The intracellular signaling pathway of octopamine upregulating immune resistance functions in Penaeus monodon.

Octopamine (OA), a biogenic monoamine, is known to mediate several immune responses. This study analyzed the effects of OA on immunological regulation in the tiger shrimp Penaeus monodon. The immune parameters including total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and phagocytic activity and clearance efficiency in response to the pathogen, Photobacterium damselae, were determined when shrimp were individually injected with saline or OA at 100 or 1000 pmol shrimp-1. In addition, the intracellular second messengers in haemocyte such as Ca2+ and adenosine 3',5'-cyclic monophosphate (cAMP) were examined in shrimp receiving saline or OA at 1 or 10 nmol shrimp-1. Results showed that all of the immune parameters significantly increased at 2-4 h in OA-injected shrimp except hyaline cells in 100 pmol shrimp-1-injected shrimp at 4 h, but phenoloxidase activity per granulocyte significantly decreased at 2-4 h. However, these had returned to saline control levels after receiving OA for 8 h except differential haemocyte count and phenoloxidase activity per granulocyte for 16 h. An injection of OA also significantly increased the survival rate of shrimp challenged with Pho. damselae. Shrimp receiving OA at 1 and 10 nmol shrimp-1 significantly increased the intracellular Ca2+ concentration ([Ca2+]i) at 30-60 min and 30 min, and cAMP concentration [cAMP]i) at 5-15 min and 15 min, respectively. However, [Ca2+]i at 50-60 min, and [cAMP]i at 30-60 min returned to saline control when the shrimp received OA at 10 nmol shrimp-1, and at 1 and 10 nmol shrimp-1, respectively. These results suggest that OA administration by injection at ≤1000 pmol shrimp-1 mediates transient upregulation of immunity together with the increased resistance of P. monodon to Pho. damselae, which are modulated through intracellular Ca2+ and cAMP second messenger pathways.

The known two types of transglutaminases regulate immune and stress responses in white shrimp, Litopenaeus vannamei.

Transglutaminases (TGs) play critical roles in blood coagulation, immune responses, and other biochemical functions, which undergo post-translational remodeling such as acetylation, phosphorylation and fatty acylation. Two types of TG have been identified in white shrimp, Litopenaeus vannamei, and further investigation on their potential function was conducted by gene silencing in the present study. Total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, transglutaminase (TG) activity, haemolymph clotting time, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when shrimps were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or TG dsRNAs. In addition, haemolymph glucose and lactate, and haemocytes crustin, lysozyme, crustacean hyperglycemic hormone (CHH), transglutaminaseI (TGI), transglutaminaseII (TGII) and clotting protein (CP) mRNA expression were determined in the dsRNA injected shrimp under hypothermal stress. Results showed that TG activity, phagocytic activity and clearance efficiency were significantly decreased, but THC, hyaline cells (HCs) and haemolymph clotting time were significantly increased in the shrimp which received LvTGI dsRNA and LvTGI + LvTGII dsRNA after 3 days. However, respiratory burst per haemocyte was significantly decreased in only LvTGI + LvTGII silenced shrimp. In hypothermal stress studies, elevation of haemolymph glucose and lactate was observed in all treated groups, and were advanced in LvTGI and LvTGI + LvTGII silenced shrimp following exposure to 22 °C. LvCHH mRNA expression was significantly up-regulated, but crustin and lysozyme mRNA expressions were significantly down-regulated in LvTGI and LvTGI + LvTGII silenced shrimp; moreover, LvTGII was significantly increased, but LvTGI was significantly decreased in LvTGI silenced shrimp following exposure to 28 and 22 °C. Knockdown of LvTGI and LvTGI + LvTGII also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. The same consequences have been confirmed in LvTGII silenced shrimp in our previous study. These results indicate that LvTGI and LvTGII not only reveal a complementary effect in gene expression levels but also play a key function in the immune defence mechanism of shrimp, by regulating the haemolymph coagulation, immune parameters and immune related gene expression, and in the regulation of carbohydrate metabolism.

Immune responses of prophenoloxidase and cytosolic manganese superoxide dismutase in the freshwater crayfish Cherax quadricarinatus against a virus and bacterium.

Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cytMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of the above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus. A phylogenetic analysis of the amino acid sequences showed that C. quadricarinatus proPO and cytMnSOD were more closely related to the proPO and cytMnSOD of other crayfish than to those of penaeids, crabs, lobsters, or freshwater prawns. A tissue distribution analysis revealed that proPO was primarily expressed in hemocytes, gills, and the heart, while cytMnSOD was detected in all tissues examined. All of the crayfish artificially infected with white spot syndrome virus (WSSV) died within 4 days. According to a non-lethal dose, there was no mortality in crayfish when infected deliberately with Aeromonas hydrophila. Total hemocyte counts (THCs) had significantly decreased in crayfish at 48 and 72 h after infection with WSSV compared to the control group. In contrast, THCs of crayfish after A. hydrophila challenge had recovered by 48 and 72 h from a lower level at 24 h. There were similar responses in enzyme activities toward WSSV and A. hydrophila infection. Phenoloxidase (PO) and superoxide dismutase (SOD) activities per hemocyte significantly increased from 48 to 72 h compared to the control group. After WSSV challenge, expressions of proPO and cytMnSOD transcripts in hemocytes significantly decreased at 12h, then had respectively recovered and increased at 24 h. At 48-72 h, transcript levels were finally downregulated. No significant differences in the expression profiles of proPO and cytMnSOD were observed between the A. hydrophila-infected and control groups, besides the significant upregulation at 24h post-infection. These results implicate proPO and cytMnSOD in the immune response, and they presented similar expression patterns, although different defense mechanisms may exist for crayfish induced by WSSV and A. hydrophila.

Identification and cloning of a selenophosphate synthetase (SPS) from tiger shrimp, Penaeus monodon, and its transcription in relation to molt stages and following pathogen infection.

Complementary (c)DNA encoding selenophosphate synthetase (SPS) messenger (m)RNA of the tiger shrimp Penaeus monodon, designated PmSPS, was obtained from the hepatopancreas by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The 1582-bp cDNA contained an open reading frame (ORF) of 1248 bp, a 103-bp 5'-untranslated region (UTR), and a 231-bp 3'-UTR, which contained a conserved selenocysteine insertion sequence (SECIS) element, a conventional polyadenylation signal, and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (416 aa) was 45.5 kDa with an estimated pI of 4.85. It contained a putative selenocysteine residue which was encoded by the unusual stop codon, (275)TGA(277), which formed at the active site with residues Sec(58) and Lys(61). A comparison of amino acid sequences showed that PmSPS was more closely related to invertebrate SPS1, such as those of Heliothis virescens and Drosophila melanogaster a and b. PmSPS cDNA was synthesized in all tested tissues, especially in the hepatopancreas. PmSPS in the hepatopancreas of shrimp significantly increased after an injection with either Photobacterium damsela or white spot syndrome virus (WSSV) in order to protect cells against damage from oxidation, and enhance the recycling of selenocysteine or selenium metabolism, indicating that PmSPS is involved in the disease-resistance response. The PmSPS expression by hemocytes significantly increased in stage C, and then gradually decreased until stage A, suggesting that the cloned PmSPS in hemocytes might play a role in viability by renewing hemocytes and antioxidative stress response for new exoskeleton synthesis during the molt cycle of shrimp.

Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp, Penaeus monodon, and its transcription following pathogen infection and related to the molt stages.

Complementary (c)DNA encoding glutathione peroxidase (GPx) messenger (m)RNA of the tiger shrimp Penaeus monodon was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. The 1321-bp cDNA contained an open reading frame (ORF) of 564bp, a 69-bp 5'-untranslated region (UTR), and a 688-bp 3'-UTR containing a poly A tail and a conserved selenocysteine insertion sequence (SECIS) element. The molecular mass of the deduced amino acid (aa) sequence (188 aa) was 21.05kDa long with an estimated pI of 7.68. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, (190)TGA(192), and forms the active site with residues Glu(75) and Trp(143). Comparison of amino acid sequences showed that tiger shrimp GPx is more closely related to vertebrate GPx1, in accordance with those in Litopenaeus vannamei and Macrobrachium rosenbergii. GPx cDNA was synthesised in lymphoid organ, gills, heart, haemocytes, the hepatopancreas, muscles, and intestines. After injected with either Photobacterium damsela or white spot syndrome virus (WSSV), the respiratory bursts of shrimp significantly increased in order to kill the pathogen, and induced increases in the activities of superoxide dismutase and GPx, and regulation in the expression of cloned GPx mRNA to protect cells against damage from oxidation. The GPx expression significantly increased at stage D(0/1), and then gradually decreased until stage C suggesting that the cloned GPx might play a role in the molt regulation of shrimp.

Identification and cloning of a selenium dependent glutathione peroxidase from giant freshwater prawn, Macrobrachium rosenbergii.

A selenium dependent glutathione peroxidase (Se-GPx) cDNA was cloned from haemocyte by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA (RACE). The 913 bp cDNA contained an open reading frame (ORF) of 558 bp encoded a deduced amino acid sequence of 186 amino acids. The prawn Se-GPx sequence contains a selenocysteine (Sec) residue which is encoded by the unusual stop codon, (115)TGA(117). According to the molecular modeling analysis, the active site Sec residue, located in the loop between beta3 and alpha2 in a pocket on the protein surface, and hydrogen bonded to Gln(73) and Trp(141). A GPx signature motif 2, (63)LAFPCNQF(70) and active site motif, (151)WNFEKF(156), two arginine (R) residues, R(89) and R(167) contribute to the electrostatic architecture that directs the glutathione donor substrate, and two putative N-glycosylation site, (75)NNT(77) and (107)NGS(109) were observed in the prawn Se-GPx sequence. In addition, the eukaryotic selenocysteine insertion sequence element is conserved in the 3'-UTR. Comparison of amino acid sequences showed that prawn Se-GPx is more closely related to vertebrate GPx 1. The prawn Se-GPx was synthesized in haemocyte, hepatopancreas, muscle, stomach, gill, intestine, eyestalk, heart, epidermis, lymph organ, ventral nerve cord, testis and ovary. The increase of respiratory burst in haemocyte was observed in pathogen, Debaryomyces hansenii-injected prawn in order to kill the pathogen, and the up-regulation in SOD and GPx acitivity, and prawn Se-GPx mRNA transcription were involved with the protection against damage from oxidation.