An E3 ubiquitin ligase TRIM9 is involved in WSSV infection via interaction with ß-TrCP.

The TRIpartite Motif (TRIM) proteins are known to play key roles in cell differentiation, apoptosis, development, autophagy and innate immunity. In the present study, a TRIM9 homolog (named LvTRIM9) was identified from the transcriptome of the Pacific whiteleg shrimp Litopenaeus vannamei. The deduced amino acid sequence of LvTRIM9 possessed typical features of TRIMs, consisting of a RING domain, two B-boxes, a coiled-coil domain, a FN3 domain, and a SPRY domain. The transcript of LvTRIM9 was detected in most tissues of the shrimp. Its expression level was obviously up-regulated at 3, 12 and 24 h post white spot syndrome virus (WSSV) infection. Knockdown of LvTRIM9 gene expression by double-strand RNA mediated interference could lead to a decrease of virus copy number in WSSV-infected shrimp. Yeast two-hybrid analysis showed that LvTRIM9 could directly interact with beta-transducin repeat-containing protein of shrimp (Lvß-TrCP), an inhibitor of NF-κB pathway. Meanwhile, knockdown of LvTRIM9 could also up-regulate the expression levels of LvRelish and downstream production of antimicrobial peptides in the intestine of shrimp. These data indicated that WSSV might hijack the LvTRIM9 for its propagation through inhibition of NF-κB pathway and downstream antimicrobial peptides production via interaction of LvTRIM9 with Lvß-TrCP in shrimp. The study improved our understanding about the impact of E3 ubiquitin ligases on the innate immune signaling pathway of shrimp and its role during WSSV infection.

Penaeid shrimp genome provides insights into benthic adaptation and frequent molting.

Crustacea, the subphylum of Arthropoda which dominates the aquatic environment, is of major importance in ecology and fisheries. Here we report the genome sequence of the Pacific white shrimp Litopenaeus vannamei, covering ~1.66 Gb (scaffold N50 605.56 Kb) with 25,596 protein-coding genes and a high proportion of simple sequence repeats (>23.93%). The expansion of genes related to vision and locomotion is probably central to its benthic adaptation. Frequent molting of the shrimp may be explained by an intensified ecdysone signal pathway through gene expansion and positive selection. As an important aquaculture organism, L. vannamei has been subjected to high selection pressure during the past 30 years of breeding, and this has had a considerable impact on its genome. Decoding the L. vannamei genome not only provides an insight into the genetic underpinnings of specific biological processes, but also provides valuable information for enhancing crustacean aquaculture.

Identification and characterization of two novel vascular endothelial growth factor genes in Litopenaeus vannamei.

Vascular endothelial growth factor (VEGF) signaling pathway induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis. Although three VEGF and two VEGF receptor genes have been identified in Litopenaeus vannamei and demonstrated their roles in WSSV infection, another two novel VEGF genes (LvVEGF4, LvVEGF5) were isolated and their involvements in the WSSV infection of shrimp were studied in the present study. The deduced amino acid sequences of both LvVEGF4 and LvVEGF5 contained a signal peptide, a typical PDGF/VEGF domain and a cysteine knot motif (CXCXCX). Tissue distribution analysis showed that LvVEGF4 was predominantly expressed in gill and hemocytes, while LvVEGF5 was mainly detected in hemocytes and intestine. WSSV infection could cause up-regulation of the transcriptional levels of LvVEGF4 and LvVEGF5. Their functions were studied by double-strand RNA interference. The results showed that knock-down of LvVEGF4 and LvVEGF5 led to a decrease of the viral copy number in WSSV infected shrimp. Yeast two-hybrid analysis showed that both LvVEGF4 and LvVEGF5 could interact with LvVEGFR1 rather than LvVEGFR2. In addition, knock-down of LvVEGF4 and LvVEGF5 could reduce the expressional levels of downstream genes FAK and PI3K. The present study provides new clues in demonstrating that the VEGF signaling pathway is involved in the process of WSSV infection in shrimp.

CRISPR/Cas9-mediated deletion of EcMIH shortens metamorphosis time from mysis larva to postlarva of Exopalaemon carinicauda.

The recently emerged CRISPR/Cas9 technology is the most flexible means to produce targeted mutations at the genomic loci in a variety of organisms. In Crustaceans, molt-inhibiting hormone (MIH) is an important negative-regulatory factor and plays a key role in suppressing the molting process. However, whether precise disruption of MIH in crustacean can be achieved and successfully used to improve the development and growth has not been proved. In this research, the complementary DNA (cDNA) and genomic DNA, including flanking regions of the MIH gene (EcMIH) of ridgetail white prawn Exopalaemon carinicauda, were cloned and sequenced. Sequence analysis revealed that EcMIH was composed of three exons and two introns. Analysis by RT-PCR showed that EcMIH mainly expressed in eyestalks. During different development periods, EcMIH was highest in juvenile stage and extremely low in others but adult prawns eyestalks. In addition, we applied CRISPR/Cas9 technology to generate EcMIH knock-out (KO) prawns and then analyzed the changes in their phenotypes. We efficiently generated 12 EcMIH-KO prawns out of 250 injected one-cell stage embryos and the mutant rate reached 4.8% after embryo injection with one sgRNA targeting the second exon of EcMIH. The EcMIH-KO prawns exhibited increased the body length and shortened the metamorphosis time of larvae from mysis larva to postlarva. Meanwhile, EcMIH-KO did not cause the health problems such as early stage death or deformity. In conclusion, we successfully obtained EcMIH gene and generated EcMIH-KO prawns using CRISPR/Cas9 technology. This study will certainly lead to a wide application prospect of MIH gene in prawns breeding.

Identification and characterization of a doublesex gene which regulates the expression of insulin-like androgenic gland hormone in Fenneropenaeus chinensis.

The doublesex and its homologue genes are important regulators of sexual differentiation which are conserved among animal kingdom. In the present study, we reported a doublesex gene (designated as FcDsx) identified from the Chinese shrimp F. chinensis. The gene structure, nucleotide and deduced amino acid sequences of FcDsx were characterized. The results showed that the deduced amino acid sequence of FcDsx had the common features of Dsx proteins, including a doublesex/male abnormal 3 (DM) domain, an oligomerization domain and a predicted monopartite nuclear localization signal. The expression patterns of FcDsx in different tissues and developmental stages were detected. FcDsx exhibited a sex-biased expression patterns in different tissues and its expression level increased along with developmental stages. In addition, its regulation on the expression of FcIAG, a gene important for sexual differentiation of male crustacean, was also analyzed. Putative Dsx binding site was identified on the promoter region of FcIAG and knockdown of FcDsx could reduce the expression of FcIAG, which suggested that FcDsx might be the upstream regulator of FcIAG. The present data indicated that FcDsx gene might involve in shrimp sexual differentiation process.

A Novel Vascular Endothelial Growth Factor Receptor Participates in White Spot Syndrome Virus Infection in Litopenaeus vannamei.

Vascular endothelial growth factor (VEGF) signaling pathway is known to play key roles in endothelial cell proliferation, migration, angiogenesis, vascular permeability, inhibition of apoptosis, and virus infection. In the present study, a novel VEGFR gene (LvVEGFR2) was identified and characterized from Litopenaeus vannamei. The deduced amino acid sequence of LvVEGFR2 possessed typical features of VEGFRs reported in other species, including six IG-like domains, a transmembrane motif, a protein kinase (PK) domain, and one tyrosine-PK active site. The transcripts of LvVEGFR2 were mainly detected in hemocytes and lymphoid organ (Oka). Subcellular localization analysis showed that LvVEGFR2 was a membrane protein. Its expression level was obviously upregulated in hemocytes and Oka of the shrimp after white spot syndrome virus (WSSV) infection. Knockdown of LvVEGFR2 gene expression by double-strand RNA mediated interference could lead to a decrease of virus copy number in WSSV-infected shrimp. The interaction between LvVEGFR2 and different LvVEGFs (LvVEGF1, LvVEGF2, and LvVEGF3) in shrimp was analyzed at the transcription level and protein level, respectively. Knockdown of LvVEGF2 or LvVEGF3 could downregulate the expression level of LvVEGFR2, and injection of the recombinant LvVEGF2 or LvVEGF3 could upregulate the expression level of LvVEGFR2. Yeast two-hybrid analysis showed that LvVEGFR2 could interact with LvVEGF2 and LvVEGF3 directly. The study improved our understanding on the VEGF signaling pathway of shrimp and its role during WSSV infection.

Acute toxic effects of zinc and mercury on survival, standard metabolism, and metal accumulation in juvenile ridgetail white prawn, Exopalaemon carinicauda.

Ridgetail white prawn (Exopalaemon carinicauda) is widely distributed in Chinese coastal zones, especially in the Yellow Sea and Bohai Sea. It is not only considered as an important economic species in China, but also taken as a potential indicator species for the environmental pollution in the estuaries. At present, the responses of this species to environmental toxicants, including trace metal are not well understood. In this study, the acute toxic effects of zinc (Zn) and mercury (Hg) on the survival, oxygen consumption, ammonia-N excretion, and metal accumulation were investigated in the juveniles of E. carinicauda. The median lethal concentrations (LC50) of Zn were 76.4, 44.0, 30.2, and 17.2mg/L, respectively, after the juveniles were exposed in for 24, 48, 72, and 96h, and the LC50 of Hg was 0.212, 0.096, 0.084, and 0.065mg/L under the same exposure duration. The juveniles decreased the oxygen consumption by 51.4%, and increased ammonia-N excretion by 129% when they were exposed in Zn at the concentration of 76.4mg/L compared with their controls without Zn exposure, therefore the O:N ratio decreased by 82.9% compared with the control. Hg exposure with the concentration of 0.212mg/L caused the inhibition of oxygen consumption by 48.1% and increasement of ammonia-N excretion by 161%, and the atomic ratio of consumed oxygen to excreted ammonia-nitrogen (O:N ratio) decreased by 80.6% in the juveniles in comparison with the control. A concentration-dependent accumulation of heavy metals was observed in the gills, hepatopancreas and muscles of the experimental animals, with a maximum accumulation of 16.3 folds for Zn and 72.8 fold for Hg in the gills of the juveniles after 24h exposure. The data obtained from the present study would provide useful information for help further understanding on the toxicological responses of this species to trace metals.

CRISPR/Cas9-Mediated Genome Editing and Mutagenesis of EcChi4 in Exopalaemon carinicauda.

The development of the type II clustered regularly interspaced short palindromic repeats (CRISPR) system has resulted in the revolution of genetic engineering, and this technology has been applied in the genome editing of various species. However, there are no reports about target-specific genome editing in shrimp. In this research, we developed a microinjection method for the ridgetail white prawn Exopalaemon carinicauda and successfully applied CRISPR/Cas9 technology to the genome editing of E. carinicauda Through coinjection of mRNA of Cas9 nuclease and gRNA specialized for E. carinicauda chitinase 4 (EcChi4), shrimps with indel mutations were obtained. Further analysis showed that the mutations could be transmitted to the next generation. This is the first time that site-specific genome editing has been successfully demonstrated in a decapod, and will further contribute to the study of functional genomics in decapods.

Recombinant Expression of a Modified Shrimp Anti-Lipopolysaccharide Factor Gene in Pichia pastoris GS115 and Its Characteristic Analysis.

Anti-lipopolysaccharide factors (ALFs) with a LPS-binding domain (LBD) are considered to have broad spectrum antimicrobial activities and certain antiviral properties in crustaceans. FcALF2 was one isoform of ALFs isolated from the Chinese shrimp Fenneropenaeus chinensis. Our previous study showed that a modified LBD domain (named LBDv) of FcALF2 exhibited a highly enhanced antimicrobial activity. In the present study, a modified FcALF2 gene (mFcALF2), in which the LBD was substituted by LBDv, was designed and synthesized. This gene was successfully expressed in yeast Pichia pastoris GS115 eukaryotic expression system, and the characteristics of the recombinant protein mFcALF2 were analyzed. mFcALF2 exhibited apparent antibacterial activities against Gram-negative bacteria, including Escherichia coli, Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus, and Gram-positive bacteria, including Bacillus licheniformis and Staphylococcus epidermidis. In addition, mFcALF2 could reduce the propagation of white spot syndrome virus (WSSV) in vivo by pre-incubation with virus. The present study paves the way for developing antimicrobial drugs in aquaculture.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization.

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp.

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis.

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm.

Microsatellite-centromere distances and microsatellite diversity in different ploidy classes of Chinese shrimp (Fenneropenaeus Chinensis).

This is the first report of microsatellite-centromere mapping in this commercial species Fenneropenaeus Chinensis, and will be important for providing fixed points in the linkage groups of genetic maps. Triploid Chinese shrimp was induced by heat shock. The fertilized eggs were treated either by retention of the first polar body or the second polar body to produce Meiosis I (MI) or Meiosis II (MII) triploid. The triploidy status in each Chinese shrimp could be confirmed by nine polymorphic microsatellite loci, in which the parents with different alleles and the female parents were each heterozygous. The nine loci were mapped in relation to their centromeres in three MII triploid families, which were induced by retention of the second polar bodies after fertilization with sperm. Microsatellite-centromere (M-C) distances ranged from 9.6 cM to 37 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in F. chinensis. Twelve polymorphic microsatellites were used to assess the heterozygosity and allelic diversity in different ploidy classes. As expected, triploids were significantly more polymorphic than diploids. The diploids had an average heterozygosity and allelic diversity value of 0.86, whereas the triploids heterozygosity averaged 0.93 and had allelic diversity value of 1.29. However, MI triploids were not significantly more polymorphic than MII in the microsatellite loci.

Ciona intestinalis as an emerging model organism: its regeneration under controlled conditions and methodology for egg dechorionation.

The ascidian Ciona intestinalis is a model organism of developmental and evolutionary biology and may provide crucial clues concerning two fundamental matters, namely, how chordates originated from the putative deuterostome ancestor and how advanced chordates originated from the simplest chordates. In this paper, a whole-life-span culture of C. intestinalis was conducted. Fed with the diet combination of dry Spirulina, egg yolk, Dicrateria sp., edible yeast and weaning diet for shrimp, C. intestinalis grew up to average 59 mm and matured after 60 d cultivation. This culture process could be repeated using the artificially cultured mature ascidians as material. When the fertilized eggs were maintained under 10, 15, 20, 25 degrees C, they hatched within 30 h, 22 h, 16 h and 12 h 50 min respectively experiencing cleavage, blastulation, gastrulation, neurulation, tailbud stage and tadpole stage. The tadpole larvae were characterized as typical but simplified chordates because of their dorsal nerve cord, notochord and primordial brain. After 8 - 24 h freely swimming, the tadpole larvae settled on the substrates and metamorphosized within 1- 2 d into filter feeding sessile juvenile ascidians. In addition, unfertilized eggs were successfully dechorionated in filtered seawater containing 1% Tripsin, 0.25% EDTA at pH of 10.5 within 40 min. After fertilization, the dechorionated eggs developed well and hatched at normal hatching rate. In conclusion, this paper presented feasible methodology for rearing the tadpole larvae of C. intestinalis into sexual maturity under controlled conditions and detailed observations on the embryogenesis of the laboratory cultured ascidians, which will facilitate developmental and genetic research using this model system.