The Pacific white shrimp (Litopenaeus vannamei) is the world’s most prevalent cultured crustacean species. However, the supply of high-quality broodstocks is limited and baseline information related to its reproductive activity and molecular issues related to gonad development are scarce. In this study, we performed transcriptome sequencing on the gonads of adult male and female L. vannamei to identify sex-related genes. A total of 25.16 gigabases (Gb) of sequences were generated from four L. vannamei gonadal tissue libraries. After quality control, 24.11 Gb of clean reads were selected from the gonadal libraries. De-novo assembly of all the clean reads generated a total of 65,218 unigenes with a mean size of 1021 bp and a N50 of 2000 bp. A search of all-unigene against Nr, SwissProt, KEGG, COG and NT databases resulted in 26,482, 23,062, 20,659, 11,935 and 14,626 annotations, respectively, providing a total of 30,304 annotated unigenes. Among annotated unigenes, 12,320 unigenes were assigned to gene ontology categories and 20,659 unigenes were mapped to 258 KEGG pathways. By comparing the ovary and testis libraries, 19,279 testicular up-regulated and 3,529 ovarian up-regulated unigenes were identified. Enrichment analysis of differentially expressed unigenes resulted in 1060 significantly enriched GO terms and 34 significantly enriched KEGG pathways. Nine ovary-specific, 6 testis-specific, 45 testicular up-regulated and 39 ovarian up-regulated unigenes were then confirmed by semi-quantitative PCR and quantitative real-time PCR. In addition, using all-unigenes as a reference, a total of 13,233 simple sequence repeats (SSRs) were identified in 10,411 unigene sequences. The present study depicts the first large-scale RNA sequencing of shrimp gonads. We have identified many important sex-related functional genes, GO terms and pathways, all of which will facilitate future research into the reproductive biology of shrimp. We expect that the SSRs detected in this study can then be used as genetic markers for germplasm evaluation of breeding and imported populations.

Crassostrea hongkongensis (C. hongkongensis) is one of the three most commonly cultivated oyster species in China. Seasonal hypoxia is one of the most serious threats to its metabolism, reproductive behavior, and survival. To investigate the effects of hypoxia stress on the antioxidant capacity and energy metabolism of C. hongkongensis, the total antioxidant capacity (T-AOC), glycogen content, and enzyme activities (phosphofructokinase, PFK; pyruvate kinase, PK; phosphoenolpyruvate carboxykinase, PEPCK) of oysters were determined under normoxic (DO 6 ± 0.2 mg/L) and hypoxic (DO 1.5 mg/L) conditions at 0 h, 6 h, 48 h, and 72 h. We also determined the T-AOC, glycogen content, and enzyme activities of oysters under reoxygenation (recovered to normoxia for 24 h). To further examine the potential molecular regulatory mechanism of hypoxic adaptation, a transcriptome analysis was conducted on the gill of C. hongkongensis under normoxia (N, 72 h), hypoxia (H, 72 h), and reoxygenation (R). After being exposed to hypoxia for 6 h, the T-AOC, glycogen content, and enzyme activities of PK, PFK, and PEPCK in C. hongkongensis were significantly decreased. However, after prolonging the duration of hypoxia exposure for 72 h, the T-AOC, glycogen content, and enzyme activities increased compared to that of 48 h. After 24 h reoxygenation, the T-AOC, glycogen content, and enzyme activity of PK and PFK returned to close to initial levels. In addition, a transcriptome analysis discovered 6097 novel genes by mapping the C. hongkongensis genome with the clean reads. In total, 352 differentially expressed genes (DEGs) were identified in the H vs. N comparison group (235 upregulated and 117 downregulated genes). After recovery to normoxia, 292 DEGs (134 upregulated and 158 downregulated genes) were identified in the R vs. N comparison group, and 632 DEGs were identified (253 upregulated and 379 downregulated genes) in the R vs. H comparison group. The DEGs included some hypoxia-tolerant genes, such as phosphoenolpyruvate carboxykinase (PEPCK), mitochondrial (AOX), tyramine beta-hydroxylase (TBH), superoxide dismutase (SOD), glutathione S-transferase (GST), and egl nine homolog 1 isoform X2 (EGLN1). Additionally, DEGs were significantly enriched in the KEGG pathways that are involved in hypoxia tolerance, including the metabolism of xenobiotics by cytochrome P450 pathways and the HIF-1 signaling pathway. Then, we selected the five hypoxic-tolerant candidate DEGs for real-time quantitative polymerase chain reaction (RT-qPCR) validation, and the results were consistent with the transcriptome sequencing data. These discoveries have increased our understanding of hypoxia tolerance, recovery ability after reoxygenation, and molecular mechanisms governing the responses to hypoxia in C. hongkongensis.

To understand the environmental adaptations among sessile bivalves lacking adaptive immunity, a series of analyses were conducted, with special emphasis on the widely distributed C. ariakensis. Employing Pacbio sequencing and Hi-C technologies, whole genome for each of a C. ariakensis (southern China) and C. hongkongensis individual was generated, with the contig N50 reaching 6.2 and 13.0 Mb, respectively. Each genome harbored over 30,000 protein-coding genes, with approximately half of each genome consisting of repeats. Genome alignment suggested possible introgression between C. gigas and C. ariakensis (northern China), and re-sequencing data corroborated this result and indicated significant gene flow between C. gigas and C. ariakensis. These introgressed candidates, well-represented by genes related to immunity and osmotic pressure, may be associated with environmental stresses. Gene family dynamics modeling suggested immune-related genes were well represented among the expanded genes in C. ariakensis. These outcomes could be attributed to the spread of C. ariakensis.

Although the expression of miRNAs has been widely applied to investigate on gonads, the role of miRNAs in the gonadal development of white Pacific shrimp (Litopenaeus vannamei) remains unknown. In this study, we performed high-throughput sequencing to identify the sex-related microRNAs (miRNAs) that elucidated the regulatory mechanisms on the gonadal differentiation of L. vannamei. We obtained a total of 29,671,557 and 28,526,942 raw reads from the ovaries and testes library, respectively. We then mapped 26,365,828 (92.73%) of the ovarian clean sequences and 23,694,294 (85.65%) of the testicular clean sequences for a transcriptome reference sequence of L. vannamei. After blasting the miRNA sequences against the miRBase database, we identified 153 significantly differentially expressed miRNAs between the ovaries and testes. To confirm the high-throughput sequencing results, we used a reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) to verify the expression patterns of the seven most differentially expressed miRNAs (i.e., novel_mir23, miR-92b-3p_3, miR-12-5p_2, novel_mir67, miR-279_1, let-7-5p_6, miR-263a-5p_1). According to the results of RT-qPCR, most of the miRNAs were expressed consistently with the high-throughput sequencing results. In addition, the target genes significantly enriched several Kyoto Encyclopedia of Genes and Genome (KEGG) pathways that were closely related to gonadal differentiation and development, including extracellular matrix–receptor interaction, Hedgehog signaling pathway, protein digestion and absorption and cell adhesion molecules (CAMs). This study revealed the first miRNAs sequencing of L. vannamei gonads. We identified sex-related differentially expressed miRNAs and KEGG pathways, which will be helpful to facilitate future research into the regulatory mechanism on the gonadal differentiation of L. vannamei.

The tri-spine horseshoe crab, Tachypleus tridentatus, is an evolutionarily ancient marine chelicerate of considerable ecological and conservation importance in the Indo-West Pacific. Although several mitochondrial genomes have been reported, comprehensive comparative mitogenomic analyses encompassing all extant limulid genera remain limited. In this study, we sequenced and characterized the complete mitochondrial genome of an F1 Tachypleus tridentatus individual derived from wild-caught broodstock from the Beibu Gulf, northern South China Sea, and subsequently maintained at an aquaculture facility, using high-throughput sequencing, and performed an integrated comparative mitogenomic analysis across the three extant genera of Limulidae. The circular mitochondrial genome was 15,006 bp in length and contained the typical set of 37 mitochondrial genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The newly sequenced Tachypleus tridentatus mitogenome showed highly conserved genome organization and nucleotide composition relative to previously published conspecific mitogenomes, confirming established mitochondrial characteristics of the species. The principal contribution of this study is the addition of a complete Beibu Gulf mitogenome and its integration with 12 additional complete mitochondrial genomes representing all extant limulid species, together with four mitochondrial markers, K2P genetic-distance analyses, and independent BI and ML phylogenetic reconstruction. The concordance among these mitochondrial datasets provides an integrated assessment of genetic divergence and mitochondrial phylogenetic relationships within Limulidae and expands the geographic representation of complete mitochondrial genomic resources for Tachypleus tridentatus.