Deletion of ArmPT, a LamB-like protein, increases cell membrane permeability and antibiotic sensitivity in Vibrio alginolyticus.

Vibrio bacterial species are dominant pathogens in mariculture animals. However, the extensive use of antibiotics and other chemicals has increased drug resistance in Vibrio bacteria. Despite rigorous investigative studies, the mechanism of drug resistance in Vibrio remains a mystery. In this study, we found that a gene encoding LamB-like outer membrane protein, named ArmPT, was upregulated in Va under antibiotic stress by RT-qPCR. We speculated that ArmPT might play a role in Va's drug resistance. Subsequently, using ArmPT gene knockout and gene complementation experiments, we confirmed its role in resistance against a variety of antibiotics, particularly kanamycin (KA). Transcriptomic and proteomic analyses identified 188 and 83 differentially expressed genes in the mutant strain compared with the wild-type (WT) before and after KA stress, respectively. Bioinformatic analysis predicted that ArmPT might control cell membrane permeability by changing cadaverine biosynthesis, thereby influencing the cell entry of antibiotics in Va. The higher levels of intracellular reactive oxygen species and the infused content of KA showed that antibiotics are more likely to enter the Va mutant strain. These results uncover the drug resistance mechanism of Va that can also exist in other similar pathogenic bacteria.

The krüppel-like factor of Penaeus vannamei negatively regulates transcription of the small subunit hemocyanin gene as part of shrimp immune response.

Hemocyanin is a multifunctional respiratory glycoprotein, which has also been implicated in other biological functions in shrimp. Moreover, recent studies have revealed that hemocyanin is also involved in a broad range of immune-related activities in shrimp. However, in spite of the considerable interest in unraveling the reasons behind the multiple immune-related functions of hemocyanin, little is known about its transcriptional regulation. Here, DNA pull-down and Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) analyses were used to isolate and identify the putative transcription factor(s) that are involved in the transcriptional regulation of the small subunit hemocyanin gene of Penaeus vannamei (PvHMCs). Krüppel-like factor (designated PvKruppel), a zinc finger transcription factor homolog in P. vannamei, was identified among the putative transcription factors, while bioinformatics analysis revealed the presence of Krüppel-like factor binding site (KLF motif) on the core promoter region of PvHMCs. Mutational analysis and electrophoretic mobility shift assay (EMSA) confirmed that PvKruppel could bind to the KLF motif on the core promoter region of PvHMCs. Moreover, in response to lipopolysaccharide (LPS), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenge, transcript levels of PvKruppel and PvHMCs were negatively correlated. Furthermore, overexpression of PvKruppel significantly reduced the promoter activity of PvHMCs, while PvKruppel knockdown by RNA interference or lipopolysaccharides (LPS) stimulation resulted in a significant increase in the transcript level of PvHMCs. Taken together, our present study provides mechanistic insights into the transcriptional regulation of PvHMCs by PvKruppel during shrimp immune response to pathogens.

Litopenaeus vannamei Src64B restricts white spot syndrome virus replication by modulating apoptosis.

The Src family kinases (SFK) are involved in signaling transductions that regulate numerous biological activities including host-virus interaction. These features of SFK have been well explored in vertebrates, however, in shrimp, the invertebrate SFK family member Src64B, has not been characterized and therefore its role in shrimp-virus interaction remains unknown. In this study, two Litopenaeus vannamei Src64B isoforms (designated LvSrc64B1 and LvSrc64B2) were first cloned and their role in white spot syndrome virus (WSSV) infection was explored. Bioinformatics analysis revealed that LvSrc64B1 and LvSrc64B2 were similar to other Src64B family members, with high homology in primary and tertiary structures, and contained the conserved SFK functional domains, as well as the putative myristylation and phosphorylation sites. Tissue distribution analysis showed that both LvSrc64B isoforms were ubiquitously expressed, albeit distinctively in the tested tissues. In addition, transcript levels of LvSrc64B1 and LvSrc64B2 were significantly induced following WSSV challenge and had similar expression patterns. Furthermore, siRNA-mediated knockdown of LvSrc64B1 and LvSrc64B2 followed by WSSV infection resulted in increased expression of viral genes, enhanced viral DNA replication, and elevation of hemocytes apoptosis. Depletion of LvSrc64B1 and LvSrc64B2 also reduced shrimp survival upon WSSV infection. In conclusion, the current data strongly suggest that Src64B is a host factor that inhibits WSSV replication by modulating apoptosis in shrimp.

Identification of novel sRNAs involved in oxidative stress response in the fish pathogen Vibrio alginolyticus by transcriptome analysis.

Vibrio alginolyticus as an important pathogen in aquaculture can encounter the oxidative stress produced by the immune system during infection. Previous studies showed that sRNAs have important functions in response to oxidative stress in bacteria; however, less of sRNAs related to oxidative stress response were identified in V. alginolyticus. In this study, a total of 749 novel sRNAs were identified by RNA sequencing; among them, 128 sRNAs were up- or downregulated in response to oxidative stress. In addition, 1,870 genes exhibited variation on mRNA levels in oxidative stress response. By analysing the target genes of the sRNAs, we concluded that these sRNAs could regulate expressions of genes responsible for iron transport, catalase, GSH-dependent defence system, electron transferred and stress response. Moreover, the functions of the sRNAs are also seemed related to the pathogenicity in V. alginolyticus. Based on the results, we constructed the oxidative stress model in V. alginolyticus. This study provides us the first outlook of sRNAs function in oxidative stress response in V. alginolyticus. Furthermore, this study can help us to prevent and control this important opportunistic pathogen in aquaculture.

Isolation and characterization of a marine testosterone-degrading bacterium: Vibrio sp. N3.

Steroids, including testosterone, estrone, 17ß-estradiol, estriol and 17ß-ethinyl estradiol, are harmful not only to the population dynamics of aquatic life forms but also to public health. In this study, a marine testosterone-degrading bacterium (strain N3) was isolated from Nanao Island in the South China Sea. In addition, the strain could also use 17ß-estradiol (E2), 17ß-ethinyl estradiol (EE2), estriol (E3) or cholesterol as a sole carbon source. According to the 16S rRNA gene sequence analysis, strain N3 was identified as Vibrio sp. Further characterization showed that the strain is aerobic, gram-negative, and mobile and exhibits resistance to ampicillin, carbenicillin, penicillin and spectinomycin. For enhancing its capacity of testosterone degradation, the Plackett-Burman factorial design and the central composite design were used to optimize the culture condition. Under optimal conditions, 92% of testosterone was degraded by Vibrio sp. N3 in 48h.

Acute Hepatopancreatic Necrosis Disease (AHPND) related microRNAs in Litopenaeus vannamei infected with AHPND-causing strain of Vibrio parahemolyticus.

BACKGROUND: Acute hepatopancreatic necrosis disease (AHPND) has emerged as a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. Given that several diseases and infections have been associated with microRNAs (miRNAs), we conducted a comparative transcriptomic analysis using the AHPND (VA) and non-AHPND (VN) strains of Vibrio parahemolyticus to identify miRNAs potentially involved in AHPND pathogenesis in Litopenaeus vannamei. RESULTS: A total of 83 miRNAs (47 upregulated and 36 downregulated) were significantly differentially expressed between the VA and VN challenged groups, while 222 target genes of these miRNAs were predicted. Functional enrichment analysis revealed that the miRNAs target genes were involved in multiple biological processes including metabolic pathways, amoebiasis, Vibrio cholerae infection etc. Finally, interaction network and qPCR (Real-time Quantitative PCR) analysis of 12 potential key AHPND-related miRNAs and their predicted target genes, revealed their possible involvement in modulating several immune-related processes in the pathogenesis of AHPND. CONCLUSIONS: We have shown using comparative transcriptomic analysis, miRNAs and their target genes that are responsive to AHPND V. parahemolyticus infection in shrimp, therefore suggesting their possible role in defense response to AHPND V. parahemolyticus infection.

Functional domains of Litopenaeus vannamei transglutaminase and their involvement in immunoregulation in shrimp.

Shrimps, which mainly rely on their innate immune system to response to infectious pathogens, have clottable proteins as an important component of this system. While transglutaminases (TGase) are found in Litopenaeus vannamei and constitute part of the coagulation system, the specific immune-related roles played by its functional domains in the immunoregulation of shrimp has not been well understood. In the present study, we report that the Ig-like domain of L. vannamei transglutaminase (TGase-C) is the main immune-related domain among the three functional domains, as it had higher bacterial agglutinative activity against Vibrio parahaemolyticus and Streptococcus iniae. Using Co-immunoprecipitation and LC-MS/MS analysis, TGase-C was shown to interact with 474 proteins, of which 52 proteins were annotated to L. vannamei. More than half of the L. vannamei annotated proteins have immune-related functions, including apoptosis. Further analysis using pull-down assay revealed that TGase-C interacted with CAP-3 (a homologue of caspase 3). In addition, siRNA-mediated knockdown of LvTGase significantly (p < 0.01) increased the expression level of LvCAP-3 coupled with a significant (p < 0.01) increase in caspase 3/7 activity, suggesting that probably LvTGase participates in shrimp immune response by modulating the activity of LvCAP-3. These findings thus suggest the Ig-like functional domain of L. vannamei's transglutaminase is the domain that is involved in immunoregulation in shrimp.

A Litopenaeus vannamei Hemocyanin-Derived Antimicrobial Peptide (Peptide B11) Attenuates Cancer Cells' Proliferation.

Antimicrobial peptides play important roles in the immune response to pathogens and tumor cells; for this reason, they are being exploited for therapeutic use. In this study, we describe a Litopenaeus vannamei hemocyanin-derived peptide, denoted B11, which shares similar features with other anticancer peptides and attenuates the proliferation of cancer cells. Cell viability assay revealed that B11 significantly inhibited the proliferation of human cervical (HeLa), human hepatocellular carcinoma (HepG2), and human esophageal cancer (EC109) cancer cell lines, but not normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE) cells or THLE-3), by inducing morphological changes, nuclear condensation, and margination, features which are indicative of apoptosis. Besides, peptide B11-induced apoptosis was confirmed by isothiocyanate-labeled Annexin V/propidium iodide (Annexin V-FITC/PI) double staining of HeLa cells. Moreover, cell uptake studies, confocal microscopy, and Western blot analysis revealed that rhodamine-labeled B11 permeated HeLa cells and localized to the mitochondria, causing mitochondria dysfunction through lost mitochondrial membrane potential, which consequently triggered the induction of apoptosis. Increased expression levels of caspase-9, caspase-3, and Bax (Bcl-2-associated X) proteins, coupled with a decrease in Bcl-2 (B-cell lymphoma 2) protein, confirmed that peptide B11 induced apoptosis via the mitochondrial pathway. Thus, the hemocyanin-derived peptide, B11, inhibits the proliferation of cancer cells by causing mitochondrial dysfunction and inducing apoptotic cell death, for which reason it could be explored as an anticancer peptide.

[Immunological cross-reactivity and cross-protection of outer membrane protein OmpU among Vibrio species].

Objective: To study the immunological cross-reactivity and cross-protection characteristics of OmpU in Vibrio species. Methods: The ompU genes from 10 Vibrio strains were cloned, sequenced, followed by bioinformatics analysis. Western blot and whole-cell ELISA assay were used respectively to determine immunological cross-reaction feature and subcellular location of OmpU with rabbit serum against recombinant OmpU from V. parahaemolyticus ATCC17802, V. alginolyticus ATCC33787, V. vulnificus ATCC27562, V. mimicus ATCC33653 and V. cholera Vb0. Finally, the cross-protective property of recombinant OmpU (V. cholera-derived) was evaluated through vaccination and subsequent challenge with heterogeneous virulent Vibrio strains in mice. Results: The similarities of OmpU proteins of Vibrio ranged from 73.0 to 100% intra-species, and from 58.6 to 89.0% inter-species. Furthermore, homologous epitopes were found in OmpU and shared by different species of Vibrios. Western blot of rabbit serum against recombinant OmpU showed cross-recognition intra- and inter-species. Bands were observed ranging from 35 to 40 kDa. Whole-cell ELISA assay further confirmed that the antiserum of recombinant OmpU from V. parahaemolyticus ATCC17802, V. vulnifgicus ATCC27562 and V. mimicus ATCC33653 recognized the tested Vibrio species, implying that epitopes of OmpU were located on the cell surface. Recorded relative percent survival of the vaccinated group varied from 43.0 to 100%, showing that mice were protected from Vibrio infection after immunization with OmpU protein. Conclusion: OmpU was a conserved antigen among tested Vibrio species and might be a universal vaccine candidate for the prevention of Vibriosis.

Assembly mechanism of microbial community under different seasons in Shantou sea area.

Coastal areas are often affected by a variety of climates, and microbial composition patterns are conducive to adaptation to these environments. In this study, the composition and pattern of microbial communities in the Shantou sea from four seasons were analyzed. The diversity of microbial community was significant differences under different seasons (p < 0.01). Meanwhile, dissolved oxygen levels, temperature were key factors to shift microbial communities. The assembly mechanism of microbial communities was constructed by the iCAMP (Infer community assembly mechanism by the phylogenetic bin-based null). Interestingly, the analyses revealed that drift was the predominant driver of this process (44.5 %), suggesting that microbial community assembly in this setting was dominated by stochastic processes. For example, Vibrio was found to be particularly susceptible to stochastic processes, indicating that the pattern of bacterial community was governed by stochastic processes. Thus, these results offering novel insight into the regulation of microbial ecology in marine environments.

Evidences of SNPs in the variable region of hemocyanin Ig-like domain in shrimp Litopenaeus vannamei.

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.

Characterization of a novel ß-agarase from an agar-degrading bacterium Catenovulum sp. X3.

An agar-degrading bacterium, Catenovulum sp. X3, was isolated from the seawater of Shantou, China. A novel ß-agarase gene agaXa was cloned from the strain Catenovulum sp. X3. The gene agaXa consists of 1,590 bp and encodes a protein of 529 amino acids, with only 40 % amino acid sequence identity with known agarases. AgaXa should belong to the glycoside hydrolase family GH118 based on the amino acid sequence similarity. The molecular mass of the recombinant AgaXa (rAgaXa) was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a maximal agarase activity at 52 °C and pH 7.4 and was stable over pH 5.0 ~ 9.0 and at temperatures below 42 °C. The K m and V max for agarose were 10.5 mg/ml and 588.2 U/mg, respectively. The purified rAgaXa showed endolytic activity on agarose degradation, yielding neoagarohexaose, neoagarooctaose, neoagarodecaose, and neoagarododecaose as the end products. The results showed that AgaXa has potential applications in agar degradation for the production of oligosaccharides with various bioactivities.

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata).

Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 µg/ml. Agglutination was inhibited by 50-200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ▵OmpA and ▵OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.

Nuclear receptor E75 is a transcription suppressor of the Litopenaeus vannamei small subunit hemocyanin gene.

Hemocyanin is a respiratory protein that possesses multiple physiological and immunological functions in shrimp. However, the transcriptional regulation of the hemocyanin gene is still poorly understood. Here, the nuclear receptor E75 of Litopenaeus vannamei (LvE75) was identified as one of the transcriptional regulators that modulates the transcription of the small molecular weight hemocyanin gene of L. vannamei (LvHMCs) by inhibiting its core promoter activity in a Dual-luciferase assay. In silico analysis revealed that the core promoter (designated HsP3), which is located at +1517/+1849 bp of LvHMCs contained a putative E75 binding motif ("ACGGAAT", spanning +1812/+1818 bp). Further, LvE75 was shown to inhibit the core promoter activity by direct binding. Importantly, in vivo silencing of LvE75 resulted in a significant upregulation in the mRNA and protein expression of LvHMCs gene. Taken together, our present results provide direct evidence that LvE75 is a transcriptional suppressor of the LvHMCs gene expression.

Molecular cloning and functional characterization of a homolog of the transcriptional regulator CSL in Litopenaeus vannamei.

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.

The outer membrane protein, LamB (maltoporin), is a versatile vaccine candidate among the Vibrio species.

Maltoporin (LamB) is a family of outer membrane proteins. There has been no report of immunological characteristics of LamB in the Vibrio species so far. In this study, lamB genes from eight Vibrio strains were cloned and sequenced. The bioinformatics analysis indicated that sequence similarities of LamB proteins were ranged from 46.7% to 81.1%. Further, the result showed that their antigenic epitopes were highly conserved implying that LamB might be a shared antigen among Vibrios. The Western blot of rabbit sera against recombinant LamB from V. alginolyticus ATCC 33787 with cell lysate of 18 Vibrio strains showed cross-recognition. Bands observed on cell lysate of Vibrio strains immunoblotted with the anti-LamB sera ranged between 40 and 49 kDa. The Whole-cell ELISA assay further confirmed that the antisera of recombinant LamB recognized the tested Vibrio strains indicating the surface-exposed of LamB. Finally, the cross-protective property of recombinant LamB was evaluated through vaccination and subsequent challenge with heterogeneous virulent Vibrio strains in zebrafish. Recorded relative percent survival (RPS) of the vaccinated group varied from 54.1% to 77.8%, showing that zebrafish were protected from Vibrio infection after immunization with LamB protein. The cumulative evidences in this study suggested that LamB was a conserved antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of Vibriosis.

Aislamiento y caracterización de una bacteria marina degradadora de testosterona: Vibrio sp. N3 / Isolation and characterization of a marine testosterone-degrading bacterium: Vibrio sp. N3

Steroids, including testosterone, estrone, 17β-estradiol, estriol and 17β-ethinyl estradiol, are harmful not only to the population dynamics of aquatic life forms but also to public health. In this study, a marine testosterone-degrading bacterium (strain N3) was isolated from Nanao Island in the South China Sea. In addition, the strain could also use 17β-estradiol (E2), 17β-ethinyl estradiol (EE2), estriol (E3) or cholesterol as a sole carbon source. According to the 16S rRNA gene sequence analysis, strain N3 was identified as Vibrio sp. Further characterization showed that the strain is aerobic, gram-negative, and mobile and exhibits resistance to ampicillin, carbenicillin, penicillin and spectinomycin. For enhancing its capacity of testosterone degradation, the Plackett-Burman factorial design and the central composite design were used to optimize the culture condition. Under optimal conditions, 92% of testosterone was degraded by Vibrio sp. N3 in 48 h.

Los esferoides-que incluyen la testosterona, la estrona, el 17 β-estradiol, el estriol y el 17 p-etinilestradiol-son nocivos no solo para la población dinámica de las formas de vida acuática, sino también para la salud pública. En este estudio se aisló una bacteria marina degradadora de testosterona de la isla de Nanao, en el Mar del Sur de China, a la que se denominó cepa N3. Se determinó que esta cepa también podría usar 17 β-estradiol (E2), 17 p-etinilestradiol (EE2), estriol (E3) o colesterol como únicas fuentes de carbono. De acuerdo con el análisis de la secuencia del gen 16S rRNA, la cepa N3 se identificó como Vibrio sp. La caracterización adicional mostró que dicha bacteria es un organismo aerobio, gram negativo y móvil, y que presenta resistencia a ampicilina, carbenicilina, penicilina y espectinomicina. Para optimizar la condición de cultivo en relación con su capacidad de degradar la testosterona, se utilizaron el diseño factorial Plackett-Burman y el diseno compuesto central. En condiciones óptimas, el 92% de la testosterona fue degradada por Vibrio sp. N3 en 48 h.

Identification of a novel alternative splicing variant of hemocyanin from shrimp Litopenaeus vannamei.

Recent evidences suggest that invertebrates express families of immune molecules with high levels of sequence diversity. Hemocyanin is an important non-specific immune molecule present in the hemolymph of both mollusks and arthropods. In the present study, we characterized a novel alternative splicing variant of hemocyanin (cHE1) from Litopenaeus vannamei that produced mRNA transcript of 2579 bp in length. The isoform contained two additional sequences of 296 and 267 bp in the 5'- and 3'-terminus respectively, in comparison to that of wild type hemocyanin (cHE). Sequence of cHE1 shows 100% identity to that of hemocyanin genomic DNA (HE, which does not form an open reading frame), suggesting that cHE1 might be an alternative splicing variant due to intron retention. Moreover, cHE1 could be detected by RT-PCR from five tissues (heart, gill, stomach, intestine and brain), and from shrimps at stages from nauplius to mysis larva. Further, cHE1 mRNA transcripts were significantly increased in hearts after 12h of infection with Vibrio parahemolyticus or poly I: C, while no significant difference in the transcript levels of hepatopancreas cHE was detected in the pathogen-treated shrimps during the period. In summary, these studies suggested a novel splicing variant of hemocyanin in shrimp, which might be involved in shrimp resistance to pathogenic infection.

SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei.

In this study, we identified a variable region in the C-terminus of hemocyanin from the shrimp Litopenaeus vannamei (2288-2503bp, HcSC) by sequence alignments. A total of 13 SNPs were identified by PCR-SSCP and HcSC clone sequencing. The SSCP patterns of HcSC could be modulated in Vibro parahaemolyticus-treated shrimps. A novel SSCP band with four SNP sites was identified in V. parahaemolyticus-resistant shrimps. More importantly, three of these four SNPs introduced variations in amino acid sequence and possibly secondary structure of the HcSC polypeptide and resulted in a higher agglutinative activity against seven pathogenic bacteria. These results suggest that the C-terminus of shrimp L. vannamei hemocyanin possesses SNPs, which may be related to shrimp resistance to different pathogens.