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Ubiquitination is a critical posttranslational modification that regulates host immune responses to pathogens. In this study, we investigated the ubiquitination of hemocyanin (PvHMC [Penaeus vannamei hemocyanin]) mediated by the mitochondrial E3 ubiquitin ligase (PvMulan) in shrimp Penaeus vannamei. We characterized distinct ubiquitination patterns of PvHMC in response to different pathogen challenges, both in vitro and in vivo. Specifically, we found that Vibrio parahaemolyticus infection led to an increase in PvMulan, which resulted in K48-linked ubiquitination and subsequent proteasomal degradation of PvHMC. In contrast, PvMulan primarily enhanced the SUMOylation of PvHMC, bolstering its immune functions against white spot syndrome virus challenges. Inhibition of PvMulan-mediated PvHMC ubiquitination significantly affected the proliferation of V. parahaemolyticus and the survival rate of infected shrimps. This study sheds light on the role of hemocyanin ubiquitination in immune regulation, illustrating its dual function in response to distinct pathogens., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Litopenaeus vannamei is a widely distributed euryhaline aquatic animal, affected by low salinity, which can impact its disease resistance and immunity. However, there is a limited understanding of the adaptation mechanisms of L. vannamei with different genetic backgrounds to low salinity. Therefore, the present study aimed to compare the immunity characteristics and transcriptomics of L. vannamei low salt-tolerant (FG I/J) and low salt-sensitive (control) families. Also, the disease resistance and immune parameters (including [THC], hemolymph cell viability, lysozyme activity [LZM], phenoloxidase content [PO], interleukin-6 [IL-6], and tumor necrosis factor-alpha [TNF-α]) of the FG I/J and control families of L. vannamei under low salinity (5‰) and ambient salinity (24‰) were examined. Additionally, hepatopancreas transcriptomics of the FG I/J and control families were analyzed at a salinity of 5‰. The results showed that the FG I/J family had higher disease resistance to Vibrio parahaemolyticus and stronger immunological capacity than the control family. Transcriptomic analysis showed significantly enriched energy metabolism and immune regulation pathways. Therefore, we speculated that energy metabolism provides sufficient energy for immunological modulation in the FG I/J family to deal with long-term low-salt stress and achieve high growth and survival rates., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Spray-dried animal plasma (SDP) in feed for several animal species provides health benefits, but research about use of SDP in shrimp feed is very limited. The objectives of the present study were to investigate the effects of dietary SDP on growth performance, feed utilization, immune responses, and prevention of Vibrio parahaemolyticus infection in Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, the post-larvae were divided into five groups (four tank/group and 80 shrimp/tank) and fed four times daily diets with porcine SDP at 0, 1.5, 3, 4.5, and 6% of the diet for 45 days. In Experiment 2, the surviving shrimp from Experiment 1 were redistributed into six groups: four SDP groups as in Experiment 1 plus the positive and negative controls (four tank/group and 30 shrimp/tank). They were then challenged with V. parahaemolyticus by immersion at 105 colony-forming units (CFU)/mL and were fed with the same diets for another 4 days. In Experiment 1, shrimp fed 4.5% or 6% SDP diets had significantly higher body weight, survival rate, and improved feed conversion ratio. The immune parameters (total hemocyte count and phagocytic, phenoloxidase, and superoxide dismutase activities) of the shrimp fed 3-6% SDP diets also showed significant enhancement compared to the control. In Experiment 2, the survival rates of the 3-6% SDP groups were significantly higher than the positive control at day 4 after the immersion challenge. Likewise, the histopathological study revealed milder signs of bacterial infection in the hepatopancreas of the 3-6% SDP groups compared to the challenged positive control and 1.5% SDP groups. In conclusion, shrimp fed diets with SDP, especially at 4.5-6% of the diet, showed significant improvement in overall health conditions and better resistance to V. parahaemolyticus infection., Competing Interests: APC provided support in the form of salary to JC. The specific roles of this author are articulated in the ‘author contributions’ section. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acute hepatopancreatic necrosis disease (AHPND) is a lethal disease in marine shrimp that has caused large-scale mortalities in shrimp aquaculture in Asia and the Americas. The etiologic agent is a pathogenic Vibrio sp. carrying binary toxin genes, pir A and pir B in plasmid DNA. Developing AHPND tolerant shrimp lines is one of the prophylactic approaches to combat this disease. A selected genetic line of Penaeus vannamei was found to be tolerant to AHPND during screening for disease resistance. The mRNA expression of twelve immune and metabolic genes known to be involved in bacterial pathogenesis were measured by quantitative RT-PCR in two populations of shrimp, namely P1 that showed susceptibility to AHPND, and P2 that showed tolerance to AHPND. Among these genes, the mRNA expression of chymotrypsin A (ChyA) and serine protease (SP), genes that are involved in metabolism, and crustin-P (CRSTP) and prophenol oxidase activation system 2 (PPAE2), genes involved in bacterial pathogenesis in shrimp, showed differential expression between the two populations. The differential expression of these genes shed light on the mechanism of tolerance against AHPND and these genes can potentially serve as candidate markers for tolerance/susceptibility to AHPND in P. vannamei . This is the first report of a comparison of the mRNA expression profiles of AHPND tolerant and susceptible lines of P. vannamei ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mai, Caro, Cruz-Flores, White and Dhar.)

Fibrinogen-related lectins are carbohydrate-binding proteins of the innate immune system that recognize glycan structures on microbial surfaces. These innate immune lectins are crucial for invertebrates as they do not rely on adaptive immunity for pathogen clearance. Here, we characterize a recombinant fibrinogen-related lectin PmFREP from the black tiger shrimp Penaeus monodon expressed in the Trichoplusia ni insect cell. Electron microscopy and cross-linking experiments revealed that PmFREP is a disulfide-linked dimer of pentamers distinct from other fibrinogen-related lectins. The full-length protein binds N-acetyl sugars in a Ca 2+ ion-independent manner. PmFREP recognized and agglutinated Pseudomonas aeruginosa. Weak binding was detected with other bacteria, including Vibrio parahaemolyticus, but no agglutination activity was observed. The biologically active PmFREP will not only be a crucial tool to elucidate the innate immune signaling in P. monodon and other economically important species, but will also aid in detection and prevention of shrimp bacterial infectious diseases.

The Toll family of receptors are a group of conserved pattern recognition receptors (PRRs) essentially controlling the initiation of innate immune responses. The white spot syndrome virus (WSSV) and Vibrio parahaemolyticus are major pathogens of aquaculture shrimp. Previous study has suggested that expression of the Toll2 receptor in Pacific white shrimp Penaeus vannamei was up-regulated by white spot syndrome virus (WSSV) infection but did not significantly changed upon infection with the bacterial pathogen Vibrio parahaemolyticus. The current study intends to investigate the role of P. vannamei Toll2 in antibacterial and antiviral immunity. We demonstrated that compared with the control, the Toll2-silenced shrimp was more susceptible to V. parahaemolyticus infection, suggesting that Toll2 may play a positive role in antibacterial immunity. However, silencing of Toll2 significantly enhanced survivorship of shrimp infected with WSSV and reduced the viral load in shrimp tissues. The expression of WSSV structural protein VP28 was also inhibited in Toll2-silenced shrimp. Histologic pathology analysis further showed that the WSSV infection was attenuated in stomach tissues from Toll2-silenced shrimp. These suggested that Toll2 could promote WSSV infection in shrimp. In Toll2-silenced shrimp, expression of antimicrobial peptides ALFs and PENs was significantly changed, which may contribute to the role of Toll2 in antibacterial immunity and WSSV infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

When the aquaculture water environment deteriorates or the temperature rises, shrimp are susceptible to viral or bacterial infections, causing a large number of deaths. This study comprehensively evaluated the effects of the oral administration of a chitosan-gentamicin conjugate (CS-GT) after Litopenaeus vannamei were infected with Vibrio parahaemolyticus, through nonspecific immunity parameter detection, intestinal morphology observation, and the assessment of microbial flora diversification by 16S rRNA gene sequencing. The results showed that the oral administration of CS-GT significantly increased total hemocyte counts and reduced hemocyte apoptosis in shrimp ( p < 0.05). The parameters (including superoxide dismutase, glutathione peroxidase, glutathione, lysozyme, acid phosphatase, alkaline phosphatase, and phenoloxidase) were significantly increased ( p < 0.05). The integrity of the intestinal epithelial cells and basement membrane were enhanced, which correspondingly alleviated intestinal injury. In terms of the microbiome, the abundances of Vibrio (Gram-negative bacteria and food-borne pathogens) in the water and gut were significantly reduced. The canonical correspondence analysis (CCA) showed that the abundances of Vibrio both in the water and gut were negatively correlated with CS-GT dosage. In conclusion, the oral administration of CS-GT can improve the immunity of shrimp against pathogenic bacteria and significantly reduce the relative abundances of Vibrio in aquaculture water and the gut of Litopenaeus vannamei .

Leucine-rich repeat (LRR) is a vital structure in some pattern recognition receptors such as TLRs, NLRs and newly reported LRR-containing proteins. Apart from some limited reported LRR-containing proteins, most of LRR proteins, especially immune-related proteins, remain uncharacterized functionally. In the present study, a transmembrane protein containing several LRR motifs, designated as LvLRRm, was identified from the shrimp Litopenaeus vannamei. LvLRRm contained a long signal peptide, one LRRNT region, 12 LRR motifs, one LRRCT region and a transmembrane region. The transcripts of LvLRRm were widely distributed in all tested tissues of shrimp and they were responsive to Vibrio parahaemolyticus infection in several immune-related tissues including Oka, intestine, gill and hemocytes. Knockdown of LvLRRm by dsRNA interference led to a decreased survival rate of shrimp infected by Vibrio parahaemolyticus and an increased in vivo Vibrio propagation. Meanwhile, knockdown of LvLRRm also down-regulated the expression levels of genes involved in antibacterial immune signaling pathways, including the transcription factors LvDorsal and LvRelish, and several antimicrobial peptides. These data suggested that LvLRRm played important roles in shrimp against Vibrio infection, which was probably functioning through activation of antibacterial immune signaling pathways. The present study provided new evidence to elucidate the immune function of LRR-containing proteins in invertebrates., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

While Vibrio parahaemolyticus (VP AHPND ) has been identified as the cause of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, mechanisms of host response remain unknown. Understanding these processes is important to improve farming practices because this understanding will help to develop methods to enhance shrimp immunity. Pre-treatment of shrimp with 5-minute chronic non-lethal heat stress (NLHS) for 7 days was found to significantly increase Litopenaeus vannamei survival against VP AHPND infection. To elucidate the mechanism involved, mRNA and miRNA expression profiles from the hemocyte of L. vannamei challenged with VP AHPND after NLHS with corresponding control conditions were determined by RNA-Seq. A total of 2,664 mRNAs and 41 miRNAs were differentially expressed after the NLHS treatment and VP AHPND challenge. A miRNA-mRNA regulatory network of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) was subsequently constructed and the interactions of DEMs in regulating the NLHS-induced immune-related pathways were identified. Transcriptomic data revealed that miRNA and mRNA interactions contribute to the modulation of NLHS-induced immune responses, such as the prophenoloxidase-activating system, hemocyte homeostasis, and antimicrobial peptide production, and these responses enhance VP AHPND resistance in L. vannamei.

Our previous work showed that using Lactobacillus pentosus HC-2 as a probiotic could improve the growth performance, immune response, gut bacterial diversity and disease resistance of Litopenaeus vannamei. However, the probiotic mechanism had not been fully characterized. In the present study, histology and proteomic analysis were performed to explore the influence of HC-2 surface protein on its probiotic effects on L. vannamei after feeding either the intact surface proteins, the probiotic treated with lithium chloride (LiCl) to remove noncovalently bound surface proteins or no probiotic for four weeks. Histological observation found that feeding with normal HC-2 obviously improved the intestinal histology and enhanced the protective effect against pathogen damage, but feeding with LiCl-treated HC-2 did not improve the intestinal environment. A total of over 2764 peptides and 1118 uniproteins were identified from the L. vannamei midgut; 211 proteins were significantly differentially expressed in the normal HC-2 group compared with the control group; 510 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the control group, and 458 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the normal HC-2 group. GO/KEGG enrichment analysis of the significantly different proteins demonstrated that feeding normal HC-2 mainly induced immune response, metabolic, cell adhesion and cell-cell signaling-related protein upregulation, which contributed to bacterial adhesion and colonization in the midgut to improve the shrimp immune system and growth, but these proteins were suppressed after the shrimp were fed bacteria deprived of surface proteins. Taken together, these results indicate that the surface proteins were indispensable for HC-2 to execute probiotic effects in the shrimp midgut., (Copyright © 2019. Published by Elsevier Ltd.)

White spot syndrome, which is caused by white spot syndrome virus (WSSV), is a highly contagious disease of penaeid shrimp. However, there is currently incomplete understanding of the infection mechanism and pathogenesis of WSSV. In this study, a novel gene of a previously uncharacterized WSSV response protein (LvWRP) in Litopenaeus vannamei was identified and characterized. The LvWRP gene has an open reading frame (ORF) of 879 bp encoding a putative protein of 292 amino acids. Sequence analysis revealed that LvWRP shared 24.9% identity with an uncharacterized protein of Penaeus monodon nudivirus. Real-time qPCR analysis showed that LvWRP was ubiquitously expressed in shrimp tissues, with transcript levels induced in hemocytes upon immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and WSSV. In addition, RNA interference-mediated knockdown of LvWRP followed by WSSV challenge revealed significant decrease in the transcript levels of WSSV IE1 and VP28 genes coupled with a reduction in WSSV copies in shrimp hemocytes. Moreover, depletion of LvWRP followed by WSSV challenge significantly increased the transcript levels of Vago4 and Vago5 as well as increased the phosphorylation of STAT, while hemocytes apoptosis in terms of caspase 3/7 activity was decreased. These results suggest that LvWRP is important for WSSV replication in shrimp, and therefore one of the vital host factors in WSSV infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hemocyanin is primarily a respiratory copper-containing glycoprotein present in the hemolymph of mollusks and arthropods. Recently, hemocyanin has attracted huge research interest due to its multifunctionality and polymorphism. Most previous immune-related studies on shrimp hemocyanin have focused on the C-terminal. Moreover, we previously reported that the C-terminal domain of Litopenaeus vannamei hemocyanin possesses single nucleotide polymorphisms (SNPs), but little is known about the molecular diversity of the N-terminal domain. In the current study, diversity within the N-terminal domain of L. vannamei hemocyanin (LvHMC-N) was explored using bioinformatics and molecular biology techniques as well as immune challenge. Twenty-five LvHMC-N variants were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing, with multiple sequence alignment showing that the 25 variants shared 87%-99 % sequence homology with LvHMC (AJ250830.1). In different shrimp individuals and different shrimp tissues (i.e., hemocytes, stomach, muscle and hepatopancreas), the LvHMC-N variants were expressed differently. Pathogen challenge could modulate the molecular diversity of LvHMC-N, as three LvHMC-Nr variants (LvHMC-Nr1, LvHMC-Nr2 and LvHMC-Nr3) were identified by sequencing following Vibrio parahaemolyticus challenge. Most importantly, recombinant proteins of these three variants (rLvHMC-Nr1, rLvHMC-Nr2 and rLvHMC- Nr3) had relatively high in vitro agglutinative activities against V. parahaemolyticus, Vibrio alginolyticus and Streptoccocus iniae. Our present data indicates that the N-terminus of L. vannamei hemocyanin also possess molecular diversity, which seems to be associated with immune resistance to pathogenic infections., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The present study reveals purification and characterization of the lectin from the haemolymph of Metapenaeus dobsoni. The Md-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 68 kDa in SDS-PAGE. Furthermore, the molecular mass was confirmed by MALDI-TOF and functional groups present were analysed by FTIR. The surface morphology of purified Md-Lec displays the homogeneous nature of protein. The X-ray diffraction (XRD) analysis expresses three peaks at 10.7716̊, 21.6258̊ and 31.7523̊which indicate the crystalline nature of the protein and the retention time of 3.068 min evident from HPLC reveals the purity of the sample. Functional analysis of purified Md-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy. It also exhibited phenoloxidase activation, encapsulation and phagocytic activities. In addition, purified Md-Lec showed the broad spectrum of bacterial agglutination activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila, important fish pathogens. Antiviral potential and anticancer activity of purified Md-Lec against CyHV-2 virus and MDA-MB-231 breast cancer cell lines were also evaluated in this study., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In aquaculture, fighting infectious diseases is a necessity. This study measured the immuno-stimulating effect of live macroalgae consumption on Litopenaeus vannamei against Vibrio parahaemolyticus and WSSV infection in two independent bioassays. Shrimps and macroalgae were cultivated in a co-culture with two species of macroalgae separately (Gracilaria vermiculophylla and Dictyota dichotoma), and later, shrimp were infected with V. parahaemolyticus. In another bioassay, shrimp and macroalgae (G. vermiculophylla, D. dichotoma and Ulva lactuca) were grown and subsequently infected with WSSV. For both bioassays, survival after 120 h was determined, the total hemocyte count (TCH) was measured and the activity of superoxide dismutase (SOD) and catalase (CAT) in tissue were measured. The results indicate that the use of macroalgae in co-culture with L. vannamei provides a nutritional benefit that achieves higher growth than the control organisms, as well as improvements of the ammonium concentration and immune response after infection with V. parahaemolyticus and WSSV. A better immune response was obtained in organisms cultured with macroalgae in both bioassays at a ratio of 1.6-1.9 for organisms infected with bacteria and 1.4 to 1.6 times for organisms infected with the virus. In turn, the enzymatic activity of SOD and CAT were higher in the treated organisms relative to the controls in both experiments., (Copyright © 2018. Published by Elsevier Ltd.)

B. subtilis and S. algae effects in growth, survival and innate immunity were assessed on L. vannamei juveniles. During 60 days, shrimp were reared in three treatments: Bs, fed with 10 6  CFU of B. subtilis per gram of commercial feed, Sa, fed with 10 6  CFU of S. algae per gram of commercial feed and Control (without bacterial addition). Then, the animals were subjected to a V. parahaemolyticus challenge. For this purpose, four treatments were established: Control (shrimp not submitted to probiotic treatments), Vibrio (Vibrio challenged shrimp), Vibrio + Bs (Bs challenged shrimp) and Vibrio + Sa (Sa challenged shrimp). Shrimp hemolymph was sampled 45-days after rearing and 24 h post-challenge for quantification of prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) and hemocyanin (HEM) transcripts by qPCR. Moreover, shrimp final weight and survival were also verified. B. subtilis administration enhanced shrimp growth and improved proPO, LGBP and HEM expression levels before and after challenge. After 60-days of feeding, Sa final weight was higher than the Control, whereas Vibrio + Sa cumulative mortality after 48 h of Vibrio challenge was lower than Vibrio group. These results could be correlated with the proPO and LGBP up regulation in Vibrio + Sa compared to Vibrio group, protecting L. vannamei from the bacterial infection. Together, these results suggest the probiotic potential of B. subtilis e S. algae in the modulation of immune-related genes as a tool to control V. parahaemolyticus infection inside shrimp., (Copyright © 2018. Published by Elsevier Ltd.)

Lactic acid bacteria (LAB) are group of beneficial bacteria that have been proposed as relevant probiotics with immunomodulatory functions. In this study, we initially isolated and identified host-derived LAB from the gut of the Pacific white shrimp Litopenaeus vannamei. Analysis of the bacterial 16S rRNA gene sequence revealed two candidate LAB, the Lactobacillus plantarum strain SGLAB01 and the Lactococcus lactis strain SGLAB02, which exhibited 99% identity to the L. plantarum strain LB1-2 and the L. lactis strain R-53658, which were isolated from bee gut, respectively. The two LAB displayed antimicrobial activities against gram-positive and gram-negative bacteria, including the virulent acute hepatopancreatic necrosis disease (AHPND)-causing strain of Vibrio parahaemolyticus (VP AHPND ). Viable colony count and SEM analysis showed that the two candidate LAB, administered via oral route as feed supplement, could reside and adhere in the shrimp gut. Double-stranded RNA-mediated gene silencing of LvproPO1 and LvproPO2 revealed a significant role of two LvproPOs in the proPO system as well as in the immune response against VP AHPND infection in L. vannamei shrimp. The effect of LAB supplementation on modulation of the shrimp proPO system was investigated in vivo, and the results showed that administration of the two candidate LAB significantly increased hemolymph PO activity, the relative mRNA expression of LvproPO1 and LvproPO2, and resistance to VP AHPND infection. These findings suggest that administration of L. plantarum and L. lactis could modulate the immune system and increase shrimp resistance to VP AHPND infection presumably via upregulation of the two LvproPO transcripts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Notch signaling pathway transcriptional regulator, CSL (also called as CBF1, Suppressor of Hairless or Lag-1 in different species, generally designated as CSL1), is not only associated with cell proliferation and differentiation but also involved in tumorigenesis, inflammation and immune regulation in vertebrates. We recently showed that Notch signaling was involved in the immune response of Litopenaeus vannamei shrimp. However, as an important transcriptional regulator of this pathway, whether or not shrimp CSL was also involved in immune response had not been explored. Here, we cloned and characterized the CSL gene in L. vannamei (LvCSL), which has a 2271 bp open reading frame (ORF) encoding a putative protein of 756 amino acids, and contains two conserved Lag1-DNA bind as well as beta trefoil domains (BTD). LvCSL clustered with invertebrates in the phylogenetic tree and closely related to the RBP Jk X1 of Parasteatoda tepidariorum. The transcript level of LvCSL analyzed by quantitative polymerase chain reaction (qPCR) showed that LvCSL was widely expressed in all tissues tested, with induced levels observed in the hepatopancreas and hemocytes following immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), therefore, suggesting LvCSL involvement in shrimp immune response to pathogens. Besides, LvCSL knockdown decreased the expression of proliferation-related genes (LvHey2 and LvAstakine), and attenuated the expression of immune-related genes L. vannamei hypoxia inducible factor alpha (LvHIF-α), LvLectin and L. vannamei small subunit hemocyanin (LvHMCS) in shrimp hemocytes, as well as significantly decreased total hemocyte count. Moreover, high cumulative mortality was observed in LvCSL depleted shrimp challenged with V. parahaemoliticus. In conclusion, our present data strongly suggest that LvCSL is an important factor in shrimp, vital for shrimp survival and contributing to immune resistance to pathogens., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chitinases are a group of hydrolytic enzymes that hydrolyze chitin and widely exist in organisms. Studies in mammals have demonstrated that chitinases play important roles in regulation of humoral and cellular immune responses. In arthropods, although it is well known that chitinases are involved in growth, molting and development, the current knowledge on the role of chitinases in immunity, especially in immune regulation, remains largely unknown. In this study, a chitinase (LvChi5) from Litopenaeus vannamei was representatively selected for studying its immune function. The start codon of LvChi5 was corrected by 5'RACE analysis and its protein sequence was reanalyzed. LvChi5 contains a catalytic domain and a chitin binding domain and shows no inhibitory effect on growth of bacteria in vitro. However, in vivo experiments demonstrated that silencing of LvChi5 increased the mortality of shrimp infected with white spot syndrome virus (WSSV) and Vibro parahaemolyticus and significantly upregulated the load of pathogens in tissues. The expression of various immune related genes, including transcription factors, antimicrobial peptides and other functional proteins with antibacterial and antiviral activities, was widely changed in LvChi5 silencing shrimp. Moreover, the recombinant LvChi5 protein could enhance the phagocytic activity of hemocytes against bacteria. These suggested that shrimp chitinase could play a role in regulation of both humoral and cellular immune responses in shrimp., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific strain of Vibrio parahaemolyticus (VP AHPND ), results in great loss of global shrimp production. Despite this, studies on shrimp defense mechanisms protecting against AHPND are few. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes from white shrimp Litopenaeus vannamei hepatopancreas upon VP AHPND infection at the early stages: 3 and 6 h post challenge and in the late stage at 48 h post challenge. Hemocyanin (HMC) is the most abundant gene identified as the up-regulated gene in the SSH library. Various hemocyanin subunits such as hemocyanin (HMC), hemocyanin subunit L1 (HMCL1), L2 (HMCL2), L3 (HMCL3), and L4 (HMCL4) were analyzed for their expression levels upon VP AHPND infection and in response to challenge with partially purified toxin of VP AHPND by qRT-PCR. Only HMC was highly up-regulated at 3 and 6 h post challenge in response to VP AHPND challenge. Two HMC subunits, HMCL3 and HMCL4, were up-regulated in the early phase of VP AHPND toxin injection. Furthermore, all subunits were down-regulated in the late phase of VP AHPND and toxin challenges. The native hemocyanin protein purified from shrimp hemolymph, identified as mixture of HMC and HMCL1, exhibited agglutination activity on VP AHPND . Injecting the purified native hemocyanin along with VP AHPND into shrimp decreased the number of bacteria in the hemolymph as compared to the VP AHPND challenged control. Moreover, pre-incubation of the purified native hemocyanin and VP AHPND toxin prior to injection into shrimp resulted in the decrease of cumulative mortality of shrimp when compared to the control. In addition, protein-protein interaction analysis carried out by ELISA technique indicated that hemocyanin exhibited VP AHPND toxin-neutralizing activity through direct interaction with PirA subunit with a dissociation constant of 6.83 × 10 -6  M. Our results indicated that upon VP AHPND infection the expression of hemocyanin was induced and hemocyanin functions might involve agglutination of invading VP AHPND and also neutralization of VP AHPND secreted toxin via direct interacting with the PirA protein., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

LvMKK4, a homologue of the mammalian mitogen-activated protein kinase kinase 4 (MKK4), was isolated and identified from Litopenaeus vannamei in the present study. The full-length cDNA of LvMKK4 is 1947 bp long, with an open reading frame (ORF) of 1185 bp encoding a putative protein of 388 amino acids. LvMKK4 contains several characteristic domains such as D domain, SIAKT motif and kinase domain, all of which are conserved in MAP kinase kinase family. Like mammalian MKK4 but not Drosophila MKK4, LvMKK4 could bind to, phosphorylate and activate p38 MAPK, which provided some insights into the signal transduction mechanism of MKK4-p38 cascade in invertebrates. Our real-time PCR data indicated that LvMKK4 was ubiquitously expressed in all tested tissues and extraordinarily abundant in muscle. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvMKK4 activated the transcription of antimicrobial peptide genes (AMPs), including Drosophila Attacin A, Drosomycin, and shrimp Penaeidins. Additionally, LvMKK4 was up-regulated in both intestine and hepatopancreas by a variety of inflammatory stimuli including LPS, Vibrio parahaemolyticus, Staphhylococcu saureus, Poly (I: C) and white spot syndrome virus. Furthermore, RNAi-mediated knockdown of LvMKK4 enhanced the sensitivity of L. vannamei to V. parahaemolyticus infection. These findings suggested that LvMKK4 played an important role in anti-bacterial response and could be a potential target for inflammation treatment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

V. parahaemolyticus are bacteria that cause the Acute Hepatopancreatic Necrosis Disease (AHPND), or Early Mortality Syndrome (EMS), in shrimp. To further understand the pathogenesis mechanisms of V. parahaemolyticus infection in shrimp, the spreading of this bacterium in various tissues was investigated. The spread of infection in shrimp that were exposed to seawater bacteria was studied by PCR and histopathology at 1 min, 1, 6, 12, 24, 48 and 72 h after exposure. The PCR results showed that V. parahaemolyticus was at its most widespread at 6 h after exposure, at which point V. parahaemolyticus was found in the gills, hepatopancreas, intestine, muscles, and hemolymph. However, examinations after 6 h of infection found only small amounts of V. parahaemolyticus in hepatopancreas and intestines. Histopathology of the hepatopancreas showed abnormalities on gross examination at 1 min-72 h after exposure. This study indicates that V. parahaemolyticus can spread quickly by using the hepatopancreas as the target tissue. After 6 h of infection, V. parahaemolyticus was eliminated by immune system while their toxins still caused damage to shrimp tissues., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A shrimp disease, the so-called acute hepatopancreatic necrosis disease (AHPND) is caused by a specific strain of Vibrio parahaemolyticus (VP) and it has resulted in significant losses to the global shrimp farming industry. In our previous study, three of tachylectin-like genes were cloned and characterized from the intestine of Penaeus monodon, designated as Penlectin5-1 (PL5-1), Penlectin5-2 (PL5-2) and Penlectin5-3 (PL5-3). These three genes all contain fibrinogen-related domain (FReD). The expression level of PL5-1, PL5-2 and PL5-3 was elevated in the stomach after oral administration with AHPND-causing V. parahaemolyticus 3HP (VP 3HP ). A polyclonal antibody to PL5-2 was successfully produced in a rabbit using the purified recombinant PL5-2 as an immunogen, and this because only the predominant protein PL5-2 could be successfully purified from shrimp plasma by affinity chromatography using a N-Acetyl-d-glucosamine column allowed us to perform functional studies of this lectin. The native purified PL5-2 protein had binding and agglutination activities towards VP 3HP . To further understand the functions and the involvements of this lectin in response to AHPND in shrimp, RNAi-mediated knockdown of PL5-1, PL5-2 or PL5-3 was performed prior to an oral administration of VP 3HP . As a result, Penlectin5-silencing in shrimp challenged with VP 3HP showed higher mortality and resulted in more severe histopathological changes in the hepatopancreas with typical signs of AHPND. These results therefore suggest a role for crustacean fibrinogen-related proteins (FRePs) in innate immune response during the development of AHPND, and maybe also during other infections., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Interleukins are a group of cytokines that play essential roles in immune regulation. Almost all interleukin genes are only found in vertebrates. In this study, an interleukin-16-like gene (LvIL-16L) was identified from Pacific white shrimp, Litopenaeus vannamei. LvIL-16L was predicted to encode a precursor (pro-LvIL-16L) with 1378 amino acids, sharing similarities with predicted pro-IL-16-like proteins from insects. The C-terminus of pro-LvIL-16L protein contained two PDZ domains homologous to the mature IL-16 cytokine of vertebrates. In tissues, LvIL-16L could be processed into a ∼36 kDa mature peptide through a caspase-3 cleavage site, which was verified by in vitro site mutation analysis and in vivo RNA interference (RNAi) experiments. The LvIL-16L mRNA could be detected in all the analyzed tissues and the expression of LvIL-16L was significantly up-regulated after immune stimulation. Using RNAi strategy, the role of LvIL-16L in immune responses was initially investigated. Interestingly, knockdown of LvIL-16L could significantly increase the mortality of the Vibro parahaemolyticus infected shrimps but reduce that of the WSSV infected shrimps, suggesting that LvIL-16L could have opposite effects on the antiviral and antibacterial immune responses in shrimp. To our knowledge, this is the first study of an IL-16-like gene in invertebrates, which could help to elucidate interleukin evolution and regulatory mechanisms of shrimp immune responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defences of various invertebrates. Whether or not laccase exists in shrimp and its function is still poorly understood. In this study, a laccase (LvLac) was cloned and identified from Litopenaeus vannamei for the first time. The full length of LvLac is 3406 bp, including a 2034 bp open reading frame (ORF) coding for a putative protein of 677 amino acids with a signal peptide of 33 aa. LvLac contains three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine, respectively. Phylogenetic analysis revealed that LvLac was close to insects laccase 1 family. LvLac expression was most abundant in heart and the crude LvLac protein could catalyze the oxidation of hydroquinone. Real-time PCR showed that LvLac expression was responsive to Vibrio parahaemolyticus, Micrococcus lysodeikticus and white spot syndrome virus (WSSV) infection. Knockdown of LvLac enhanced the sensitivity of shrimps to V. parahaemolyticus and M. lysodeikticus challenge, suggesting that LvLac may play a positive role against bacterial pathogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Thioester-containing proteins (TEPs) are present in a wide range of species from deuterostomes to protostomes and are thought to be involved in innate immunity. In the current study, a TEP gene homologous to insect TEPs (iTEP) from the crustacean Litopenaeus vannamei, named LvTEP1, is cloned and functionally characterized. The open reading frame (ORF) of LvTEP1 is 4383 bp in length, encoding a polypeptide of 1460 amino acids with a calculated molecular weight of 161.1 kDa LvTEP1, which is most similar to other TEPs from insects, contains some conserved sequence features, including a N-terminal signal peptide, a canonical thioester (TE) motif, and a C-terminal distinctive cysteine signature. LvTEP1 is expressed in most immune-related tissues, such as intestine, epithelium, and hemocytes, and the mRNA level of LvTEP1 is upregulated in hemocytes after bacterial and viral challenges, indicating its involvement in the shrimp innate immune response. An expression assay in Drosophila S2 cells shows LvTEP1 to be a full-length secretory protein, and processed forms are present in the supernatant. Of note, only the processed form of LvTEP1 protein can bind to both the gram-negative bacterium Vibrio parahaemolyticus and the gram-positive bacterium Staphylococcus aureus in vitro, and its abundance can be induced after bacterial treatment. Moreover, knockdown of LvTEP1 renders shrimps more susceptible to both V. parahaemolyticus and S. aureus, as well as white spot syndrome virus (WSSV) infection, suggesting its essential defensive role against these invading microbes. We also observe that the expression of LvTEP1 is regulated in a manner dependent on both NF-κB and AP-1 transcription factors in naive shrimps and in vitro, suggesting that LvTEP1 could be poised in the body cavity prior to infection and thus play an important role in basal immunity. Taken together, our findings provide some in vitro and in vivo evidence for the involvement of LvTEP1 in shrimp innate immunity and provide some insight into its expression regulation mediated by multiple transcription factors or signaling pathways., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

It has been proposed that invertebrate stimulators of interferon genes (STINGs) do not take part in the innate immune response to infection. Herein, we identified a new STING homolog from pacific white shrimp Litopenaeus vannamei (LvSTING). Some amino acids crucial for recognizing cyclic dinucleotides in mammals are highly conserved in LvSTING. Moreover, LvSTING expression can be robustly induced by challenge with the Gram-negative bacteria Vibrio parahaemolyticus. Silencing of LvSTING contributes to decreased expression of the antimicrobial peptide PEN4 and renders shrimp more susceptible to V. parahaemolyticus infection, while coinjection with the recombinant LvSTING protein can rescue PEN4 expression in vivo and confer shrimp with more resistance to infection. Taken together, these results suggest that LvSTING is involved in the innate immune response to bacterial infection., (© 2017 Federation of European Biochemical Societies.)

Acute Hepatopancreatic Necrosis Disease (AHPND) is an emerging disease in aquacultured shrimp caused by a pathogenic strain of Vibrio parahaemolyticus. As with several pathogenic bacteria, colonization of the stomach appeared to be the initial step of the infection for AHPND-causing Vibrio. To understand the immune responses in the stomach of black tiger shrimp (Penaeus monodon), differentially expressed transcripts (DETs) in the stomach during V. parahaemolyticus strain 3HP (VP3HP) infection was examined using Ion Torrent sequencing. From the total 42,998 contigs obtained, 1585 contigs representing 1513 unigenes were significantly differentially expressed with 1122 and 391 unigenes up- and down-regulated, respectively. Among the DETs, there were 141 immune-related unigenes in 10 functional categories: antimicrobial peptide, signal transduction pathway, proPO system, oxidative stress, proteinases/proteinase inhibitors, apoptotic tumor-related protein, pathogen recognition immune regulator, blood clotting system, adhesive protein and heat shock protein. Expression profiles of 20 of 22 genes inferred from RNA sequencing were confirmed with the results from qRT-PCR. Additionally, a novel isoform of anti-lipopolysaccharide factor, PmALF7 whose transcript was induced in the stomach after challenge with VP3HP was discovered. This study provided a fundamental information on the molecular response in the shrimp stomach during the AHPND infection that would be beneficial for future research., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Innate immunity in shrimp is important in resisting bacterial infection. The NF-κB pathway is pivotal in such an immune response. This study cloned and functionally characterized the solute carrier family (SLC) 15 member A 4 (LvSLC15A4) gene in Litopenaeus vannamei. The open reading frame of LvSLC15A4 is 1, 902 bp long and encodes a putative 633-amino acid protein, which is localized in the plasma membrane and intracellular vesicular compartments. Results of the reporter gene assay showed that LvSLC15A4 upregulated NF-κB target genes, including the immediate-early gene 1 of white spot syndrome virus, as well as several antimicrobial peptide genes, such as pen4, CecA, AttA, and Mtk in S2 cells. Moreover, knocked-down expression of LvSLC15A4 reduced pen4 expression in L. vannamei. LvSLC15A4 down-regulation also increased the cumulative mortality of Vibrio parahemolyticus-infected L. vannamei. Furthermore, LvSLC15A4 expression was induced by unfolded protein response (UPR) in L. vannamei hematocytes. These results suggest that LvSLC15A4 participates in L. vannamei innate immunity via the NF-κB pathway and thus may be related to UPR., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Growing evidence indicates that activator protein-1 (AP-1) plays a major role in stimulating the transcription of immune effector molecules in cellular response to an incredible array of stimuli, including growth factors, cytokines, cellular stresses and bacterial and viral infection. Here, we reported the isolation and characterization of a cDNA from Litopenaeus vannamei encoding the full-length c-Fos protein (named as Lvc-Fos). The predicted amino acid sequences of Lvc-Fos contained a basic-leucine zipper (bZIP) domain, which was characteristic of members of the AP-1 family. Immunoprecipitation and native-PAGE assays determined that Lvc-Fos could interact with the Lvc-Jun, a homolog of c-Jun family in L. vannamei, in a heterodimer manner. Further investigation demonstrated that Lvc-Fos and Lvc-Jun were expressed in all tested tissues and located in the nucleus. Real-time RT-PCR analysis showed both Lvc-Fos and Lvc-Jun in gills were up-regulated during Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges. In addition, reporter gene assays indicated Lvc-Fos and Lvc-Jun could activate the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp, as well as WSSV immediate early (IE) genes wsv069 and wsv249, in a different manner. Knockdown of Lvc-Fos or Lvc-Jun by RNA interference (RNAi) resulted in higher mortalities of L. vannamei after infection with V. parahaemolyticus, suggesting that Lvc-Fos and Lvc-Jun might play protective roles in bacterial infection. However, silencing of Lvc-Fos or Lvc-Jun in shrimp caused lower mortalities and virus loads under WSSV infection, suggesting that Lvc-Fos and Lvc-Jun could be engaged for WSSV replication and pathogenesis. In conclusion, our results provided experimental evidence and novel insight into the roles of L. vannamei AP-1 in bacterial and viral infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cactin (Cactus interactor) is a conserved protein which was initially discovered as a novel interactor of Drosophila IkB protein Cactus. Cactin was always characterized as a negative regulator of many different developmental processes, but only found to play an immune role in humans. To better know the immune function of Cactin gene, Litopenaeus vannamei Cactin (LvCactin) was identified and characterized in this study. The full-length cDNA of LvCactin is 3, 150 bp long, with an open reading frame (ORF) encoding a Cactin&#95;mid domain in the N-terminus 356–547 residues and a CactinC&#95;cactus domain in the C-terminal 731–855 residues. The LvCactin protein was located in the cytoplasm and LvCactin mRNA was constitutively expressed in healthy L. vannamei, with the highest expression level in the eyestalk. LvCactin could be regulated in hemocytes after lipopolysaccharide (LPS), poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV) challenges. Dual-luciferase reporter assays in Drosophila Schneider 2 cells revealed that LvCactin inhibited the promoters of Drosophila and shrimp antimicrobial peptide (AMP) genes. Knockdown of LvCactin by RNA interference (RNAi) increased the expression of shrimp AMP genes PEN4, crustin and ALF2 but not Lyz2. However, the mortality rates of LvCactin-knockdown shrimp in response to V. parahaemolyticus or WSSV infections were not significantly different from those of the control group. Taken together, all the results suggested that LvCactin may play a role in innate immune in L. vannamei.

C-type lectins (CTLs) play crucial roles in innate immune responses in invertebrates by recognizing and eliminating microinvaders. In this study, a CTL from pacific white shrimp Litopenaeus vannamei (LvCTL3) was identified. LvCTL3 contains a single C-type lectin-like domain (CTLD), which shows similarities to those of other shrimp CTLs and has a mutated 'EPD' motif in Ca(2+)-binding site 2. LvCTL3 mRNA can be detected in all tested tissues and expression of LvCTL3 in gills was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges, suggesting activation responses of LvCTL3 to bacterial, virus and immune stimulant challenges. The 5'flanking regulatory region of LvCTL3 was cloned and we identified a NF-κB binding motif in the LvCTL3 promoter region. Dual-luciferase reporter assays indicated that over-expression of L. vannamei dorsal can dramatically up regulate the promoter activity of LvCTL3, suggesting that LvCTL3 expression could be regulated through NF-κB signaling pathway. As far as we know, this is the first report on signaling pathway involve in shrimp CTLs expression. The recombinant LvCTL3 protein was expressed in Escherichia coli and purified by Ni-affinity chromatography. The purified LvCTL3 can agglutinate Gram-negative microbe Vibrio alginolyticus and V. parahaemolyticus and Gram-positive bacteria Bacillus subtilis in the presence of calcium ions, but cannot agglutinate Gram-positive bacteria Streptococcus agalactiae. The agglutination activity of LvCTL3 was abolished when Ca(2+) was chelated with EDTA, suggesting the function of LvCTL3 is Ca(2+)-dependent. In vivo challenge experiments showed that the recombinant LvCTL3 protein can significantly reduce the mortalities of V. parahemolyticus and WSSV infection, indicating LvCTL3 might play significant roles in shrimp innate immunity defense against bacterial and viral infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The polysaccharide fucoidan from brown seaweed Sargassum wightii was extracted and it was incorporated with pellet diets at three concentrations (0.1, 0.2 & 0.3%). The fucoidan incorporated diets were fed to shrimp Penaeus monodon for 60 days and the growth performance was assessed. The weight gain and SGR of control group was 6.83 g and 9.72%, respectively, but the weight gain and SGR of various concentrations (0.1-0.3%) of fucoidan incorporated diets fed groups of shrimp was increased from 7.30 to 8.20 g and 9.83 to 10.03%, respectively. After 60 days of feeding experiment, the relative quantification of prophenoloxidase gene of experimental groups over control group was analysed by RT-PCR and it was ranged between 2.13 and 7.95 fold increase within 33.52-34.61 threshold cycles, respectively at 0.1-0.3% concentrations of fucoidan. After 60 days of feeding experiment, the P. monodon were challenged with shrimp pathogen Vibrio parahaemolyticus and the mortality percentage was recorded daily up to 21 days. The reduction in mortality percentage of experimental groups over control group was recorded from 44.56 to 72.79%, respectively in 0.1-0.3% of fucoidan incorporated diets fed groups. During challenge experiment, all the immunological parameters such as THC, prophenoloxidase activity, respiratory burst activity, superoxide dismutase activity, phagocytic activity, bactericidal activity and bacterial clearance ability of experimental groups were significantly (P < 0.05) increased than control group. The V. parahaemolyticus load was enumerated from the infected shrimp at every 10 days intervals during challenge experiment. In control group, the Vibrio load was increased in hepatopancreas and muscle tissues from 10th to 21st days of challenge test. But in the experimental groups, the Vibrio load in both the tissues decreased positively from 10th to 21st days of challenge duration. It is concluded that the S. wightii fucoidan had enhanced the innate immunity and increased resistance to V. parahaemolyticus infection in P. monodon., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The toll-like receptor (TLR)/NF-κB signaling pathways play critical roles in the innate immune system. The intracellular signal transduction of most TLR pathways in invertebrate cells is triggered by formation of a heterotrimeric complex composed of MyD88, Tube and Pelle. In this study, we identified a Litopenaeus vannamei Pelle (LvPelle) and an isoform of L. vannamei Tube (LvTube) designated as LvTube-1. The interactions among LvPelle, LvTube/LvTube-1 and LvMyD88/LvMyD88-1 were elucidated and their functions during pathogen infections were investigated. Knockdowns of LvPelle and LvTube/LvTube-1 using RNAi strategy led to higher mortalities of shrimps during Vibrio parahemolyticus infection, and could reduce the genome copy number of white spot syndrome virus (WSSV) in the infected muscle tissue but did not affect the mortality caused by WSSV infection. The effects of LvPelle and LvTube/LvTube-1 on promoters containing NF-κB binding motifs were analyzed by dual-luciferase reporter assays and the results demonstrated that LvTube-1 could activate the NF-κB activity to significantly higher level than LvTube did. Moreover, tissue distributions of LvTube and LvTube-1 mRNAs and their expression profiles during pathogen and immune stimulant challenges were different, indicating that they could play different roles in immune responses. This is the first report of Tube isoforms in invertebrates. Together with our previous study on LvMyD88 isoforms, our results suggest that various isoforms of adaptor components may be involved in various regulatory patterns of signal transduction in invertebrate TLR/NF-κB pathway and this could be a strategy adopted by invertebrates to modulate immune responses., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Single nucleotide polymorphisms (SNPs) are the commonest mode of genetic variation in invertebrate immune-related genes. Hemocyanin presents in the hemolymph of both mollusks and arthropods and functions as an important antigen non-specific immune protein. But people know very little about its gene polymorphism so far. In current study, bioinformatics, molecular biology and environmental challenge approaches were used to identify the SNPs within hemocyanin Ig-like domain in shrimp Litopenaeus vannamei. A total of 11 SNPs were found in a variable region of Ig-like domain from L. vannamei hemocyanin large subunit (1258-1460 bp, HcLV1), 5 of which (1272, 1315, 1380, 1410 and 1450) were confirmed present in both genomic DNA and cDNA by clone sequencing. Furthermore, HcLV1 showed 3, 5 and 5 SSCP bands, respectively, in 16, 25 and 30 °C-treated shrimps, suggesting that the SSCP pattern of HcLV1 could be modulated by environmental stress. In addition, HcLV1 displayed two extra bands with different mobility when shrimps treated with Vibrio parahaemolyticus for 6-24 h, which was not observed in the control group. In conclusion, our data suggest that shrimp L. vannamei hemocyanin Ig-like domain possesses SNPs, which may be associated with environmental stress or pathogenic challenge., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The present study investigated the in vivo hemocytic and hepatopancreatic response to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) injection in shrimp Litopenaeus vannamei. The proliferation of bacteria and virus in shrimp, animal mortality, total hemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst, and gene expression of immune factors associated with immune recognition (lectin), prophenoloxidase (proPO) activation, and the anti-microorganism (lysozyme) and active oxygen defense response (including respiratory burst, cytosolic manganese superoxide dismutase [C-MnSOD], and catalase [CAT]) were quantified. Shrimp death rate increased significantly and was time-dependent after V. parahaemolyticus or WSSV injection. The production of superoxide anion, and the gene expression including lectin in hemocytes, proPO in the hepatopancreas, lysozyme, C-MnSOD and CAT could be induced by injection with V parahaemolyticus and WSSV. The highest value of lysozyme was in the hemocytes with 66.59 times (at 3 h) greater expression than in the control group after WSSV injection and 3.69 times (24 h) greater than in the control group after V parahaemolyticus injection. In the hepatopancreas, CAT expression showed a significant increase, with up to 16 times greater expression than in the control group at 6 h postinjection with WSSV and 7.02 times greater expression than in the control group at 48 h postinjection with V parahaemolyticus (p < 0.05). However, significant decreases in PO activity and proPO transcripts in hemocytes and lectin transcripts in the hepatopancreas were detected after V parahaemolyticus and WSSV injection (p < 0.05). The results suggest that lysozyme, the antioxidase system, and reactive oxygen species might play a crucial role in shrimp defense against bacterial and viral infection.