Long non-coding RNA profile in banana shrimp, Fenneropenaeus merguiensis and the potential role of lncPV13 in vitellogenesis.

The long non-coding RNAs (lncRNAs) have been known to play important roles in several biological processes as well as in reproduction. This study aimed to identify lncRNA in ovary female banana shrimp, Fenneropenaeus merguiensis, and investigate the potential role of lncPV13 in the vitellogenesis. After the in silico identification of the ovarian transcriptome, a total of 24,733 putative lncRNAs were obtained, and only 147 putative lncRNAs were significantly differentially expressed among the ovarian development stages. To validate the in silico identification of lncRNAs, the 16 lncRNAs with the highest differential expression in the transcriptome analysis were evaluated by RT-qPCR. The 6 lncRNAs showed higher expression levels in the mature stage than in the previtellogenic stage and were found in several tissues such as in eyestalks, brains, thoracic ganglia, gills, and muscle. Furthermore, most candidate lncRNAs were amplifiable in Litopenaeus vannamei's and Penaeus monodon's DNA but not in Macrobrachium rosenbergii's DNA, suggesting some lncRNAs are expressed in a species-specific manner among penaeid shrimp. In this study, the lncPV13 was investigated for its vitellogenin regulating function by RNA interference. The result indicates that the lncPV13 expression was suppressed in the ovary on day 7 after the injection of double-stranded RNA specific to lncPV13 (dslncPV13), while vitellogenin (Vg) expression was significantly decreased. In contrast, the gonad inhibiting hormone (GIH) expression was significantly increased in the lncPV13 knockdown shrimp. However, the oocyte proliferation was not significantly different between control and lncPV13 knockdown shrimp. This suggests that lncPV13 regulate Vg synthesis through GIH inhibition. Finally, our findings provide lncRNA information and potential lncRNAs involved in the vitellogenesis of female banana shrimp.

Trachysalambria aspera (Alcock, 1905) (Crustacea, Decapoda, Penaeidae) from the southeastern Arabian Sea, India.

Trachysalambria aspera (Alcock, 1905) was first reported and described in detail by Alcock (1905) as Trachypeaneus asper Alcock, 1905, from the Ganjam coast (Odisha, India) and Andaman Sea. Chan et al. (2016) revised the species under the genus Trachysalambria based on molecular genetic data and resolved the confusion in their taxonomy in literature. However, they could not examine the types of T. aspera due to their non-availability (Chan et al. 2016). So we discuss the characters of the species based on the photograph of the type specimen from the Zoological Survey of India, Kolkata with that of Chan et al. (2016) and also with the specimens collected from southwest coast of India where the species has not been reported earlier, but only Trachysalambria curvirostris (George, 1967). Earlier, among Trachysalambria species, T. curvirostris was reported from both the east (Bay of Bengal) and west coasts (Arabian Sea) of Indian waters (Kunju, 1960; George, 1967; Muthu, 1971; Thomas, 1976). Chan et al. (2016) states that T. curvirostris may have a restricted distribution to northern Pacific Ocean.

Characterization and Expression Analysis of Insulin Growth Factor Binding Proteins (IGFBPs) in Pacific White Shrimp Litopenaeus vannamei.

The insulin signaling (IIS) pathway plays an important role in the metabolism, growth, development, reproduction, and longevity of an organism. As a key member of the IIS pathway, insulin-like growth factor binding proteins (IGFBPs) are widely distributed a family in invertebrates and vertebrates that are critical in various aspects of physiology. As an important mariculture species, the growth of Pacific white shrimp, Litopenaeus vannamei, is one of the most concerning characteristics in this area of study. In this study, we identified three IGFBP genes in the genome of L. vannamei and analyzed their gene structures, phylogenetics, and expression profiles. LvIGFBP1 was found to contain three domains (the insulin growth factor binding (IB) domain, the Kazal-type serine proteinase inhibitor (Kazal) domain, and the immunoglobulin C-2 (IGc2) domain), while LvIGFBP2 and LvIGFBP3 only contained a single IB domain. LvIGFBP1 exhibited high expression in most tissues and different developmental stages, while LvIGFBP2 and LvIGFBP3 were only slightly expressed in hemocytes. The RNA interference of LvIGFBP1 resulted in a significantly smaller increment of body weight than that of control groups. These results will improve our understanding of the conservative structure and function of IGFBPs and show potential applications for the growth of shrimp.

Characterization of the complete mitochondrial genomes of two species of Penaeidae (Decapoda: Dendrobranchiata) and the phylogenetic implications for Penaeoidea.

In the present study, mitogenomes of the species Trachypenaeus curvirostris and Parapenaeus fissuroides (Decapoda: Dendrobranchiata: Penaeidae) were sequenced. The total lengths of the two species were 15,956 bp and 15,937 bp in length with A + T biases of 67.08% and 67.69%, respectively. Both two species showed positive AT skews (0.016, 0.058) and negative GC skews (-0.254, -0.310). Both mitogenomes contained 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. Results of phylogenetic analyses support close relationships among Aristeidae, Benthesicymidae and Solenoceridae. The family Sicyoniidae was observed to be deeply nested within Penaeidae. Within Penaeidae, T. curvirostris and P. fissuroides were most closely related to the genus Parapenaeopsis and Metapenaeopsis, respectively, indicated that these two species belong to Penaeidae. These results will help to better understand the evolutionary position of Penaeidae and provide reference for further phylogenetic research on Penaeoidea species.

A Novel Forkhead Box Protein P (FoxP) From Litopenaeus vannamei Plays a Positive Role in Immune Response.

The forkhead box protein P (FoxP) family members have been known to be important for regulation of immune responses in vertebrates, but their roles in invertebrate immunity remain unclear. In this study, a novel FoxP gene (LvFoxP) was identified from Pacific white shrimp Litopenaeus vannamei and functionally studied in the context of immune response. Possessing a conserved FoxP coiled-coil domain and a forkhead domain, LvFoxP shared homology to vertebrate FoxP family members, in particular FoxP1. Expression of LvFoxP was detectable in all the examined tissues and could be up-regulated by immune challenge in gill and hemocytes. The LvFoxP protein was present in both the cytoplasm and nucleus of hemocytes and could be nuclear-translocated upon immune stimulation. Silencing of LvFoxP increased the susceptibility of shrimp to infections by Vibrio parahaemolyticus and white spot syndrome virus (WSSV) and down-regulated the expression of multiple components of NF-κB and JAK-STAT pathways and almost all the examined immune effector genes. Moreover, the phagocytic activity of hemocytes from LvFoxP-silenced shrimp against V. parahaemolyticus was decreased. These suggested that LvFoxP could play a positive role in immune response. The current study may provide novel insights into the immunity of invertebrates and the functional evolution of the FoxP family.

Novel Allergen Discovery through Comprehensive De Novo Transcriptomic Analyses of Five Shrimp Species.

Shellfish allergy affects 2% of the world's population and persists for life in most patients. The diagnosis of shellfish allergy, in particular shrimp, is challenging due to the similarity of allergenic proteins from other invertebrates. Despite the clinical importance of immunological cross-reactivity among shellfish species and between allergenic invertebrates such as dust mites, the underlying molecular basis is not well understood. Here we mine the complete transcriptome of five frequently consumed shrimp species to identify and compare allergens with all known allergen sources. The transcriptomes were assembled de novo, using Trinity, from raw RNA-Seq data of the whiteleg shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), banana shrimp (Fenneropenaeus merguiensis), king shrimp (Melicertus latisulcatus), and endeavour shrimp (Metapenaeus endeavouri). BLAST searching using the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. The analyses revealed up to 39 unreported allergens in the different shrimp species, including heat shock protein (HSP), alpha-tubulin, chymotrypsin, cyclophilin, beta-enolase, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Multiple sequence alignment (Clustal Omega) demonstrated high homology with allergens from other invertebrates including mites and cockroaches. This first transcriptomic analyses of allergens in a major food source provides a valuable resource for investigating shellfish allergens, comparing invertebrate allergens and future development of improved diagnostics for food allergy.

Morphological description of the first protozoeal stage of the deep-sea shrimps Aristeus antennatus and Gennadas elegans, with a key.

Accurate information on commercial marine species larvae is key to fisheries science, as their correct identification is the first step towards studying the species' connectivity patterns. In this study, we provide a complete morphological description of the first protozoeal stage of the valued deep-sea blue and red shrimp Aristeus antennatus and of the small mesopelagic shrimp Gennadas elegans. These two larval morphologies previously posed a risk of misidentification, thus hindering the study of A. antennatus larval ecology and dynamics in the context of fisheries science. Using specimens caught in the plankton at various locations in the Northwestern Mediterranean Sea and identification confirmed by molecular methods, the larvae of A. antennatus and G. elegans are distinguished from each other by the ornamentation of the antennula. A possible confusion in previous descriptions of Aristeidae larvae is addressed and a new key for the identification of Dendrobranchiata larvae provided.

Marketplace shrimp mislabeling in North Carolina.

Seafood mislabeling occurs in a wide range of seafood products worldwide, resulting in public distrust, economic fraud, and health risks for consumers. We quantified the extent of shrimp mislabeling in coastal and inland North Carolina. We used standard DNA barcoding procedures to determine the species identity of 106 shrimp sold as "local" by 60 vendors across North Carolina. Thirty-four percent of the purchased shrimp was mislabeled, and surprisingly the percentage did not differ significantly between coastal and inland counties. One third of product incorrectly marketed as "local" was in fact whiteleg shrimp: an imported and globally farmed species native to the eastern Pacific, not found in North Carolina waters. In addition to the negative ecosystem consequences of shrimp farming (e.g., the loss of mangrove forests and the coastal buffering they provide), North Carolina fishers-as with local fishers elsewhere-are negatively impacted when vendors label farmed, frozen, and imported shrimp as local, fresh, and wild-caught.

Litopenaeus vannamei sirtuin 6 homolog (LvSIRT6) is involved in immune response by modulating hemocytes ROS production and apoptosis.

The histone deacetylase, sirtuin 6 (SIRT6), plays an essential role in the regulation of oxidative stress, mitochondrial function and inflammation in mammals. However, the specific role of SIRT6 in invertebrate immunity has not been reported. Here, we characterized for the first time, a sirtuin 6 homolog in Litopenaeus vannamei (LvSIRT6), with full-length cDNA of 2919 bp and 1536 bp open reading frame (ORF) encoding a putative protein of 511 amino acids, which contains a typical SIR2 domain. Sequence and phylogenetic analysis revealed that LvSIRT6 shares a close evolutionary relationship with SIRT6 from invertebrates. Real-time quantitative PCR analysis of LvSIRT6 transcripts revealed that they were ubiquitously expressed in shrimp and induced in hepatopancreas and hemocytes upon challenge with Vibrio parahaemolyticus, Streptococcus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting the involvement of LvSIRT6 in shrimp immune response. Moreover, knockdown of LvSIRT6 decreased mitochondrial membrane potential and increased total ROS level in hemocytes, especially upon V. parahaemolyticus challenge. Depletion of LvSIRT6 also increased hemocytes apoptosis in terms of decreased expression of pro-survival LvBcl-2, but increased expression of pro-apoptotic LvBax and LvCytochrome C, coupled with high LvCaspase3/7 activity. Shrimp were rendered more susceptible to V. parahaemolyticus infection upon LvSIRT6 knockdown. Taken together, our present data suggest that LvSIRT6 plays an important role in shrimp immune response by modulating hemocytes ROS production and apoptosis during pathogen challenge.

Identification and characterization of a novel L-type lectin (MjLTL2) from kuruma shrimp (Marsupenaeus japonicus).

L-type lectins (LTLs) belong to the lectin family and are characterized by a conserved structural motif in their carbohydrate recognition domain. LTLs are homologous to leguminous lectins. In this study, we identified and functionally characterized an LTL from kuruma shrimp Marsupenaeus japonicus. We designated this LTL as MjLTL2. MjLTL2 contains a signal peptide, a Lectin_leg domain, a coiled coil, and transmembrane domain. MjLTL2 is distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestine; higher expression levels are seen in hemocytes and the hepatopancreas than in other tissues. MjLTL2 was upregulated following challenge of shrimp with Vibrio anguillarum and white spot syndrome virus (WSSV). MjLTL2 can agglutinate several bacteria without Ca2+. In addition, MjLTL2 could bind to several Gram-positive and -negative bacteria by binding to their lipopolysaccharide and peptidoglycan. However, MjLTL2 could not enhance the clearance of V. anguillarum in vivo. In the presence of WSSV infection, MjLTL2 knockdown by RNA interference resulted in a 7-day lower cumulative mortality of M. japonicus. Moreover, less VP19, VP24, VP26, and VP28 mRNAs were extracted from the hemocytes of MjLTL2 knockdown shrimp than from the control. These results suggest that MjLTL2 is involved in immune responses in shrimp.

Mitochondrial ATPase inhibitor factor 1, MjATPIF1, is beneficial for WSSV replication in kuruma shrimp (Marsupenaeus japonicus).

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F1F0-ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (MjATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). MjATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant MjATPIF1 protein injection. Further study found that MjATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that MjATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation.

A multigene and morphological analysis expands the diversity of the seabod shrimp Xiphopenaeus Smith, 1869 (Decapoda: Penaeidae), with descriptions of two new species.

After being stable for nearly a century, the taxonomic history of the genus Xiphopenaeus has been marked by many changes in the last three decades. The taxonomic status of the Atlantic species has a low resolution, and many species are still undefined and grouped as cryptic species. Here we employed an integrative approach to define the species of Xiphopenaeus and the morphological characters needed to differentiate them. We combined the analyses of two molecular markers (COI and 16 S rDNA), scanning electron microscopy and light microscopy. Based on specimens from 17 localities from the Atlantic and Pacific oceans, we detected five divergent genetic groups, three in the Atlantic (A1, A2, A3) and two in the Pacific (P1, P2). Male secondary sexual characters were able to differentiate four out of the five genetic groups. Group A1 corresponds to X. kroyeri, and A2 and A3 correspond to new species. We redescribed the genus and two new species are described and illustrated: Xiphopenaeus dincao nov. sp. (A2) and Xiphopenaeus baueri nov. sp. (A3). Since the holotype of X. riveti was missing and the specimen analysed from group P2 was a female, the status of the species of Xiphopenaeus from the Pacific remains unresolved.

Integrative taxonomy of commercially important deep water penaeoid shrimps from India.

The deep water penaeoid shrimp is an important commercial crustacean resource along the Indian coast. The molecular and morphological information of this group from the Indian coast is scarcely known. In this study, we investigated the identification and phylogenetic relationships of the deep water penaeoid shrimps using three mitochondrial (cytochrome oxidase subunit I (COI), cytochrome b, 16S rRNA) genes, which were compared with 54 morphological characters and further used to evaluate character evolution. Our study revealed remarkable molecular divergence (3.3-33.0%) in nine species from three genera of Solenoceridae, four species from three genera of Penaeidae and one species from Aristeidae using COI. Phylogenetic analysis using maximum likelihood and Bayesian approaches revealed that all species from these families are monophyletic. The present analysis revealed the existence of subgroups in the genus Solenocera suggesting the slow reduction of postrostral carina which corresponds to the increase in distributional depth during the evolutionary process which further indicates the origin of the genus in the continental shelf and extending up to the continental slope. In addition, we generated the DNA barcode database involving these species which can help further to investigate the detailed evolution and biogeography of these valuable crustacean resources.

Morphometric variation in pink shrimp populations at Rio de Janeiro coast (SE Brazil): are they really similar in closer areas?

Farfantepenaeus brasiliensis and F. paulensis are the most exploited shrimps of SE-S Brazilian coast. Our aim was to verify if adjacent nursery areas with different environmental condition (Sepetiba and Guanabara bays, SE Brazil) influence on shrimp populations (eg, CPUE) and body shapes. Samplings were carried out during 12 months in those bays ca. 85 Km far from each other. Carapace length (CL), total body length (TL), wet weight, abdomen size and TL/CL ratio were used to analyze variations in shape through regressions. In general, F. brasiliensis was 4 to 6 times more abundant than F. paulensis. The sex ratio differed from 1:1 in F. brasiliensis in both bays, with dominance of females, largest catches occur in autumn. However, differences in size and morphology were found between bays, mainly regarding the TL/CL ratio. Shrimps in Sepetiba Bay have higher TL/CL showing a more "elongated shape" (larger abdomen) when compared to those from Guanabara Bay. Results suggest the existence of an estuary vs shrimp morphology relationship which results in differences in body shape even in spatially close areas. TL/CL ratio has proven useful for assessing shrimp populations differences and might be tested for tracking the origin of adult shrimps stocks at the coast.

Morphometrics and Mitochondrial DNA Genes Analysis Suggest a New Species of Penaeus (Crustacea: Penaeidae) from the Persian Gulf.

There are two morphotypes of Penaeus semisulcatus described hitherto in the Persian Gulf, namely the banded and non-banded antennae morphotypes. In this study, we used morphometric measurements and two mitochondrial genes (16S rRNA and cytochrome oxidase subunit I-COI) to assess relationships between the two morphotypes of P. semisulcatus. Out of 25 morphological characters examined, 10 characters were found significantly different between the two morphotypes when tested against separate sexes or both sexes combined. Results from the 16S rRNA and COI sequence analysis of two morphotypes of P. semisulcatus morphotype showed up to 6% and 17% sequence divergence, respectively. The 16S rDNA and COI sequences of the non-banding morphotype were not only very different to those of the banding morphotype but was also very different to all other Penaeus species (i.e., P. monodon, P. merguiensis, and P. indicus) included in the study. Both parsimony and Neighbor-Joining trees based on 16S rDNA and COI sequences provide similar tree topology that clearly separated the two morphotypes into two distinct groups. Based on these findings, we propose the two morphotypes of P. semisulcatus to be relegated as two sympatric species.

Comparative sequence analysis of crustin isoform MjCRS7 and MjWFDC-like gene from kuruma shrimp Marsupenaeus japonicus shows variant of the WFDC domain.

Crustins are well known cysteine-rich cationic antimicrobial peptides (AMPs) in crustaceans that have WFDC [WAP (whey acidic protein) four-disulfide core] domain at the carboxyl terminus. Proteins containing a WFDC domain have been discovered in many invertebrates and vertebrates. Although, there have been many WFDC domain containing nucleotide sequences found in NCBI GenBank database, their distinct sequential characteristics and their role in the innate immune system is not well understood. Here, we identified a new crustin isoform from Marsupenaeus japonicus by transcriptome analysis. The full-length cDNA of this isoform (MjCRS7) consists of 537 bp that include a 489 bp open reading frame (ORF) encoding 162 deduced amino acids (aa). The sequence contains the eight conserved cysteine residues characteristic of the WFDC domain. A phylogenetic analysis showed that MjCRS7 is a type II crustin. We also identified the full-length cDNA of a M. japonicus MjWFDC-like gene. MjWFDC-like has a 543 bp ORF encoding 180 aa. In an RT-PCR analysis, MjCRS7 and MjWFDC-like transcripts were mainly detected in gill tissue. An alignment of MjCRS7 and MjWFDC-like with previously reported M. japonicus crustin isoform 1-5 (MjCRS1-5) showed variation in the WFDC-like domain. Neither of the genes was responsive to Vibrio parahaemolyticus, Vibrio penaeicida or white spot syndrome virus (WSSV) either by immersion or injection challenge test. Although crustins are mainly antimicrobial peptides, the present results suggest that MjCRS7 may have other roles in M. japonicus.

A comparative integrated gene-based linkage and locus ordering by linkage disequilibrium map for the Pacific white shrimp, Litopenaeus vannamei.

The Pacific whiteleg shrimp, Litopenaeus vannamei, is the most farmed aquaculture species worldwide with global production exceeding 3 million tonnes annually. Litopenaeus vannamei has been the focus of many selective breeding programs aiming to improve growth and disease resistance. However, these have been based primarily on phenotypic measurements and omit potential gains by integrating genetic selection into existing breeding programs. Such integration of genetic information has been hindered by the limited available genomic resources, background genetic parameters and knowledge on the genetic architecture of commercial traits for L. vannamei. This study describes the development of a comprehensive set of genomic gene-based resources including the identification and validation of 234,452 putative single nucleotide polymorphisms in-silico, of which 8,967 high value SNPs were incorporated into a commercially available Illumina Infinium ShrimpLD-24 v1.0 genotyping array. A framework genetic linkage map was constructed and combined with locus ordering by disequilibrium methodology to generate an integrated genetic map containing 4,817 SNPs, which spanned a total of 4552.5 cM and covered an estimated 98.12% of the genome. These gene-based genomic resources will not only be valuable for identifying regions underlying important L. vannamei traits, but also as a foundational resource in comparative and genome assembly activities.

Sperm ultrastructure of shrimps from the family Penaeidae (Crustacea: Dendrobranchiata) in a phylogenetic context.

We describe the sperm ultrastructure of six penaeid species, including at least one member of each tribe (Penaeini, Parapenaeini and Trachypenaeini). Fragments of the vas deferens of the Penaeidae Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Litopenaeus schmitti, Parapenaeus americanus, Rimapenaeus constrictus and Xiphopenaeus kroyeri were fixed and processed according to the routine for transmission electron microscopy. The morphological results were contextualized in an evolutionary perspective using molecular markers for the phylogenetic reconstruction of this group. A phylogram was proposed by Bayesian inference based on 1007 bp of 33 sequences of the combined genes (16S rDNA and COI mtDNA) from 27 dendrobranchiate specimens. Our findings show that morphological differences in the sperm ultrastructures of members among the tribes of Penaeidae can be used as a baseline to understand their evolutionary relationships. Individuals from the Penaeini tribe show plesiomorphic characteristics in the sperm ultrastructure compared to the Trachypenaeini tribe from which they were derived, such as shrimp from family Sicyoniidae. The morphological complexity of the sperm of the different penaeid members corroborated with the genetic phylogeny, which showed different clades for each tribe and the close relationship with Sicyoniidae. The sperm features of the selected species studied here reflected their evolutionary history. These features confirm the previous phylogenetic hypothesis and question the monophyly of Penaeidae, which should be verified in the future with a more complete set of representative members of each tribe.

cDNA cloning and expression analysis of a phosphopyruvate hydratase gene from the chinese shrimp Fenneropenaeus chinensis.

In the present study a cDNA encoding a phosphopyruvate hydratase (enolase) was cloned from the muscle of the Chinese shrimp (Fenneropenaeus chinensis) and named as FcEnolase. The cDNA of FcEnolase encoded a protein of 434 amino acid residues with a molecular mass 47.22 kDa. The residues 342-355 constituted the signature motif "LLLKVNQIGSVTES". A SNP locus (C96T) in the ORF at 96 bp was identified. The results showed that the FcEnolase was a conserved gene. In the normal F. chinensis, the mRNA level in the muscle was much higher (P < 0.05) than the mRNA level in the gill and hepatopancreas. To verify the mRNA level of FcEnolase in the F. chinensis post WSSV infection, a real-time RT-PCR was performed. In the WSSV-infected F. chinensis, the FcEnolase mRNA level was significantly (P < 0.05) up-regulated in the muscle at 12 and 24 h post challenge (hpc) to approximately 2.7-fold and 2.7-fold the mRNA level in the controls, respectively. The FcEnolase mRNA level in the gill was significantly (P < 0.05) down-regulated at 6 hpc to approximately 0.3-fold the mRNA level in the control, followed by a significant (P < 0.05) up-regulation at 12 hpc to approximately 2.8-fold the mRNA level in the control. There was no obvious change of FcEnolase mRNA level in the hepatopancreas during the infection process. The expression profile coincided with the fact that WSSV primarily infects the tissues of muscle and gill, but hardly infects hepatopancreas. To verify the protein level of FcEnolase post WSSV infection, a Western blot was performed. The FcEnolase protein level in the muscle at 24 hpc significantly (P < 0.05) increased to approximately 2.1-fold the level in the control. These results showed the characterization of FcEnolase and suggested that the FcEnolase might be involved in the response of F. chinensis to WSSV infection.

Death associated protein 1 (DAP 1) positively regulates virus replication and apoptosis of hemocytes in shrimp Marsupenaeus japonicus.

Death-associated protein 1 (DAP1) is a small proline-rich cytoplasmic protein that functions both in the apoptosis and autophage process of mammalian and in the clinical cancer of human. However, little knowledge is known about the homologue gene of DAP1 and its roles in the physiological process of invertebrates. In this paper, we report a novel function of DAP1 in the antivirus immunity of shrimp. A homologue gene of DAP1 was cloned from Marsupenaeus japonicus and named as Mjdap-1. The full-length of Mjdap-1 was 1761 bp with a 309 bp open reading frame that encoded 102 amino acids. Reverse transcription-PCR results showed that Mjdap-1 was expressed in all tested tissues, including hemocytes, gills, intestines, stomach, heart, hepatopancreas, testes, and ovaries. In shrimp, Mjdap-1 transcripts were up-regulated by white spot syndrome virus (WSSV) infection; Mjdap-1 knockdown decreased the virus copy in vivo and the mortality of M. japonicus to WSSV challenge. Conversely, injecting the purified recombinant MjDAP1 protein promoted the amplification of virus in shrimp. Flow cytometric assay showed, the virus infection-induced apoptosis of hemocytes was enhanced by MjDAP1 protein injection and inhibited in MjDAP1 knockdown shrimp. Furthermore, the expression of apoptosis-inducing factor (AIF) was regulated by Mjdap-1, but the caspase transcripts were not affected. Our results suggested that MjDAP1 facilitated the amplification of virus in shrimp, which may be attributed to the promotion of hemocyte apoptosis in an AIF-dependent manner. These results provided a new insight into the function of this protein that may be used for virus disease control.