Cloning and characterization of DOPA decarboxylase in Litopenaeus vannamei and its roles in catecholamine biosynthesis, immunocompetence, and antibacterial defense by dsRNA-mediated gene silencing.

Catecholamines (CAs) play critical roles in regulating physiological and immunological homeostasis in invertebrates and vertebrates under stressful environments. DOPA decarboxylase (DDC), an enzyme responsible for the decarboxylation step of dopamine synthesis, participates in neurotransmitter metabolism and innate immunity. In shrimp, two genes encoding CA-related enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, were further identified and characterized as neuroendocrine-immune regulators. In this study, full-length complementary DNA of DDC cloned from the thoracic ganglia of shrimp, Litopenaeus vannamei, (LvDDC) was predicted to encode a 452-amino acid protein with a pyridoxal-dependent decarboxylase-conserved domain, and this deduced protein of LvDDC was phylogenetically closely related to insect DDC. LvDDC messenger RNA expression was analyzed by a semiquantitative RT-PCR and a real-time quantitative RT-PCR and found to be abundant in the hepatopancreas and nervous system but at low levels in haemocytes, heart, stomach, and gills. To determine the role of LvDDC, double-stranded (ds)RNA was used for in vivo assessments. LvDDC-depleted shrimp revealed significant increases in the total haemocyte count, hyaline cells, granular cells, phenoloxidase activity, and respiratory bursts of haemocytes per unit of haemolymph, and phagocytic activity and clearance efficiency toward Vibrio alginolyticus. Further, decreased LvDDC mRNA expression was accompanied by decreases in dopamine, glucose, and lactate levels in haemolymph. In shrimp that received LvDDC-dsRNA for 3 days and were then challenged with V. alginolyticus, the survival rate of LvDDC-depleted shrimp was significantly higher than that of shrimp that received diethyl pyrocarbonate-water or non-targeted dsRNA. In conclusion, the cloned LvDDC was responsible for controlling dopamine synthesis, which then regulated physiological and immune responses in L. vannamei.

Correction: The New Face of the Old Molecules: Crustin Pm4 and Transglutaminase Type I Serving as RNPs Down-Regulate Astakine-Mediated Hematopoiesis.

[This corrects the article DOI: 10.1371/journal.pone.0072793.].

High genetic diversities between isolates of the fish parasite Cryptocaryon irritans (Ciliophora) suggest multiple cryptic species.

The ciliate protozoan Cryptocaryon irritans parasitizes marine fish and causes lethal white spot disease. Sporadic infections as well as large-scale outbreaks have been reported globally and the parasite's broad host range poses particular threat to the aquaculture and ornamental fish markets. In order to better understand C. irritans' population structure, we sequenced and compared mitochondrial cox-1, SSU rRNA, and ITS-1 sequences from 8 new isolates of C. irritans collected in China, Japan, and Taiwan. We detected two SSU rRNA haplotypes, which differ at three positions, separating the isolates into two main groups (I and II). Cox-1 sequences also support the division into two groups, and the cox-1 divergence between these two groups is unexpectedly high (9.28% for 1582 nucleotide positions). The divergence is much greater than that detected in Ichthyophthirius multifiliis, the ciliate protozoan causing freshwater white spot disease in fish, where intraspecies divergence on cox-1 sequence is only 1.95%. ITS-1 sequences derived from these eight isolates and from all other C. irritans isolates (deposited in the GenBank) not only support the two groups, but further suggest the presence of a third group with even greater sequence divergence. Finally, a small Ka/Ks ratio estimated from cox-1 sequences suggests that this gene in C. irritans remains under strong purifying selection. Taken together, the C. irritans species may consists of many subspecies and/or syngens. Further work is needed to determine if there is reproductive isolation between the groups we have defined.

Immobilization antigen vaccine adjuvanted by parasitic heat shock protein 70C confers high protection in fish against cryptocaryonosis.

The immobilization antigen (iAg) has been demonstrated as a protective immunogen against Cryptocaryon irritans infection. In this study, C-terminal domain of heat shock protein 70 cloned from C. irritans (Hsp70C) was tested for its immuno-stimulatory effects. The iAg and Hsp70C cDNAs were constructed independently in secretory forms and were encapsulated in chitosan nanoparticles. In the first immunization trial, grouper fingerlings orally intubated with iAg and iAg:Hsp70C presented 96% and 100% relative percent survival (RPS), respectively, after a lethal challenge. In the second trial, both iAg and iAg:Hsp70C groups showed 100% RPS and the skin trophont burden was significantly lowered. The iAg:Hsp70C still provides a significantly high protection of 51% RPS at 49 days post immunization, when an even more serious lethal infection occurs. RT-qPCR results showed that Hsp70C could up-regulate the expression of i) T cell markers: Cluster of Differentiation 8 alpha (CD8α) and CD4, ii) cytokine genes: Interferon gamma (IFNγ), Tumor Necrosis Factor alpha (TNFα) and Interleukin 12 p40 (IL-12/P40), iii) antibody genes: Immunoglobulin M heavy chain (IgMH) and IgTH, and iv) major histocompatibility complex (MHC-I & MHC-II), in the spleen of iAg:Hsp70C group. Furthermore, significantly high levels of iAg-specific IgM was detected in skin mucus which efficiently immobilized live theronts in iAg- and iAg:Hsp70C-immunized fish at 5 weeks post immunization. Hsp70C significantly increased the number of nonspecific CD8(+) skin leucocytes which exerted cytotoxicity against theronts, although cytotoxic activity showed no difference among the various groups. Because of this complementary cooperation of cellular and humoral immune responses, Hsp70C enhances the efficacy of iAg vaccine and constrains C. irritans infection. In view of the severe loss caused by cryptocaryonosis, application of this parasitic vaccine in farmed and ornamental fish, is worthy to be considered.

Grouper interleukin-12, linked by an ancient disulfide-bond architecture, exhibits cytokine and chemokine activities.

Interleukin-12 (IL-12) is a pleiotropic cytokine which bridges innate and adaptive immunity in defense against pathogens. IL-12 proved to be an effective and successful adjuvant to enhance both the innate and adaptive immune responses and could be applicable for a rationale vaccine formulation in fish against pathogen infection. We have cloned the p35 and p40 cDNAs of IL-12 from orange-spotted grouper (Epinephelus coioides). Grouper IL-12 most resembles with sea bass orthologues; moderate to low identity with other teleost and mammalian counterparts. The structural model of grouper IL-12 heterodimer revealed NC(141)F three amino acid patch of grouper p35, which is present in teleost p35 but absent in mammalian and avian p35, and is spatially nearby the conserved cysteine residue located at A-helix of p35 to form a disulfide bond when the 14aa peptide located at loop 1 of grouper p35 was aligned with human corresponding exon 4, instead of exon 5. The results indicated that the loss of this 3aa patch during evolution was compensated by the duplication of exon 4 in mammalian p35 to gain another cysteine residue to form a disulfide bond, evidenced by chicken p35 which does not contain NCF corresponding 3-aa patch nor exon 4 duplication. Accordingly, the inter-chain disulfide bond of IL-12 heterodimer is conserved from teleost to mammalian IL-12. A single chain grouper IL-12 (scgIL-12) construct linked by (G4S)3 was successfully expressed in baculovirus-insect cell system; its identity has been confirmed by LC/MS/MS. In addition, the biological activity of recombinant scgIL-12 (rscgIL-12) are demonstrated for its stimulation of PBL proliferation, chemotactic migration, induction of TNF-α gene expression and a plausible adjuvant effect of prolonged protection against parasite infection in fish. We illustrated the first time in lower vertebrate that grouper IL-12 possesses both cytokine and chemokine activities.

The new face of the old molecules: crustin Pm4 and transglutaminase type I serving as rnps down-regulate astakine-mediated hematopoiesis.

Astakine is an important cytokine that is involved in crustacean hematopoiesis. Interestingly, the protein levels of astakine increased dramatically in plasma of LPS-injected shrimp while mRNA levels remained unchanged. Here, we investigated the involvement of astakine 3'-untranslated region (UTR) in its protein expression. The 3'-UTR of astakine down-regulated the expression of reporter protein but the mRNA stability of reporter gene was unaffected. We identified the functional regulatory elements of astakine 3'-UTR, where 3'-UTR242-483 acted as repressor. The electrophoresis mobility shift assay (EMSA), RNA pull-down assay and LC/MS/MS were performed to identify the protein association. We noted that crustin Pm4 and shrimp transglutaminase I (STG I) were associated to astakine 3'-UTR242-483, while two other proteins have yet to be revealed. Depletion of hemocytic crustin Pm4 and STG I significantly increased the protein level of astakine while astakine mRNA level remained unaffected. Lipopolysaccharide (LPS) stimulated the secretion of crustin Pm4 and STG I from hemocytes to plasma and increased the astakine level to stimulate the hemocytes proliferation. Altogether, we identified the shrimp crustin Pm4 and STG I as novel RNA binding proteins that play an important role in down-regulating astakine expression at post-transcriptional level and are crucial for the maintenance of hematopoiesis.

An evolutionarily ancient NO synthase (NOS) in shrimp.

Nitric oxide (NO) is a well known essential molecule that is involved in multiple functions such as neuron transduction, cardiac disease, immune responses, etc.; nitric oxide synthase (NOS) is a critical enzyme that catalyzes the synthesis of it. A very few crustacean NOS molecules were biochemically characterized so far. In the present study, we cloned and characterized a NOS cDNA from haemocytes of tiger shrimp (Penaeus monodon) (PmNOS). The full-length of PmNOS cDNA contained 3997 bp, including a 5'UTR of 249 bp, ORF of 3582 bp and a 3'UTR of 166 bp. The putative peptide was 1193 amino acid residues in length, with an estimated molecular weight of 134.7 kDa and pI 6.7. Structurally, PmNOS contained oxygenase and reductase domains at N-terminal and C-terminal, respectively, and connected with a calmodulin binding motif. The deduced amino acid sequence of PmNOS shared 98% identical to the Chinese shrimp (Fenneropenaeus chinensis) NOS. Phylogenetically, PmNOS clustered with invertebrate NOS, but not clustered with iNOS, eNOS or nNOS found in vertebrates. PmNOS mRNA was expressed in many tissues or organs including thoracic and ventral nerves, midgut, gill, eyestalk, haemocytes, subcuticular epithelium and heart, but not found in hepatopancreas, muscle and lymphoid organ. But there was no significant difference in PmNOS mRNA expression after stimulation with LPS either by different concentration or time course or against CpG-ODN 2006. The enzyme activities of rPmNOS or crude homogenates from different tissues were detected, and were shown its highest activity in thoracic and ventral nerves, moderate in midgut and haemocytes but the lowest activity were seen in muscle. The addition of NOS antibody against NADPH binding domain leads to less activity which suggested that NADPH was an essential cofactor for PmNOS catalytic activity. The calcium dependency of PmNOS was ascertained using calmodulin inhibitor, Trifluroperazine. To confirm the population of haemocyte which produce NOS, the florescence test was assayed, and it implicated that the production of NO was catalyzed by subset of granulocytic NOS. Since the MW range, inducible/noninducible transcript, calcium-dependent activity and tissue distribution, we suggest that PmNOS may recognize as an ancient NOS evolutionarily.

Codon changed immobilization antigen (iAg), a potent DNA vaccine in fish against Cryptocaryon irritans infection.

The immobilization antigen (iAg) DNA sequence from Chiayi isolate of Cryptocaryon irritans was computationally reviewed to replace the stop codons with suitable amino acids and its GC content was intensified. The plasmid construct comprising the codon changed iAg (optiAg/optimized iAg) was successfully expressed in the bacterial strain BL21 and also in grouper fin cells (GF-1). Results of immobilization assay, ELISA and western blot of C. irritans theront and recombinant iAg by grouper antiserum against optiAg DNA indicated that the codon changed iAg retains the native conformation. The DNA vaccine construct pcDNA3.1-optiAg was encapsulated in water-oil-water triple layer emulsions measuring 19 µm diameters and was used for the immunization experiment. In trial I experiment, grouper fish were immunized twice via intramuscular injection with the pcDNA3.1-optiAg and were challenged with C. irritans at 8-day post immunization (dpi), which resulted in 46% relative percent survival (RPS). In trial II, single immunization with pcDNA3.1-optiAg boosted with recombinant iAg protein, resulted in 40% RPS. The data from this study reveal that codon change in iAg not only accomplished the expression of iAg protein in both prokaryotic and eukaryotic cell systems, but also optiAg was proved as immunogenic due to the protection it confers to the immunized fish against C. irritans infection. Hence, it is concluded that iAg can be a potent DNA vaccine in fish against infection of the ciliated protozoan, C. irritans.

Cytotoxic CD8α+ leucocytes have heterogeneous features in antigen recognition and class I MHC restriction in grouper.

CD8 is a membrane glycoprotein found primarily on the surface of T lymphocytes such as cytotoxic T lymphocytes (CTL), natural killer cells (NK) and γδ T lymphocytes. It helps T lymphocytes to kill the infected cells that presents microbial antigen on the cell surface. However, analysis of fish cellular immunity has been limited because of the lack of CD8 antibodies in grouper. In this present study, we cloned full-length CD8α cDNAs from orange-spotted grouper (Epinephelus coioides), an important fish species economically. The deduced protein of CD8α contained 227 amino acid residues in length and included one signal peptide, Ig superfamily V domain, hinge region, transmembrane domain, cytoplasmic tail and conserved binding motif associated with tyrosine kinase p56(lck). The molecular weight of the mature protein was estimated at 22.5 kDa and pI at 9.55. Phylogenetically, the predicted grouper CD8α protein was similar to CD8α from other marine fish species in which the identity was 50-60%. Real-time PCR revealed that CD8α transcript was constitutively expressed in thymus, head kidney, gill, spleen, gut and peripheral blood leucocyte (PBL); and the highest expression in thymus. CD8α transcript in the spleen of fish injected with nervous necrosis virus (NNV) was significantly up-regulated at 4 days post-injection compared to the untreated fish. Rabbit antiserum prepared against recombinant CD8α protein was able to recognize specifically the subset lymphocytes which have a diameter of 7 µm, a high nucleus/cytoplasm ratio and a ring-shaped cytoplasm. The cytotoxicity of CD8α(+) lymphocytes at one-week post-NNV infection was enhanced significantly against NNV-infected autologous fin cells in comparison with NNV-infected allogeneic or RSIV-infected autologous fin cells. Flow cytometry analysis revealed that both the number and mean fluorescence intensity (MFI) of CD8α(+) PBL were significantly increased at 7 days post-NNV infection. The specific cytotoxicity and MHC class I restriction of the lymphocytes sorted by rCD8α antibody are properties that can be attributed to CTL. In addition, low level of cytotoxicity was found in PBL against allogeneic targets as well as CD8α(+) effectors killed autologous targets nonspecifically, implicated presence of cytotoxic T subsets, possibly nonspecific cytotoxic cells (NCC) and γδ T lymphocytes, without MHC class I restriction. In conclusion, grouper cytotoxic CD8α(+) PBL have heterogeneous features in specific antigen recognition and class I MHC restriction.

Proline-rich domain of penaeidin molecule exhibits autocrine feature by attracting penaeidin-positive granulocytes toward the wound-induced inflammatory site.

Antimicrobial peptides (AMPs) are amphipathic structures of low molecular weight that are generally positively charged. Penaeidins are shrimp-specific AMPs that are synthesized and stored in granulocytes and released after stimulation. Penaeidin is composed of an N-terminal proline-rich domain (PRD) and a C-terminal cysteine-rich domain (CRD). Penaeidin and PRD were approved as a cytokine in vitro, but how penaeidin regulates hemocyte adhesion in vivo is unclear. The present study examines penaeidin immunomodulation function in the wound-induced inflammation response in tiger shrimp (Penaeus monodon). Both penaeidin transcript and protein decreased in peripheral hemocytes but increased in the wound tissue. The wounded tissue sections were compared in penaeidin normal and penaeidin knockdown shrimps. Only the shrimps at normal penaeidin expression level present the concentration of hemocytes phenomenon in the wounded tissue at 2h post-wound. This phenomenon was recovered in the penaeidin knockdown shrimps by adding recombinant penaeidin or PRD to the wounded tissue. Penaeidin was also found to be simultaneous expression of integrin in vivo. Penaeidin-positive granulocytes decreased in the peripheral hemolymph post-wound. The hemocytes that concentrated in the wounded tissue were 80% penaeidin-positive granulocytes. We propose that penaeidin acts as a pro-inflammatory cytokine and attracts penaeidin-positive granulocytes toward the inflammatory site by autocrine activity through integrin-dependent cell migration.

A long form of shrimp astakine transcript: molecular cloning, characterization and functional elucidation in promoting hematopoiesis.

Hemocytes play important roles in crustacean immune responses. Generation of new hemocytes (hematopoiesis) is thus necessary to maintain homeostasis which is vital to crustaceans. In vertebrates, certain cytokines have been demonstrated to regulate hematopoiesis and immune responses. In invertebrates, however, little is known about cytokines related to hematopoiesis. In the present study, we cloned an astakine molecule from hemocytic cDNA of tiger shrimp (Penaeus monodon) which was 1509 bp in length with a 5'-UTR of 143 bp, a coding region of 375 bp and a 3'-UTR of 991 bp. The present clone (GenBank accession no. EU980444) showed to be a longer form of astakine transcript with an extra insert of 671 bp in the 3'-UTR than the NCBI-recorded shrimp astakine cDNA sequence (GenBank accession no. AY787657). The deduced protein had 124 amino acid residues, including a signal peptide and one prokineticin domain. The calculated molecular weight (MW) of the mature peptide was 11,295 Da and pI was 5.2. Phylogenetically, this molecule is most similar to astakine-related molecules of arthropod including tiger shrimp astakine, crayfish astakines 1, 2a and 2b, aphid astakine-like molecule and parasitic wasp astakine-like molecule. Nested RT-PCR showed that astakine mRNA is expressed in many tissues and organs of the shrimp such as eyestalk, subcuticular epithelium, gills, heart, hepatopancreas, lymphoid organ, intestine, muscle, nerve and hemocytes. Real-time PCR further revealed that astakine mRNA is expressed mainly in the hemocytes. The astakine transcript is not inducible in the hemocytes until 24 h post LPS injection of shrimp. The recombinant protein of shrimp astakine (rPmAst) was synthesized using insect cell-baculovirus expression system. The authenticity of rPmAst protein was examined by MALDI-MS/MS spectrometry. Using ESI-MS it was determined that the MW of C-terminally histidine-tagged recombinant protein is 12,107 Da. It is 10 Da less than the computer-predicted MW (12,117 Da), allowing the formation of five pairs of disulfide bonds. Using BrdU incorporation assay it was demonstrated that the injection of rPmAst to the shrimp promoted cell proliferation in hematopoietic tissues. Therefore, we conclude that shrimp astakine functions as a cytokine that influences cell proliferation in the hematopoietic tissues.

Tiger shrimp (Penaeus monodon) penaeidin possesses cytokine features to promote integrin-mediated granulocyte and semi-granulocyte adhesion.

Recent studies indicated that antimicrobial peptides (AMPs) play multiple roles in both innate and adaptive immune functions. The penaeidin of tiger shrimp Penaeus monodon shows an antimicrobial activity against Gram-positive bacteria and filamentous fungi. To study immunomodulation functions of the penaeidin, we transfected shrimp hemocytes in primary culture with penaeidin-specific small interfering RNA (siRNA-3) and observed a concomitant 20% reduction in adhesive hemocytes compared with mock-transfected cells. The addition of biosynthesized or chemically synthesized penaeidin or penaeidin proline-rich domain (PRD) to the culture medium of penaeidin knock-down hemocytes led to a full recovery in the number of adhesive hemocytes. The effect of penaeidin knock-down on the expression of tiger shrimp cell adhesion-associated molecules was examined using real-time Q-PCR. Results demonstrated 91% and 64% decreases in the expression of integrin-beta and collagen, respectively, and a 396% increase in the expression of collagenase. The addition of chemically synthesized penaeidin after penaeidin knock-down hemocytes normalized the expression of these genes. The addition of the integrin-beta ligand competitor RGDS to mock-transfected hemocytes decreased the number of adhesive hemocytes similar to penaeidin knock-down. In conclusion, penaeidin possesses an integrin-beta-mediated cytokine feature that promotes shrimp granulocyte and semi-granulocyte adhesion. This is the first report about functional shrimp cytokine.

Cloning of penaeidin gene promoter in tiger shrimp (Penaeus monodon).

Penaeidins belong to a family of antimicrobial peptides that are expressed in the hemocytes of penaeid shrimps. Using an extender PCR method and a nested PCR, we cloned two types of genomic fragment flanking the 5' end of penaeidin gene in tiger shrimp (Penaeus monodon): Type536 and Type411 sequences. Both fragments contained TATA box, GATA, dorsal and AP-1 motifs and were ligated to an expression vector with a luciferase reporter gene. The constructs were then delivered into Drosophila S2 cell line. The promoter functions of the two fragments were determined using a luciferase expression assay. The study demonstrated that Type411 sequence performed higher transcriptional activity than Type536. Alignment of the upstream sequences of penaeidin genes in P. monodon and Litopenaeus vannamei showed that the promoter regions were obviously more diverse than the 5'UTRs. Phylogenetic analysis indicated the presence of two types of promoters that are not species-specific in the two shrimps.

Cloning and expression analysis of heat shock cognate 70 gene promoter in tiger shrimp (Penaeus monodon).

Heat shock cognate 70 (HSC70) functions as a molecular chaperon and plays an important role in protein folding. HSC70 cDNA of tiger shrimp (Penaeus monodon) was cloned and characterized in our previous study. After shrimps were treated with the 1-hr heat shock, the HSC70 mRNA level in hemocytes increased (approximately 8 fold) using real-time quantitative PCR. An hsc70 clone was obtained from genomic library screening. The gene contains 2 exons separated by a 1557-bp intron. The 5'-flanking region sequence (approximately 1 kb) ahead of the hsc70 gene contains a putative core promoter region and transcription elements including perfect heat shock element (HSE), imperfect HSE, CAAT elements, SP1, NF-kappaB and GC box. In insect Sf21 cells, the region could drive expression of the enhanced green fluorescent protein (EGFP) and luciferase gene to verify its promoter function. In the luciferase assay system, the effects of serial deletions on the hsc70 promoter were elucidated. Autographa californica multiple nuclear polyhedrosis virus infection (MOI=0.1) on Sf21 cells significantly increased the hsc70 promoter activity. In addition, the effects of amino acid analogs and arsenic acid incubation with the cells on the hsc70 promoter activity were examined.

Cloning and characterization of a shrimp clip domain serine protease homolog (c-SPH) as a cell adhesion molecule.

Clip domain serine protease homologs (c-SPHs) are involved in various innate immune functions in arthropods such as antimicrobial activity, cell adhesion, pattern recognition, opsonization, and regulation of the prophenoloxidase system. In the present study, we cloned a c-SPH cDNA from tiger shrimp (Penaeus monodon) hemocytes. It is 1337 bp in length with a coding region of 1068 bp consisting a protein of 355 amino acid residues. The deduced protein includes one clip domain and one catalytically inactive serine protease-like (SP-like) domain. Its molecular weight is estimated to be 38 kDa with an isoelectric point of 7.9. The predicted cutting site of the signal peptide is located between Gly(21) and Gln(22). We aligned 15 single clip domain SPH protein sequences from 12 arthropod species; the identity of these clip domains is low and that of SP-like domains is from 34% to 46%. The conserved regions are located near the amino acid residues which served as substrate interaction sites in catalytically active serine protease. Phylogenetically, the tiger shrimp c-SPH is most similar to a low molecular mass masquerade-like protein of crayfish, but less similar to c-SPHs in Chelicerata and Insecta. Nested reverse transcription polymerase chain reaction (RT-PCR) revealed that c-SPH mRNA is expressed most in tissues with the highest hemocyte abundance. Antimicrobial and opsonization activities of the molecule were not detected. The expression of c-SPH mRNA in hemocytes was up-regulated at the 12-day post beta-glucan immersion. Recombinant c-SPH could significantly enhance hemocyte adhesion. The result suggests that the shrimp c-SPH protein plays a role in innate immunity.

More than one type of transglutaminase in invertebrates? A second type of transglutaminase is involved in shrimp coagulation.

Coagulation (clot formation) forms a physical barrier to prevent the loss of body fluid and dissemination of microbes into the haemocoel after injury or infection. Its quickness and efficiency are essential for the survival of invertebrates that rely solely on innate immunity. Transglutaminase (TG) catalyses intermolecular or intramolecular epsilon-(gamma-glutamyl) lysine bond formation, resulting in a protein polymerisation, and plays a role in blood coagulation and post-translational protein remodelling. In the present study, we cloned a TG from shrimp (Penaeus monodon) haemocyte cDNA. It was assigned as shrimp transglutaminase II (STG II). The STG II cDNA consists of a coding region of 2,274bp. The deduced protein has 757 amino acid residues with a calculated molecular mass of 85,000 Da and an isoelectric point of 5.48. RT-PCR results showed a significant level of STG II expression in haemocytes but not in hepatopancreas, in contrast to shrimp STG I (AY074924.1). The genetic distance between STG II and STG I is much larger than the distance between STG II and the TG of the kuruma shrimp (Marsupenaeus japonicus). Evidence based on tissue distribution and genetic distance suggests that no less than two types of shrimp TG exist that are encoded at different chromosomal locations. The recombinant STG II (rSTG II) incorporated a TG-specific substrate, dansylcadaverine (DCA), into clottable proteins (CP) in a calcium dependent manner. Other haemocyte- or plasma-derived TG substrate is not required for CP polymerisation but may be necessary for stable clot formation. The rSTG II catalysed clottable proteins into a long chain under transmission electron microscopy (TEM) observation. In conclusion, STG II is characterized as a haemocyte TG and is involved in coagulation.

Molecular cloning and characterization of tiger shrimp (Penaeus monodon) transglutaminase.

Transglutaminases (TG) are important for blood coagulation and post-translation remodeling of proteins. Using a plaque screening assay, we isolated cDNA encoding a novel TG from a shrimp (Penaeus monodon) hemocyte cDNA library. The TG cDNA consists of 2988 bp with an open reading frame of 2271 bp. The deduced protein has 757 amino acid residues, a calculated molecular mass of 84,713 Da and an isoelectric point of 5.56. Neither a typical hydrophobic leader sequence nor a transmembrane domain could be identified from the deduced sequence. Thus, shrimp TG may be a typical cytoplasmic protein. The sequence of shrimp TG was similar to crayfish, other invertebrate and vertebrate TG sequences. Enzyme activity was detected in all organs tested. This is consistent with the widespread, low-level expression of TG mRNA. However, high levels of TG expression were detected in hematopoietic tissue. TG signals were stronger in mitotic cells, indicating that cell proliferation and TG synthesis are associated. Preliminary data showed that recombinant TG existed the enzyme activity but lacked coagulation activity.

Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon).

We cloned the complementary deoxyribonucleic acid (cDNA) of the heat shock cognate 70 (hsc70) gene of tiger shrimp (Penaeus monodon). It was 2207 bp long and included a 1959-bp coding region, a 40-bp flanking region at the 5' end, and a 208-bp flanking region at the 3' end. The deduced, 652-amino acid sequence had a molecular mass of 71 481 Da and an estimated isoelectric point (pI) of 5.2. Based on phylogenetic analysis, the gene is clustered with the hsc70 proteins of invertebrates and vertebrates. In native gel electrophoresis, recombinant P. monodon hsc70 expressed in an Escherichia coli system is tightly associated with carboxymethylated alpha-lactalbumin (CMLA), which indicates that hsc70 probably functions as a chaperone. In an in vitro adenosine triphosphatase assay, recombinant hsc70 hydrolyzed adenosine triphosphate to adenosine-5'-diphosphate and increased hydrolysis activity by binding to unfolded peptide, CMLA. In situ hybridization using an antisense riboprobe revealed that the hsc70 gene was active in most tissues of unstressed shrimp. The expression of hsc70 messenger ribonucleic acid (mRNA) in hemocytes increased 2- to 3-fold at the first hour after shrimp experienced heat shock and 0.5-hour recovery. Hsc70 mRNA decreased gradually to the background level. Cloning and characterizing the P. monodon hsc70 gene is the first, crucial step in studying the relationship of heat shock proteins with the stress or immune responses of shrimp.

Characterization of Cryptocaryon irritans, a parasite isolated from marine fishes in Taiwan.

The ciliated protozoan parasite Cryptocaryon irritans infecting marine fishes in Taiwan is described. Developmental characteristics and sequences of the ribosomal DNA regions such as part of 18 S, the entire first internal transcribed spacer, and part of 5.8 S of various Taiwan isolates of C. irritans were investigated. A total of 5 isolates was obtained from different fish-host species and localities, the majority from cultured fish species. C. irritans from Taiwan is able to shift its developmental characteristics, i.e. from non-adherent to adherent tomonts, from individualistic to aggregate-forming tomonts, from infection of the gills only to infection of the gills and body. Thus, it is not possible to classify strains of C. irritans on the basis of these parameters. Premature tomonts that developed from dead fishes were able to produce theronts that could infect fish host. Isolates from Pingtung and the USA had identical nucleotide sequences while an isolate from Malaysia was identical to an Israel isolate. Percentage variation among pairs of Taiwan isolates showed a higher degree of variation than isolate sequences listed in GenBank. Sequence analysis revealed highly aberrant isolates in Taiwan, and a phylogenetic tree distinguished a marine and a low-salinity variant. C. irritans from marine fishes in Taiwan, therefore, display some characteristics not previously reported. Since manipulation of salinity in brackishwater ponds and marine cage sites is not feasible, there is a need to develop new strategies for the control and prevention of cryptocaryoniasis.

Evaluation of dorsal aorta cannulation for immunological studies of grouper (Epinephelus malabaricus).

Blood is often withdrawn to study the immune responses of fish. However, netting, handling and anaesthetising the experimental fish, and drawing blood samples cause severe stress that may alter the effects of immune study protocols and treatments. We evaluated the effect of aorta cannulation, for use in immune studies, on grouper (Epinephelus malabaricus) plasma cortisol, total red and white blood cell counts and phagocytosis. Plasma cortisol increased from 30 to 88 ng/ml 1 day after insertion of the cannula, to a maximum of 951 ng/ml 3 to 5 days after surgery, indicating the groupers were stressed by cannulation and post-cannulation inflammation. Total RBC count decreased, and total WBC count increased after surgery. Following cannulation, the phagocytic index of peripheral blood leukocytes decreased from 100% to 46%. The adverse effects of cannulation were mitigated by continuously immersing groupers in oxytetracycline (OTC), which decreased the recovery period for treated fish. In contrast, OTC-treatment did not markedly improve the recovery of groupers subjected to caudal vessel puncture. Cortisol levels in OTC-treated grouper with caudal vessel puncture were significantly higher than in OTC-treated, cannulated grouper, and remained at a high level until day 13 of the experiment. From day 7 to 13, total RBC and WBC counts in OTC-treated, cannulated groupers were significantly different from those in OTC-treated groupers with caudal vessel puncture. OTC treatment improved the phagocytic index of groupers subjected to caudal vessel puncture, but the phagocytic index was lower than that of groupers subjected to cannulation. Cannulation minimises visual and handling disturbances, and facilitates standardisation of experimental conditions and quick and easy sampling via the dorsal aorta cannula. Therefore, dorsal aorta cannulation minimises the stress of blood sampling and should prove useful for immune studies in fish.