A newly identified secretory phospholipase A2 group XIIA homolog (LcPLA2XIIA) in Larimichthys crocea exhibits antimicrobial and antitumor activities.

The phospholipase A2 (PLA2) superfamily has attracted increasing attention in recent years due to the multiple physiological and pathological functions exerted by its members. Up to date, the knowledge about the biological role of PLA2XIIA subfamily members remains limited. In this study, a new member of PLA2XIIA subfamily, LcPLA2XIIA, was characterized in large yellow croaker. Different from most members of the PLA2 superfamily with positive charge, LcPLA2XIIA encodes an anionic protein, which is similar to other members of PLA2XIIA subfamily. LcPLA2XIIA is highly expressed in the intestine, and afterwards, it is up-regulated after with Pseudomonas plecoglossicida or Staphylococcus aureus. LcPLA2XIIA exhibits strong inhibitory activity against these two bacteria. The results indicate that LcPLA2XIIA plays an important role in the antimicrobial immune responses of large yellow croaker. LcPLA2XIIA displays strong binding activity to all the tested bacteria. It specifically interacts with LTA, a unique component on the surface of Gram-positive bacteria. It also significantly promotes bacterial agglutination in the presence of Ca2+. These findings reveal that the binding and agglutinating abilities of LcPLA2XIIA to bacteria contribute greatly to its antibacterial activity. In addition, LcPLA2XIIA significantly inhibits the proliferation of infectious hematopoietic necrosis virus instead of recombinant human adenovirus type 5. It also suppresses the growth of human colorectal adenocarcinoma cells by inducing apoptosis, but it has no obvious inhibitory effect on the growth of epithelioma papulosum cyprinid cells. This study provides new insights into the antibacterial activity, and the mechanism of LcPLA2XIIA in large yellow croaker, and antiviral and antitumor functions of PLA2XIIA subfamily members.

Paospora carinifang n. gen., n. sp. (Microsporidia: Spragueidae), a parasite of the ridgetail white prawn, Palaemon carinicauda.

A new microsporidian disease of the pond-reared ridgetail white prawn, Palaemon carinicauda, was found in China. Light microscopy, pathology, and scanning electron microscopy showed that the parasite infected the host's skeletal muscle tissue and formed spherical sporophorous vesicles (SPOVs). Electron microscopy revealed that its merogonic life stages developed in direct contact with the host cytoplasm. The sporogonic life stages underwent octosporoblastic sporogony with the formation of eight uninucleate spores in each SPOV. Fresh SPOVs were 5.4 ± 0.55 µm in diameter. The octospores were oval and measured 2.3 × 1.5 µm (fresh) and 1.96 × 1.17 µm (fixed). The isofilar polar filament was coiled with 9-10 turns and arranged in two rows. Phylogenetic analysis based on the SSU rRNA gene suggests that this microsporidium has close affinities with members of the genera Potaspora and Apotaspora, but represents an independent generic taxon. We therefore propose the establishment of a new genus and species (Paospora carinifang n. gen., n. sp.) within the family Spragueidae. We also propose a taxonomic revision to transfer Potaspora macrobrachium to this new genus and reclassify it as Paospora macrobrachium comb. nov.

Comparative transcriptomic analysis of hepatopancreas reveals that more genes are involved in the exposure response of Vibrio parahaemolyticus PirAvp compared to PirBvp.

Vibrio parahaemolyticus (VP-AHPND) is regarded as one of the main pathogens that caused acute hepatopancreatic necrosis disease (AHPND) in the Pacific white shrimp Litopenaeus vannamei. PirAvp and PirBvp toxin proteins are the main pathogenic proteins of AHPND in shrimp. Knowledge about the mechanism of shrimp response to PirAvp or PirBvp toxin is very helpful for developing new prevention and control strategy of AHPND in shrimp. In this study, the pathological sections showed that after 4 h treatment, significant pathological changes were observed in the PirBvp treated group, and no obvious pathological changes was found in PirAvp treated group. In order to learn the mechanism of shrimp response to PirAvp and PirBvp, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp after treatment with PirAvp or PirBvp. A total of 9978 differentially expressed genes (DEGs) were identified between PirAvp or PirBvp-treated and PBS control shrimp, including 6616 DEGs in the PirAvp treated group and 3362 DEGs in the PirBvp treated group. There were 2263 DEGs that were commonly expressed, 4353 DEGs were only expressed in PirAvp VS PBS group and 1099 DEGs were uniquely expressed in PirBvp VS PBS group. Among these DEGs, the anti-apoptosis related pathways and immune response related genes significantly expressed in the commonly expressed DEGs of PirAvp VS PBS group and PirBvp VS PBS group, and small GTPase-mediated signaling and DNA metabolic process might relate to the host special reaction towards PirAvp and PirBvp exposure. The data suggested that the differential expression of these immune and metabolic-related genes in hepatopancreas might contribute to the pathogenicity variations of shrimp to VP-AHPND. The identified genes in this study will be useful for clarifying the response mechanism of shrimp toward different toxins of VP-AHPND and will further provide molecular basis for understanding the pathogenic mechanism of VP-AHPND.

The multi-omics analysis in the hepatopancreas of Eriocheir sinensis provides novel insights into the response mechanism of heat stress.

Under global warming, heat stress can induce the excessive production of reactive oxygen species, causing irreversible damage to aquatic animals. It is essential to predict potentially harmful impacts on aquatic organisms under heat stress. Eriocheir sinensis, a typical crustacean crab, is widely distributed in China, American and Europe. Parent E. sinensis need migrate to the estuaries to reproduce in winter, and temperature is a key environmental factor. Herein, we performed a comprehensive transcriptomic and proteomic analysis in the hepatopancreas of E. sinensis under heat stress (20 °C and 30 °C), focusing on heat shock protein family, antioxidant system, energy metabolism and immune defense. The results revealed that parent E. sinensis generated adaptative responses to maintain physiological function under 20 °C stress via the transcriptional up-regulation of energy metabolism enzymes, mRNA synthesis and heat shock proteins. The transcriptional inhibition of key enzymes related to energy metabolism implied that 30 °C stress may lead to the dysfunction of energy metabolism in parent E. sinensis. Meanwhile, parent E. sinensis also enhanced the expression of ferritin and phospholipase D at translational level, and the glutathione s-transferase and heat shock protein 70 at both transcriptional and translational levels, speculating that parent E. sinensis can strengthen antioxidant and immune capacity to resist oxidative stress under 30 °C stress. This study elucidated the potential molecular mechanism in response to heat stress of parent E. sinensis hepatopancreas. The preliminary selection of heat tolerance genes or proteins in E. sinensis can provide a reference for the population prediction and the study of evolutionary mechanism under heat stress in crabs.

A novel C-type lectin (SpccCTL) suppresses MCRV replication by binding viral protein and regulating antiviral peptides in Scylla paramamosain.

Pattern recognition receptors (PRRs) play a crucial role in the recognition and activation of innate immune responses against invading microorganisms. This study characterizes a novel C-type lectin (CTL), SpccCTL. The cDNA sequence of SpccCTL has a full length of 1744 bp encoding a 338-amino acid protein. The predicted protein contains a signal peptide, a coiled-coil (CC) domain, and a CLECT domain. It shares more than 50 % similarity with a few CTLs with a CC domain in crustaceans. SpccCTL is highly expressed in gills and hemocytes and upregulated after MCRV challenge, suggesting that it may be involved in antiviral immunity. Recombinant SpccCTL (rSpccCTL) as well as two capsid proteins of MCRV (VP11 and VP12) were prepared. Pre-incubating MCRV virions with rSpccCTL significantly suppresses the proliferation of MCRV in mud crabs, compared with the control (treatment with GST protein), and the survival rate of mud crabs is also significantly decreased. Knockdown of SpccCTL significantly facilitates the proliferation of MCRV in mud crabs. These results reveal that SpccCTL plays an important role in antiviral immune response. GST pull-down assay result shows that rSpccCTL interacts specifically with VP11, but not to VP12. This result is further confirmed by a Co-IP assay. In addition, we found that silencing SpccCTL significantly inhibits the expression of four antimicrobial peptides (AMPs). Considering that these AMPs are members of anti-lipopolysaccharide factor family with potential antiviral activity, they are likely involved in immune defense against MCRV. Taken together, these findings clearly demonstrate that SpccCTL can recognize MCRV by binding viral capsid protein VP11 and regulate the expression of certain AMPs, suggesting that SpccCTL may function as a potential PRR playing an essential role in anti-MCRV immunity of mud crab. This study provides new insights into the antiviral immunity of crustaceans and the multifunctional characteristics of CTLs.

Ecytonucleospora hepatopenaei n. gen. et comb. (Microsporidia: Enterocytozoonidae): A redescription of the Enterocytozoon hepatopenaei (Tourtip et al., 2009), a microsporidian infecting the widely cultivated shrimp Penaeus vannamei.

The microsporidian Enterocytozoon hepatopenaei from Penaeus vannamei (EHPPv) was redescribed on the basis of spore morphology, life cycle, pathology, and molecular character. Compared with the Enterocytozoon hepatopenaei isolated from Penaeus monodon (EHPPm), described by Tourtip et al. in 2009, new features were found in EHPPv. Electron microscopy demonstrated that EHPPv was closely associated with the nucleus of host cell. The merogony and sporogony phages were in direct contact with the cytoplasm of host cells, whereas some of the sporoblasts and the spores were surrounded by the interfacial envelope. Mature spores of EHPPv were oval and monokaryotic, measuring 1.65 ± 0.15 µm × 0.92 ± 0.05 µm. Spores possessed many polyribosomes around a bipartite polaroplast and the polar filament with 4-5 coils in two rows. Phylogenetic analyses showed all Enterocytozoon hepatopenaei isolates shared a common ancestor. Based on the morphological and molecular analyses, we propose the establishment of a new genus Ecytonucleospora and transferring Enterocytozoon hepatopenaei to the genus Ecytonucleospora, retaining the specific epithet hepatopenaei that Tourtip et al. proposed in recognition of their first research, as the new combination Ecytonucleospora hepatopenaei n. comb. Furthermore, it was suggested Enterospora nucleophila, Enterocytozoon sp. isolate RA19015_21, and Enterocytozoon schreckii be assigned into this new genus.

PcTrim prevents early infection with white spot syndrome virus by inhibiting AP1-induced endocytosis.

Viruses have evolved various strategies to achieve early infection by initiating transcription of their own early genes via host transcription factors, such as NF-κb, STAT, and AP1. How the host copes with this immune escape has been a topic of interest. Tripartite motif (TRIM) family proteins with RING-type domains have E3 ubiquitin ligase activity and are known as host restriction factors. Trim has been reported to be associated with phagocytosis and is also believed to be involved in the activation of autophagy. Preventing the virus from entering the host cell may be the most economical way for the host to resist virus infection. The role of TRIM in the early stage of virus infection in host cells remains to be further interpreted. In the current study, a crayfish TRIM with a RING-type domain, designated as PcTrim, was significantly upregulated under white spot syndrome virus (WSSV) infection in the red swamp crayfish (Procambarus clarkii). Recombinant PcTrim significantly inhibited WSSV replication in crayfish. RNAi targeting PcTrim or blocking PcTrim with an antibody promoted WSSV replication in crayfish. Pulldown and co-IP assays showed that PcTrim can interact with the virus protein VP26. PcTrim restricts the expression level of dynamin, which is involved in the regulation of phagocytosis, by inhibiting AP1 entry into the nucleus. AP1-RNAi effectively reduced the expression levels of dynamin and inhibited host cell endocytosis of WSSV in vivo. Our study demonstrated that PcTrim might reduce early WSSV infection by binding to VP26 and then inhibiting AP1 activation, resulting in reduced endocytosis of WSSV in crayfish hemocytes. Video Abstract.

Development of a recombinant adenovirus-vectored vaccine against both infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout Oncorhynchus mykiss, and the concurrent infection of the two viruses is very common among modern trout hatcheries, which has caused huge economic losses to the rainbow trout farming industry. To prevent and control the spread of IHNV and IPNV in juvenile trout simultaneously, in this study a bivalent recombinant adenovirus vaccine with IHNV Glycoprotein (G) and IPNV VP2 genes was developed. After immunizing juvenile trout with this bivalent vaccine via the immersion route, the expression levels of IHNV G and IPNV VP2 and the representative immune genes in vaccinated and control rainbow trout were tested to evaluate the correlation of immune responses with the expression of viral genes. The neutralizing antibody level induced by this bivalent vaccine as well as the protection efficacy of the vaccine against IHNV and IPNV was also evaluated. The results showed that IHNV G and IPNV VP2 were successfully expressed in juvenile trout, and all the innate and adaptive immune genes were up-regulated. This indicated that the level of the innate and adaptive immune responses were significantly increased, which might be induced by the high expression of the two viral proteins. Compared with the controls, high levels of neutralizing antibodies against IHNV and IPNV were induced in the vaccinated trout. Besides, the bivalent recombinant adenovirus vaccine showed high protection rate against IHNV, with the relative percent survival (RPS) of 81.25%, as well as against IPNV, with the RPS of 78.95%. Taken together, our findings clearly demonstrated that replication-defective adenovirus can be developed as a qualified vector for fish vaccines and IHNV G and IPNV VP2 were two suitable antigenic genes that could induce effective immune protection against these two pathogens. This study provided new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile trout.

SpgC1qR interacts with WSSV VP28 exhibiting antiviral activity.

Although human gC1qR is a multi-ligand binding protein with diverse biological functions, the functions of invertebrate gC1qR homologues remain largely unknown. In the present study, we characterized a novel gC1qR homologue, namely SpgC1qR, from mud crab Scylla paramamosain. SpgC1qR shared high identity and similar three-dimensional structure with human gC1qR. After challenge with White spot syndrome virus (WSSV), the transcripts of SpgC1qR were significantly increased, suggesting that SpgC1qR may be involved in antiviral immune response. To reveal the likely antiviral activity of SpgC1qR, the proliferation profile of WSSV in SpgC1qR-silenced crabs was examined. The result showed that knockdown of SpgC1qR by RNAi facilitated viral proliferation in vivo. This result was further confirmed by a SpgC1qR pre-incubation assay, in which pre-incubating WSSV particles with rSpgC1qR dramatically suppressed viral replication. Moreover, a GST pull-down assay revealed that SpgC1qR specifically bound to the viral envelope protein VP28. These findings clearly demonstrated that SpgC1qR specifically interacted with viral envelope protein VP28 and restricted WSSV replication, suggesting that it played a crucial role in anti-WSSV immune response of mud crab. This study provided new insights into the antiviral mechanism mediated by SpgC1qR and the biological functions of invertebrate gC1qR homologues.

A modification of nested PCR method for detection of Enterocytozoon hepatopenaei (EHP) in giant freshwater prawn Macrobrachium rosenbergii.

The microsporidian Enterocytozoon hepatopenaei (EHP) has become a critical threat to the global shrimp aquaculture industry, thus necessitating early detection by screening. Development of a rapid and accurate assay is crucial both for the active surveillance and for the assessment of shrimp with EHP infection. In the present study, a distinct strain of E. hepatopenaei (EHP Mr ) was found in Macrobrachium rosenbergii. The SWP1 gene analysis revealed it was a new genotype that differed with the common strain isolated from the Litopenaeus vannamei (EHP Lv ). A nested SWP-PCR method was modified to fix the bug that the original inner primers could not recognize the EHP Mr strain. The redesigned inner primers successfully amplified a product of 182 bp for both the EHP Mr strain and the EHP Lv strain. The new primers also had good specificity and high sensitivity, which may serve as an alternative for EHP genotyping. This study provided a method for detection of EHP in the biosecurity of Macrobrachium rosenbergii farming, and the developed protocol was proposed for the routine investigation and potential carrier screening, especially for molecular epidemiology.