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.
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
and successfully applied CRISPR/Cas9 technology to the genome editing of
Through coinjection of mRNA of Cas9 nuclease and gRNA specialized for
chitinase 4 (
), 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.
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.
•Two VEGF genes (LvVEGF4, LvVEGF5) from Litopenaeus vannamei were apparently regulated by WSSV infection.•Knock-down of LvVEGF4 and LvVEGF5 led to a decrease of the viral copy number in WSSV infected shrimp.•LvVEGF4 and LvVEGF5 could interact with LvVEGFR1 rather than LvVEGFR2.•Knock-down of LvVEGF4 and LvVEGF5 could reduce the expressional levels of downstream genes FAK and PI3K.
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 (
) was identified and characterized from
. The deduced amino acid sequence of
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
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
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
or
could downregulate the expression level of
, and injection of the recombinant LvVEGF2 or LvVEGF3 could upregulate the expression level of
. 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.
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.
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 (Lvβ-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 Lvβ-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.
•WSSV infection up-regulated the expression level of LvTRIM9.•Silencing of LvTRIM9 reduced virus replication in WSSV infected shrimp.•LvTRIM9 could directly interact with Lvβ-TrCP.•Silencing of LvTRIM9 up-regulated the expression levels of LvRelish and AMPs.
An emergent shrimp disease, acute hepatopancreatic necrosis disease (AHPND), has been causing great economic losses to the shrimp farming industry. The original causing agent of AHPND was regarded as ...Vibrio parahaemolyticus, with a plasmid named pVA1 carrying two Photorhabdus insect-related (Pir) toxin genes pirA and pirB. In the present study, a Vibrio owensii strain named FcYS03 was isolated from the Chinese shrimp Fenneropenaeus chinensis, which caused a high mortality in shrimp with typical AHPND signs. This strain carried the binary toxin genes pirAB and could cause AHPND both in Fenneropenaeus chinensis and Penaeus vannamei (previously named Litopenaeus vannamei). The pathogenicity of FcYS03 to P. vannamei exhibited apparent temperature and salinity dependence. Through genome sequence analysis among FcYS03 and AHPND-causing V. parahaemolyticus strain 3HP, we found that a segmental rearrangement phenomenon existed in the plasmid of FcYS03. The toxin genes pirAB happened to be located at the rearranged segment, and two transposases for transposon Tn903 were distributed at the two ends of the rearranged segment, which indicated that transposon Tn903 might be related to the transfer of the toxin genes among the bacteria. The study will provide valuable information for the understanding and prevention of AHPND in shrimp aquaculture.
•A strain of Vibrio owensii (FcYS03) causing AHPND was isolated from Fenneropenaeus chinensis.•Its pathogencity to shrimp is water temperature and salinity dependent.•A segmental rearrangement existed in the plasmid of FcYS03 compared with the known plasmid pVA1 in V. parahemolyticus 3HP.
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.
•The expression profiles of EcMIH were demonstrated.•CRISPR-Cas9 system efficiently generated indels in EcMIH loci.•EcMIH-KO prawns exhibited increased body length and shortened metamorphosis time.
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.
•An acute toxic test of Zn and Hg on Exopalaemon carinicauda is carried out.•Zn and Hg exposure significantly inhibit the oxygen consumption of E. carinicauda.•Hg shows greater toxicity to E. carinicauda than Zn.•The hepatopancrea is the main target organ for metal accumulation.
Triploid
Penaeus (
Fenneropenaeus)
chinensis was successfully produced by heat shock. Their metamorphosis, the relationship between body weight and length and difference in appearance between ...triploids and their diploid siblings under laboratory culture were studied. Hematological studies showed a smaller number of haemocytes, but larger cell volume, in triploids than in diploids. Triploid shrimp did not show higher growth during the immature stage, but exhibited superior growth during the maturation stage. Characteristics of reproductive organs indicated that triploid shrimp may be sterile and sex ratio can be changed through triploidization of shrimp. This paper summarizes the progress made in triploid shrimp research which would be helpful in understanding more about triploids of crustaceans.
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