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.

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.

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.

[Lead accumulation and elimination in juvenile Acipenser sinensis tissues].

A semi-static test of Pb exposure (0, 0.2, 0.8, and 1.6 mg L(-1)) was carried out to examine the Pb accumulation and elimination in different tissues of juvenile Chinese sturgeon (Acipenser sinensis). With increasing Pb concentration in water, the Pb concentration in juvenile A. sinensis tissues increased, showing a clear dose-response relationship. The Pb accumulation had a trend of being higher in bone (dorsalbone plate and cartilaginous bone) and muscle, followed by in stomach, intestine, and skin, and relatively low in liver, gill, and notochord. A 6-week duration test after Pb exposure showed that in low dose group (0.2 mg x L(-1)), the Pb concentration in all tissues had no significant differences to the control group, but in medium dose group (0.8 mg x L(-1)), the Pb concentration in most tissues except gill stomach, cartilaginous bone, and muscle was significantly higher than the control. In high dose group (1.6 mg x L(-1)), the Pb concentration in the tissues other than liver, intestine and skin was significantly higher than the control. Through the comparison of accumulation and elimination, it was found that after low- and medium dose exposure, the Pb concentration in skin, gill, and liver was higher than its accumulation, presuming that Pb was absorbed from gill, skin, and alimentary canal, and eliminated from gill and skin.

[Modality and structure of Japanese eel (Anguilla japonica) during spermiogenesis].

The special process and special structure which bring organelle during the spermiogenesis of Japanese eel, Anguilla japonica were observed by scanning electron microscope and transmission electron microscope. The process which spermatoblast became sperm including four special stages, the early stage, the middle stage, the later stage and the spermic stage, then came into being a normal mature sperm. In the early stage, cell nucleus became long form gradually by the oval form. In one side of the cell nucleus, there was a big and special globoid structure dyeing lower, account for 1/3 of cell nucleus cubage. It contains a little of deep dyeing grain form and the lines form material, the outside is wrapped by plasmalemma separated with the cell nucleus, the outside of that structure and cell nucleus still lay a plasmalemma. The spermiogenesis of early stage did not form independent centriolar complex and mitochondria. In the middle stage, the cell nucleus presented a long form with the globoid structure on the top of the nucleus, and the down side had no globoid structure where the flagellum primordium appears. The globoid structure changed with the spermiogenesis. The inner part of the globoid differentiated a centriolar complex and mitochondria step by step. The lysosomes distributed in the medium segment of the cell nucleus obviously. In the late stage, the cell nucleus was similar with the shape of eyebrow or crescent. The centriolar complex released from the globoid structure, then became an independent structure. There were mitochondria which had not become the independent structure still in the globoid structure. Under the karyon, there was flagellum primordium where sent a rather long flagellum. The flagellum formed a typical "9+0" microtubular structure at that time. The spermatozoa in this phase has movable ability. In the spermic stage, the cell nucleus was round in shape. The centriolar complex was inside implantation fossa. Mitochondria were under karyon. And under the mitochondria was the central space of the sleeve. The flagellum formed a typical "9+2" microtubular structure at that time. The spermatozoa of Japanese eel, A. japonica became complete mature spermatozoa must pass through four phases for abnormality.

[Development and allometric growth patterns of larval Acipenser schrenckii].

The growth and development of larval Amur sturgeon Acipenser schrenckii could be divided into two stages, i.e., pre-larval or free embryonic stage [from hatching (0 day of age and 10.17 +/- 0.63 mm of body length) to initial feeding (9 days of age and 18.93 +/- 0.74 mm of body length)], and post-larval stage [from initial feeding to juvenile stage (38 days of age and 41.89 +/- 5.09 mm of body length)]. During pre-larval stage, some organs related to feeling, feeding, breathing and swimming developed and differentiated rapidly; and during post-larval stage, dorsal, lateral and ventral scuta appeared, and the fish morphologically developed into juvenile. The growth characteristics of larval A. schrenckii presented allometric patterns, i.e., the growth inflexion points of eye diameter, mouth width, tail fin length, and pectoral fin length appeared in the ages of 2 days, 8-9 days, 10 days, and 11 days, respectively. Before the growth inflexion points, the organs grew fast; while after the growth inflexion points, the growth slowed down and approximately appeared isometric. The organs of larval A. schrenckii had the characteristics of congruous and rapid development. With the development of the organs related to feeling, feeding, breathing and swimming, larval A. schrenckii obtained the ability of escaping from predators and feeding, and consequently, its survival chance increased.