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.

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.

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.

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.

Nucleospora hippocampi n. sp., an Intranuclear Microsporidian Infecting the Seahorse Hippocampus erectus From China.

The life cycle, ultrastructure, and molecular phylogeny of a new intranuclear microsporidian, Nucleospora hippocampi n. sp., infecting the intestine of the Hippocampus erectus, were described. The histopathology revealed an extensive infection, mainly in the columnar epithelium of the intestinal mucosa layer. The enterocytes were the important target cell for Nucleospora hippocampi n. sp. infection. Transmission electron microscopy results showed that this microsporidian developed directly within the host cell nucleoplasm. In the intranuclear life cycle, the transformation from meront to sporogonial plasmodium was recognized by forming electron-dense disc structures, which were considered the polar tube precursors. The microsporidian showed the typical morphological characteristics of the family Enterocytozoonidae in the formation and development of spore organelles prior to the division of the sporogonial plasmodium. According to wet smear observation, eight spores were generally formed in a single host nucleus. Mature spores were elongated ovoids that were slightly bent and measured 1.93 × 0.97 µm. The isofilar polar tube was arranged in 7~8 coils in one row. Phylogenetic analysis of its small subunit ribosomal DNA sequences demonstrated that the parasite belonged to the Nucleospora group clade. The histological, ultrastructural, and molecular data support the emergence of a new species in the genus Nucleospora. This is the first report of Nucleospora species in Asia and threatened syngnathid fishes.

Preference for Shelters at Different Developmental Stages of Chinese Mitten Crab (Eriocheir sinensis).

A shelter is a good habitat for aquatic organisms, which could aid in avoiding cannibalism and facilitate predation. Chinese Mitten Crab (Eriocheir sinensis) is an important aquaculture species with troglodytism and nocturnal habit. To clarify the preference for shelters at different developmental stages of E. sinensis, different shelters (mud, sand, grass and rocks) were selected for comparison. These results indicated that juvenile crabs had a significant preference for grass; button-sized crabs preferred to hide in mud; and the favorite shelters for parent crabs were rocks, followed by mud. E. sinensis in three stages all showed concealing behavior. The concealing behavior of juvenile crabs was the most significant, followed by button-sized and parent crabs. Additionally, E. sinensis held a low hiding rate at night but a high hiding rate during the daytime due to nocturnal habits. These findings will help to better understand the habits of E. sinensis and provide a reference for resource restoration, habitat construction and the restoration of E. sinensis.

PvML1 suppresses bacterial infection by recognizing LPS and regulating AMP expression in shrimp.

Toll and Toll-like receptors (TLRs) play essential roles in the innate immunity of Drosophila and mammals. Recent studies have revealed the presence of Toll-mediated immune signaling pathways in shrimp. However, the recognition and activation mechanism of Toll signaling pathways in crustaceans remain poorly understood due to the absence of key recognition molecules, such as peptidoglycan recognition proteins. Here, a novel MD2-related lipid-recognition (ML) member named PvML1 was characterized in Penaeus vannamei. We found that PvML1 shared a similar 3D structure with human MD2 that could specifically recognize lipopolysaccharides (LPS) participating in LPS-mediated TLR4 signaling. PvML1 was highly expressed in hemocytes and remarkably upregulated after Vibrio parahemolyticus challenge. Furthermore, the binding and agglutinating assays showed that PvML1 possessed strong binding activities to LPS and its key portion lipid A as well as Vibrio cells, and the binding of PvML1 with bacterial cells led to the agglutination of bacteria, suggesting PvML1 may act as a potential pathogen recognition protein upon interaction with LPS. Besides, coating V. parahemolyticus with recombinant PvML1 promoted bacterial clearance in vivo and increased the survival rate of bacterium-challenged shrimp. This result was further confirmed by RNAi experiments. The knockdown of PvML1 remarkably suppressed the clearance of bacteria in hemolymph and decreased the survival rate of infected shrimp. Meanwhile, the silencing of PvML1 severely impaired the expression of a few antimicrobial peptides (AMPs). These results demonstrated the significant correlation of bacterial clearance mediated by PvML1 with the AMP expression. Interestingly, we found that PvML1 interacted with the extracellular region of PvToll2, which had been previously shown to participate in bacterial clearance by regulating AMP expression. Taken together, the proposed antibacterial model mediated by PvML1 might be described as follows. PvML1 acted as a potential recognition receptor for Gram-negative bacteria by binding to LPS, and then it activated PvToll2-mediated signaling pathway by interacting with PvToll2 to eliminate invading bacteria through producing specific AMPs. This study provided new insights into the recognition and activation mechanism of Toll signaling pathways of invertebrates and the defense functions of ML members.

SpSR-B2 functions as a potential pattern recognition receptor involved in antiviral and antibacterial immune responses of mud crab Scylla paramamosain.

Although class B scavenger receptors (SR-Bs) in mammals are multifunctional molecules, the functions of SR-Bs in invertebrates remain largely unknown. In this study, we characterized an SR-B homolog, namely SpSR-B2, from Scylla paramamosain. SpSR-B2 shared high similarity with mammalian SR-Bs, and exhibited specific binding activity to ac-LDL, indicating that it may be a new member of SR-B class in invertebrates. SpSR-B2 was upregulated after challenge with white spot syndrome virus (WSSV) or bacteria. Binding assays showed that SpSR-B2 specifically interacted with WSSV envelope protein VP24. Besides, SpSR-B2 could bind to all tested bacterial cells and agglutinate these bacteria. SpSR-B2 also exhibited a strong binding activity to LPS but weak binding activities to other tested polysaccharides. These findings indicated that SpSR-B2 was a potential recognition molecule for viral protein VP24 and bacterial LPS. Knockdown of SpSR-B2 resulted in dramatically decreased expressions of certain antimicrobial peptides (AMPs), and overexpression of SpSR-B2 led to the increased expression of the AMP of SpALF2, suggesting that SpSR-B2 could regulate the expression of AMPs. Taken together, this study revealed that SpSR-B2 functioned as a potential pattern recognition receptor participating in antiviral and antibacterial immunity, and provided new insights into the immune functions of invertebrate SR-Bs.

SpCrus2 Glycine-Rich Region Contributes Largely to the Antiviral Activity of the Whole-Protein Molecule by Interacting with VP26, a WSSV Structural Protein.

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo; moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.

Transcriptome analysis of pacific white shrimp (Penaeus vannamei) intestines and hepatopancreas in response to Enterocytozoon hepatopenaei (EHP) infection.

Penaeus vannamei is the most economically important species of shrimp cultured worldwide. Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that severely affects the growth and development of shrimps. In this study, the transcriptome differences between EHP-infected and uninfected shrimp were investigated through next-generation sequencing. The unigenes were assembled with the reads from all the four libraries. The differentially expressed genes (DEGs) of intestines and hepatopancreas were analyzed. There were 2,884 DEGs in the intestines and 2,096 DEGs in the hepatopancreas. The GO and KEGG enrichment analysis indicated that DEGs were significantly enriched in signaling pathways associated with nutritional energy metabolism and mobilizing autoimmunity. Moreover, the results suggested the downregulation of key genes in energy synthesis pathways contributed greatly to shrimp growth retardation; the upregulation of immune-related genes enhanced the resistance of shrimp against EHP infection. This study provided identified genes and pathways associated with EHP infection revealing the molecular mechanisms of growth retardation.

Bacterial Flora in the Gill Tissues and Intestinal Tracts of Male and Female Chinese Mitten Crabs (Eriocheir sinensis) with Different Diets in a Mud Pond.

The Chinese mitten crab, Eriocheir sinensis, is an economically valuable aquaculture species. Prior to sale, farmed crabs are often fattened with pellet feed or wild fish. In this study, PacBio Sequel sequencing was used to determine the bacterial flora in the intestinal tracts and gill tissues of male and female E. sinensis fed with various diets. The flora was then compared with the microorganisms found in environmental samples. The results showed that Proteobacteria was the dominant phylum in both tissue and environmental samples. The relative abundances of Proteobacteria in the water grass surface flushing samples and water grass samples were the highest, at up to 95.68% and 67.85%, respectively. Beyond that, Bacteroidetes, Firmicutes, and Tenericutes were the dominant phyla (>1%) in the intestinal samples, whereas Bacteroidetes and Actinobacteria were the dominant phyla in the gills. In addition, different environment samples contained diverse bacterial phyla, indicating some differences in the community composition between the different sample groups. Heat map clustering and principal coordinate plot analyses indicated that intestinal samples, crab gill samples, and environmental samples clustered together, respectively. Furthermore, an unweighted pair-group method with arithmetic mean technique confirmed that the intestinal and gill samples of crabs with different diets separately clustered together, suggesting the microbial assemblages of the same tissues share a greater similarity than those from crabs of different sex and eating different diets. What's more, biomarker bacteria (LDA ≥ 4) from the different groups were identified. Pathogenic agents from the genus Aeromonas were abundant in the intestinal samples of crabs fed with pellet feed, and Vibrio species were prevalent in the intestinal samples of crabs fed with wild fishes.

Application of biofertilizers increases fluoroquinolone resistance in Vibrio parahaemolyticus isolated from aquaculture environments.

Antimicrobial resistance genes in aquaculture environments have attracted wide interest, since these genes pose a severe threat to human health. This study aimed to explore the possible mechanisms of the ciprofloxacin resistance of Vibrio parahaemolyticus (V. parahaemolytiucs) in aquaculture environments, which may have been affected by the biofertilizer utilization in China. Plasmid-mediate quinolone resistance (PMQR) genes, representative (fluoro)quinolones (FNQs), and ciprofloxacin-resistance isolates in biofertilizer samples were analyzed. The significantly higher abundance of oqxB was alarming. The transferable experiments and Southern blot analysis indicated that oqxB could spread horizontally from biofertilizers to V. parahaemolyticus, and two (16.7%) trans-conjugants harboring oqxB were provided by 12 isolates that successfully produced OqxB. To the best of our knowledge, this study is the first to report PMQR genes dissipation from biofertilizers to V. parahaemolyticus in aquaculture environments. The surveillance, monitoring and control of PMQR genes in biofertilizers are warranted for seafood safety and human health.

A novel Laccase gene from Litopenaeus vannamei is involved in the immune responses to pathogen infection and oxidative stress.

Laccases (Lacs) are copper-containing oxidase enzymes that are found in various plants, fungi, and microorganisms. For invertebrates, particularly insects and crustaceans, Lacs have been shown to be involved in immune responses. In shrimp, a Lac gene has been cloned and functionally characterized, which revealed that it is involved in shrimp anti-pathogen infection. In the present study, a novel Lac gene (LvLac2) was cloned from Litopenaeus vannamei. Real-time RT-PCR analysis showed that LvLac2 is induced by white spot syndrome virus (WSSV)- or Vibrio alginolyticus infection. In addition, the downregulated expression of LvLac2 decreased the cumulative mortality of WSSV- or V. alginolyticus infected shrimps. Moreover, LvLac2 is also induced by oxidative stress. Knocking down the expression of LvLac2 decreased the severity of hepatopancreatic injury caused by oxidative stress, as well as reduced the cumulative shrimp mortality during oxidative stress. Furthermore, gene reporter assays showed that the expression of LvLac2 is regulated by NF-E2-related factor 2, which is the key transcription factor of the oxidative stress response signaling pathway. Our study revealed that LvLac2 not only participates in immune responses against infections in L. vannamei but is also involved in oxidative stress responses.

Cross-immunity in Nile tilapia vaccinated with Streptococcus agalactiae and Streptococcus iniae vaccines.

Streptococcus agalactiae and Streptococcus iniae are major bacterial pathogens of tilapia that can cause high mortality concomitant with large economic losses to aquaculture. Although development of vaccines using formalin-killed bacteria to control these diseases has been attempted, the mechanism of immunity against streptococcal infections and the cross-protective ability of these two bacteria remains unclear. To explore the immunological role of these vaccines, we compared the immune responses of tilapia after immunization with both vaccines and compared the relative percent survival (RPS) and cross-immunization protection of tilapia after separate infection with S. agalactiae and S. iniae. All results revealed that vaccinated fish had significantly higher (P < 0.05) levels of specific antibodies than control fish 14 days post secondary vaccination (PSV) and 7 days post challenge. In vaccinated fish, the mRNA expression of interleukin-8 (IL-8), interleukin-12 (IL-12), caspase-3 (C-3), tumour necrosis factor (TNF), and interferon (IFN) was significantly up regulated (P < 0.05) in the head kidney after immunized; similar results were found for IL-8, TNF and IFN in the posterior kidney, meanwhile the expression levels of C-3 and IFN were significantly increased (P < 0.05) in the spleen of vaccinated fish. Additionally, the levels of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in vaccinated fish were improved at different degree when compared to the control fish. These results showed that vaccination with formalin-killed cells (FKCs) of either S. agalactiae or S. iniae conferred protection against infection by the corresponding pathogen in Nile tilapia, resulting in RPS values of 92.3% and 91.7%, respectively. Furthermore, cross-protection was observed, as the S. agalactiae FKC vaccine protected fish from S. iniae infection, and vice versa. These results suggested that the S. agalactiae and S. iniae FKC vaccines can induce immune responses and generate excellent protective effects in Nile tilapia.