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In decapod crustaceans, eyestalk ablation is widely used to expedite ovarian maturation and spawning because of the removal of a gonad inhibiting hormone produced by the X-organ sinus gland. However, eyestalk ablation also results in negative impacts on the immunocompetence of the eyestalk-ablated females. In the current study, we investigated the impact of eyestalk ablation on the transcriptomic responses of three major nervous organs of shrimp, including the eyestalk ganglion, brain and thoracic ganglion, using the Illumina Hiseq™ 4000 platform. A total of 48,249 unigenes with an average length of 1253 bp and a N50 value of 2482 bp were obtained. Following eyestalk ablation treatment, a total of 2,983, 6325 and 6575 unigenes were detected as differentially expressed (log2Ratio >1 and FDR

Macrobrachium rosenbergii is an important aquaculture species known as the king of freshwater prawn. Macrobrachium rosenbergii displays remarkable sexual dimorphism, yet our knowledge of the molecular mechanism underpinning sex determination and differentiation of M. rosenbergii is still fragmentary. In this study, through in silico prediction and experimental characterization, we identified a new Dmrt gene (Mr‐Dsx) from M. rosenbergii. The cDNA of Mr‐Dsx included a 115 bp 5′ untranslated region (UTR), a 2,568 bp open reading frame (ORF) and a 259 bp 3′ UTR. Mr‐Dsx was expressed in a wide range of tissues, and showed dynamic expression changes at different moulting stages in the testis, androgenic gland (AG) and eyestalk. In response to unilateral eyestalk ablation (UEA), Mr‐Dsx was significantly induced in the testis and AG. Gene knockdown of Mr‐Dsx resulted in pronounced suppression of the insulin‐like androgenic hormone (IAG) gene. The results suggest that Mr‐Dsx is likely to be negatively regulated by the eyestalk neurohormones, and Mr‐Dsx may participate in the transcriptional activation of IAG. The study characterized the potential roles of a newly identified Dmrt homologue from M. rosenbergii, and would contribute to a deeper understanding of the molecular regulatory network underlying sex differentiation and moulting of prawns.

In this report, we studied the vitellogenin gene family in the whiteleg shrimp Litopenaeus vannamei by transcriptomics, bioinformatics, and molecular biology methods. At least three moderately homologous vitellogenin (Vg) genes (i.e. LvVg1, LvVg2, and LvVg3) were identified in the genome. The deduced LvVg proteins consisted of a vitellogenin_N domain, a DUF1943 domain, and a VWD domain typical of most vitellogenins from oviparous animals. LvVg1 was the most abundant Vg expressed in the hepatopancreas and ovary of maturing females. Furthermore, multiple isoforms of LvVg1 were evolved presumably due to the need for rapid Vg production during the rapid phase of vitellogenesis. LvVg transcripts were detected in different larval stages, juveniles, and subadults. During the non-reproductive cycle, LvVg expression in the hepatopancreas peaked at the intermolt stages. During the female vitellogenesis cycle, a two-phase expression pattern of LvVg1 gene was observed in the hepatopancreas and ovary. Moreover, the eyestalk optic nerve, brain, and thoracic ganglion consisted of factors that differentially regulated the expression of the three Vg genes. In addition to their reproduction-related roles, Vg may also be involved in growth and molt-related processes. Phylogenetic analysis revealed the early expansion and separation of these Vg genes, and it is most likely correlated with the expansion of Vg’s function. In conclusion, the evolution of multiple LvVg1 isoforms and the acquisition of different Vg genes (i.e. LvVg2 and LvVg3) may occur universally in most decapods. Full information on the total number of Vg genes and precise knowledge on the expression pattern and endocrine regulation of each Vg during all life cycle stages are crucial for us to understand the roles of this emerging gene family in the control of shrimp reproduction and other non-reproductive processes.

The banana shrimp, Fenneropenaeus merguiensis, is an important fishery species in the Indo-West Pacific region. As the shrimp is very sensitive to stressors such as ammonia stress in water, understanding the molecular mechanisms of stress tolerance in F. merguiensis is of pivotal importance for improving its farming performance. In the current study, by using the RNA sequencing platform and comparative transcriptomic analysis, we conducted a comprehensive study on the transcriptomic changes of F. merguiensis in response to ammonia stress. A total of 106,996 unique transcripts (or unigenes) with an average length of 672 bp and a N50 value of 1164 bp were recovered, and a large number of potential SSR loci in the transcriptome were identified. Totally, 55,529 transcripts can find significant hits when compared to known sequences in major databases including the nr, nt, SWISSPROT, GO, COG, and KEGG databases. Analysis of differential gene expression between the ammonia-challenged group and the control group revealed that 9190 annotated transcripts were differentially expressed upon ammonia exposure. Among them, 3712 were significantly induced while 5478 of them were repressed. Functional enrichment analysis of these differentially expressed genes further showed that 22 Gene Ontology terms and 62 KEGG pathways were significantly over-represented. Remarkably, many of the genes showing the largest magnitude of expression changes were related to cytoskeleton remodeling and immune response, highlighting the involvement of these biological processes in the ammonia stress response of F. merguiensis. Our study is the first comprehensive investigation on the transcriptomic response to ammonia stress in F. merguiensis. The genes and pathways identified here not only represent valuable genetic resources for development of molecular markers and genetic breeding studies, but open new avenues for studies on the molecular mechanisms of ammonia stress tolerance in penaeid shrimp.

Crustacean neuroparsins are poly-cysteine rich neuropeptides that share some similarities with the ovary ecdysteroidogenesis hormone (OEH) of mosquitoes, the N-terminal end of the growth factor binding protein region of the vertebrate and mollusk insulin-like growth factor binding protein and single insulin binding domain protein. Neuroparsins can promote reproduction and neurite outgrowth in various insects. Though many studies have been made in insects, the amount of work reported in crustaceans is still limited. This review emphasizes the neuroparsins found in decapod crustaceans with references to the neuroparsin first discovered in insects. To be more complete in identifying all the neuroparsin members and to understand the structure/function relationship within a single species, we have collected all neuroparsins from the GenBank and our transcriptome datasets. Then, we employed a comparative approach to study the sequence homology, tissue expression patterns, making predictions of their function and the evolutionary relationship particularly in decapod crustaceans. Results from alignment and phylogenetic studies indicated that crustacean neuroparsins consist of unique feature that can be used as criteria for their classification. These features include the presence of 12 cysteine residues in the mature peptide, the strict spacing between these cysteine residues and the size of the mature peptide. Because of the limited data on the expression information, the functions of most neuroparsin are unknown. The review will focus on the site of synthesis, expression, functions, the sequence homology and the evolutionary relationship of this group of neurohormones.

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway as a negative regulator in mammals and Drosophila, but the function of PIAS in crustaceans is still unclear. In this study, a PIAS termed LvPIAS was cloned and characterized from Litopenaeus vannamei. The full length of LvPIAS was 3065 bp, including a 2361 bp open reading frame (ORF) coding for a protein of 786 aa. LvPIAS expression was most abundant in muscle and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly I: C and white spot syndrome virus (WSSV). LvPIAS could be induced by the transcription factor LvSTAT, but LvPIAS could inhibit the transcriptional activity of LvSTAT to the LvPIAS promoter conversely, which indicated that there was a negative feedback loop between LvSTAT and LvPIAS. Furthermore, RNAi-mediated knockdown of LvPIAS shrimps showed higher survival rate to WSSV infection than those in the control group (dsGFP injection), suggesting that LvPIAS may play a negatively role against WSSV infection.

Ovary development of decapod crustaceans is controlled by a complex neuro-endocrine regulatory network. In crustacean aquaculture, eyestalk ablation (ESA) technique has been widely used to induce ovary development and maturation. However, this technique can cause significant hormonal imbalance leading to inferior egg quality and weak immunity of the animals. Understanding the molecular regulatory mechanism of ovary development will help to develop alternative measures to expedite ovarian maturation. In this study, we performed a comprehensive transcriptomic analysis of five important organs related to ovary development, including the eyestalk ganglion, brain, thoracic ganglion, hepatopancreas, and ovary, at different ovary developmental stages of the Pacific white shrimp Litopenaeus vannamei. A total of 698,118,770 clean reads were obtained from the Illumina HiSeq™ 4000 sequencing platform, resulting in an assembly of 48,722 unique transcripts (or unigenes) with an N50 of 2463 bp and a mean length of 1244 bp. 18,661 unigenes can be matched to homologous sequences in public databases. Pairwise comparisons revealed that a wide variety of genes were differentially expressed and several gene expression trends were significantly enriched in different organs during ovary development. Through weighted gene co-expression network analysis, six tissue- or stage-specific modules of co-expressed genes were identified. GO and KEGG analysis further disclosed that a rich set of genes, including those associated with the CHH family hormones, ecdysone signaling, juvenile hormone signaling, Insulin-PI3K-Akt signaling, EGFR signaling, and TGFβ signaling pathways, were preferentially expressed in specific tissues, implying their crucial roles in the regulation of ovary development process. Taken together, the study reports a comprehensive analysis of transcriptomic modulation in major neuro-endocrine organs related to shrimp ovary development. The data obtained sheds light on the potential coordination between the nervous system and its target organs (i.e. ovary and hepatopancreas), and contributes to a deeper understanding of the regulatory network orchestrating ovary development of shrimp.

Hypoxia is one of the most common physiological stressors in shrimp farming. Post-transcriptional regulation by microRNAs has been recognized as a ubiquitous strategy to enable transient phenotypic plasticity and adaptation to stressful environment, but involvement of microRNAs in hypoxia stress response of penaeid shrimp remains elusive. In this study, small RNA sequencing and comparative transcriptomic analysis was conducted to construct a comprehensive microRNA dataset for the whiteleg shrimp Litopenaeus vannamei exposed to hypoxia challenge. A total of 3324 known miRNAs and 8 putative novel miRNAs were identified, providing a valuable resource for future investigation on the functional mechanism of miRNAs in shrimp. Upon hypoxia, 1213 miRNAs showed significant differential expression, and many well-known miRNAs involved in hypoxia tolerance such as miR-210, let-7, miR-143 and miR-101 were identified. Remarkably, the vast majority of these miRNAs were up-regulated, suggesting that up-regulation of miRNAs may represent an effective strategy to inhibit protein translation under stressful hypoxic condition. The differentially expressed miRNAs were potentially targeting a wide variety of genes, including those with essential roles in hypoxia tolerance such as HIF1a and p53. GO and KEGG enrichment analysis further revealed that a broad range of biological processes and metabolic pathways were over-represented. Several GO terms associated with gene transcription and translation and KEGG pathways related to cytoskeleton remodeling, immune defense and signaling transduction were enriched, highlighting the crucial roles of these cellular events in the adaptation to hypoxia. Taken together, our study revealed that the differentially expressed miRNAs may regulate host response to hypoxia by modulating the expression of stress response genes such as HIF1a and p53 and affecting key cellular events involved in hypoxia adaptation. The findings would expand our knowledge of the biochemical and molecular underpinnings of hypoxia response strategies used by penaeid shrimp, and contribute to a better understanding of the molecular mechanisms of hypoxia tolerance in decapod crustaceans.

Members of activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element binding protein (ATF/CREB) family are induced by various stress signals and function as effector molecules. Consequently, cellular changes occur in response to discrete sets of instructions. In this work, we found an ATF transcription factor in Litopenaeus vannamei designated as LvATFβ. The full-length cDNA of LvATFβ was 1388 bp long with an open reading frame of 939 bp that encoded a putative 313 amino acid protein. The protein contained a basic region-leucine zipper (bZip) domain that was a common feature among ATF/CREB transcription factors. LvATFβ was highly expressed in intestines, gills, and heart. LvATFβ expression was dramatically upregulated by white spot syndrome virus (WSSV) infection. Pull-down assay revealed that LvATFβ had strong affinity to promoters of WSSV genes, namely, wsv059 and wsv166. Dual-luciferase reporter assay showed that LvATFβ could upregulate the expression of wsv059 and wsv166. Knocked down LvATFβ resulted in decreased expression of wsv059 and wsv166 in WSSV-challenged L. vannamei. Knocked down expression of wsv059 and wsv166 by RNA interference inhibited the replication and reduce the mortality of L. vannamei during WSSV challenge inoculation. The copy numbers of WSSV in wsv059 and wsv166 knocked down group were significant lower than in the control. These results suggested that LvATFβ may be involved in WSSV replication by regulating the expression of wsv059 and wsv166.

A 4-week feeding trial was conducted to determine the effects of oxidized fish oil (OFO, POV: 234.84 meq kg−1) on growth performance and oxidative stress of Litopenaeus vannamei. Five diets containing various OFO levels (0, 25, 50, 75 and 100 g kg−1) with the same dietary lipid level were fed to L. vannamei. The results showed that the body weight gain and the specific growth rate of the shrimp fed with 50, 75 and 100 g kg−1 of OFO diets decreased significantly (P