Improved genome assembly of the whiteleg shrimp Penaeus (Litopenaeus) vannamei using long- and short-read sequences from public databases.

The Pacific whiteleg shrimp Penaeus (Litopenaeus) vannamei is a highly relevant species for the world's aquaculture development, for which an incomplete genome is available in public databases. In this work, PacBio long-reads from 14 publicly available genomic libraries (131.2 Gb) were mined to improve the reference genome assembly. The libraries were assembled, polished using Illumina short-reads, and scaffolded with P. vannamei, Feneropenaeus chinensis, and Penaeus monodon genomes. The reference-guided assembly, organized into 44 pseudo-chromosomes and 15,682 scaffolds, showed an improvement from previous reference genomes with a genome size of 2.055 Gb, N50 of 40.14 Mb, L50 of 21, and the longest scaffold of 65.79 Mb. Most orthologous genes (92.6%) of the Arthropoda_odb10 database were detected as "complete," and BRAKER predicted 21,816 gene models; from these, we detected 1,814 single-copy orthologues conserved across the genomic references for Marsupenaeus japonicus, F. chinensis, and P. monodon. Transcriptomic-assembly data aligned in more than 99% to the new reference-guided assembly. The collinearity analysis of the assembled pseudo-chromosomes against the P. vannamei and P. monodon reference genomes showed high conservation in different sets of pseudo-chromosomes. In addition, more than 21,000 publicly available genetic marker sequences were mapped to single-site positions. This new assembly represents a step forward to previously reported P. vannamei assemblies. It will be helpful as a reference genome for future studies on the evolutionary history of the species, the genetic architecture of physiological and sex-determination traits, and the analysis of the changes in genetic diversity and composition of cultivated stocks.

Sex-specific role of the optic gland in octopus maya: A transcriptomic analysis.

The optic glands (OG) of cephalopods are a source of molecules associated with the control of reproductive traits and lifecycle events such as sexual maturation, reproductive behavior, feeding, parental care, and senescence. However, little is known about the role of the optic gland in Octopus maya adults during mating and egg laying. RNA sequencing, de novo transcriptome assembly, ubiquity and differential expression analysis were performed. First, we analyzed the expression patterns of transcripts commonly associated with OG regulatory functions to describe their possible role once the maturation of the gonad is complete. The transcriptomic profiles of the optic gland of both sexes were compared with emphasis on the signaling pathways involved in the dimorphism of reproductive traits. Results suggest that in the OG of males, the reproductive condition (mated or non-mated) did not affect the general expression profile. In contrast, more differentially expressed genes were observed in females. In mated females, the mRNA metabolic process and the response to norepinephrine were enriched, suggesting a high cellular activity in preparation for the laying of the embryos. Whereas in egg-laying females, energetic and metabolic processes were the most represented, including the oxidation-reduction process. Finally, the gene expression patterns in senescence females suggest a physiological response to starvation as well as upregulation of genes involved retrotransposon activity. In conclusion, more substantial fluctuations in gene expression were observed in the optic glands of the fertilized females compared to the males. Such differences might be associated with the regulation of the egg-laying and the onset of senescence.

A natural antisense transcript of the fem-1 gene was found expressed in female gonads during the characterization, expression profile, and cellular localization of the fem-1 gene in Pacific white shrimp Penaeus vannamei.

The fem-1 gene in Caenorhabditis elegans is involved in sex differentiation; it is specifically required for all aspects of male development. In this study, the full-length cDNA of the fem-1 (Pvfem-1) gene was isolated from the Pacific whiteleg shrimp Penaeus vannamei. The Pvfem-1 transcript is 3778 nt long and encodes a putative protein (PvFEM-1) of 638 amino acids that presented eight ankyrin repeats. The translated protein showed a significant (P < 0.05) structural similitude by superposition with C. elegans FEM-1 protein. Pvfem-1 expression was evaluated by qPCR and in situ hybridization (ISH) during embryogenesis, larval development, and gonads of both genders in subadult and adult life stages. Pvfem-1 was found expressed in brain, intestine, hepatopancreas, and in the gonads of both genders in subadults and adults when quantified by RT-qPCR. A significant finding was the discovery of a natural antisense transcript (NAT) of Pvfem-1 by ISH. It was present in the oocyte nucleus of subadult female shrimp gonads but was not seen within oocytes from adult females, although it was detected in follicular cells, suggesting a possible post-transcriptional regulation of Pvfem-1 in female gonad. Conversely, in males, no NAT was observed, and Pvfem-1 was found expressed in spermatogonia of both, subadult and adult shrimps indicating a function in male sexual differentiation and gametes generation. This study represents the first step for future functional analysis that is expected to contribute to clarifying the role of Pvfem-1 in sex differentiation and determination.

Sex determination and differentiation genes in a functional hermaphrodite scallop, Nodipecten subnodosus.

The lion-paw, Nodipecten subnodosus is one of three scallop species commercially exploited on the west coast of the Peninsula of Baja California. Because nothing is known about sex determination and sexual differentiation in hermaphrodite scallops, in the present work, a global transcriptomic analysis was performed in two early developmental stages, settling eyed-larvae and spat, as well as in three tissues (undifferentiated gonad, digestive gland, and adductor muscle). Over 27 million Illumina paired-end reads were obtained through the MiSeq platform. After processing the reads a total of 243,774 transcripts were assembled with an N50 of 980 and an average length of 775nt. A total of 43,252 proteins were inferred and 36,103 transcripts had at least one homolog in the SwissProt database according to a blastx search. After differential expression analyses and GO annotations it was possible to identify several sex-related genes in the scallop, including one known to be involved in the sex determination pathway of the hermaphrodite model organism Caenorhabditis elegans, N. subnodosus-sex1 (Ns-sex1). Other interesting sex determination and differentiation genes were Ns-dmrta2, Ns-sox9, Ns-wnt4, Ns-doa, Ns-ovo, Ns-vir, among others. Most of these genes were mainly expressed in the testis region, suggesting their participation in male gonad region sex differentiation. These results represent the first available information on the genetics of sex determination and differentiation in a functional hermaphrodite scallop.

Transcriptomic information from Pacific white shrimp (Litopenaeus vannamei) ovary and eyestalk, and expression patterns for genes putatively involved in the reproductive process.

The increased use of massive sequencing technologies has enabled the identification of several genes known to be involved in different mechanisms associated with reproduction that so far have only been studied in vertebrates and other model invertebrate species. In order to further investigate the genes involved in Litopenaeus vannamei reproduction, cDNA and SSH libraries derived from female eyestalk and gonad were produced, allowing the identification of expressed sequences tags (ESTs) that potentially have a role in the regulation of gonadal maturation. In the present study, different transcripts involved in reproduction were identified and a number of them were characterized as full-length. These transcripts were evaluated in males and females in order to establish their tissue expression profiles during developmental stages (juvenile, subadult and adult), and in the case of females, their possible association with gonad maturation was assessed through expression analysis of vitellogenin. The results indicated that the expression of vitellogenin receptor (vtgr) and minichromosome maintenance (mcm) family members in the female gonad suggest an important role during previtellogenesis. Additionally, the expression profiles of genes such as famet, igfbp and gpcr in brain tissues suggest an interaction between the insulin/insulin-like growth factor signaling pathway (IIS) and methyl farnesoate (MF) biosynthesis for control of reproduction. Furthermore, the specific expression pattern of farnesoic acid O-methyltransferase suggests that final synthesis of MF is carried out in different target tissues, where it is regulated by esterase enzymes under a tissue-specific hormonal control. Finally, the presence of a vertebrate type steroid receptor in hepatopancreas and intestine besides being highly expressed in female gonads, suggest a role of that receptor during sexual maturation.