transcriptome studies and repetitive sequence analyses [7][8][9].…”

Whole-genome shotgun sequence assembly enables rapid gene characterization in the tropical fish barramundi,Lates calcarifer

“…Despite an increasing availability of genetic resources for the species (e.g. microsatellite and SNP markers, linkage and BAC‐based maps and transcriptomic assemblies), the complete characterization of genes is still reliant on laborious molecular methods (e.g. genome walking/RACE PCR cloning/Sanger sequencing).…”