Activation mechanism of pre-phenoloxidase in lobster and shrimp

Phenoloxidase in shrimp and lobster is shown to exist in latent form which could be activated by trypsin and by an endogenous enzyme with tryptic activity. On Sephadex G-100 gel, three isoenzymes, differing in molecular weights, were isolated from naturally activated lobster shell extracts. Mechanism of activation of ptephenoloxidase involving limited proteolysis by the activating enzyme to form isoenzymes has been proposed.

Phenoloxidase, a marker enzyme for differentiation of the neural ectoderm and the epidermal ectoderm during embryonic development of amphioxus Branchiostoma belcheri tsingtaunese

The development of phenoloxidase during amphioxus embryogenesis was spectrophotometrically and histochemically studied for the first time in the present study. It was found that (1) PO activity initially appeared in the general ectoderm including the neural ectoderm and the epidermal ectoderm at the early neurala stage but not in the mesoderm or the endoderm, and (2) PO activity disappeared in the neural plate cells but remained unchanged in the epidermal cells when the neural plate was morphologically quite distinct from the rest of the ectoderm. It is apparent that PO could serve as a marker enzyme for differentiation of the neural ectoderm from the epidermal ectoderm during embryonic development of amphioxus. (C) 2000 Elsevier Science ireland Ltd. All rights reserved.

Differential expression profiling of components associated with exoskeletal hardening in crustaceans

Background: Exoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix. Glycoproteins have been implicated in the calcification process of many matrices. Sclerotization, on the other hand, is catalysed by phenoloxidases, which also play a role in melanization and the immunological response in arthropods. Custom cDNA microarrays from Portunus pelagicus were used to identify genes possibly associated with the activation pathways involved in these processes. Results: Two genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display molt cycle-related differential expression profiles. C-type lectin receptor up-regulation was found to coincide with periods associated with new uncalcified cuticle formation, while the up-regulation of mannose-binding protein occurred only in the post-molt stage, during which calcification takes place, implicating both in the regulation of calcification. Genes presumed to be involved in the phenoloxidase activation pathway that facilitates sclerotization also displayed molt cycle-related differential expression profiles. Members of the serine protease superfamily, trypsin-like and chymotrypsin-like, were up-regulated in the intermolt stage when compared to post-molt, while trypsin-like was also up-regulated in pre-molt compared to ecdysis. Additionally, up-regulation in pre- and intermolt stages was observed by transcripts encoding other phenoloxidase activators including the putative antibacterial protein carcinin-like, and clotting protein precursor-like. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and intermolt. Conclusion: Cuticle hardening in crustaceans is a complex process that is precisely timed to occur in the post-molt stage of the molt cycle. We have identified differential expression patterns of several genes that are believed to be involved in biomineralization and sclerotization and propose possible regulatory mechanisms for these processes based on their expression profiles, such as the potential involvement of C-type lectin receptors and mannose binding protein in the regulation of calcification.

Purification and characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera

Phenoloxidases are oxidative enzymes, which play an important role in both cell mediated and humoral immunity. Purification and biochemical characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera (Hubner) were carried out to study its biochemical properties. Prophenoloxidase consists of a single polypeptide chain with a relative molecular weight of 85 kDa as determined by SDSPAGE, MALDITOF MS and LCESI MS. After the final step, the enzyme showed 71.7 fold of purification with a recovery of 49.2%. Purified prophenoloxidase showed high specific activity and homology with phenoloxidase subunit-1 of Bombyx mori and the conserved regions of copper binding (B) site of phenoloxidase. Purified prophenoloxidase has pH optima of 6.8 and has high catalytic efficiency towards the dopamine as a substrate in comparison to catechol and L-Dopa. The PO activity was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and kojic acid.

Comparative effect of Pediococcus acidilactici and Lactococcus lactis on growth performance, survival and enzyme activity of western white leg shrimp (Litopenaeus vannamei)

This study was done in Shahid Kiani Marine Aquaculture Development Center, Choebde, Abadan in order to evaluate the effects of Pediiococcus acidilactici, Lactococcus lactis and vitamin C on growth performance, survival, enzymatic activities and immune responses of L. vannamei during three months. Treatments were included control group, Pediiococcus and Lactococcus treatments which fed with diet containing 1×10P9P cfu gP_1P bacteria and vitamin C. At the end of the experiment, the growth factors, immune parameters, digestive enzymes, intestinal, histology of intestine, carcasses and microbial flora (bacterial total count and lactic acid count) were evaluated. The results indicated that administration of lactobacillus had significant effects on the growth factors as the highest weight, increase specific growth rate, relative growth rate, feed conversion ratio and protein efficiency in the shrimps received pediococcus and then Lactococcus (P<0.05). The best immune function was also observed in the shrimps fed by probiotics, so that proteins and hemoglobin̛ hemolymph, phenoloxidase activity and challenged with V. parahaemolyticus showed a statistical difference comparing to the control group and the group received vitamin C (P<0.05). Some digestive enzymes, in pediococcus treatment showed a significant increase when compared to other treatments (P<0.05). Significant changes in bacterial intestinal flora were observed in probiotic groups compared with control and vitamin C groups (P < 0.05). Histological results showed the positive effects of probiotics in the gut (P < 0.05). While these supplements cannot caused to significant impacts on the shrimp carcass composition (P ˃ 0.05). As a result pediococcus group had the best performance among treatments.

Effect of replacement of fish meal by meat and bone meal and poultry by-product meal in diets on the growth and immune response of Macrobrachium nipponense

The potential use of poultry by-product meal (PBM) and meat and bone meal (MBM) as alternative dietary protein sources for juvenile Macrobrachium nipponense was studied by a 70-day growth trial. Triplicate groups of M. nipponense (initial body weight: 0.37 g) were fed at 20.7-22.4 degreesC on each of the five isoenergetic and isonitrogenous diets (protein content about 38%) with different replacement of fish meal by MBM or PBM. The control diet used white fish meal as the sole protein source, the other four diets were prepared with 15% or 50% fish meal protein substituted by either MBM (MBM15, MBM50) or PBM (PBM15, PBM50). The results showed that replacement of fish meal by MBM in diets did not affect growth performance of M. nipponense (P > 0.05), while specific growth rate in PBM15 was significantly higher than that in other groups (P < 0.05). Survival rates of shrimp fed with MBM15 diet were significantly higher than that in other groups (P < 0.05). No significant differences in immunological parameters, including total haemocyte count (THC), phenoloxidase activity (PO) and respiratory burst (O-2(-)), were observed between the shrimps that were fed five experimental diets, and all determined immunological parameters in control groups were slightly higher than those in replacement groups. In conclusion, either MBM or PBM investigated could replace up to 50% fish meal protein in diets for M. nipponense. (C) 2003 Elsevier Ltd. All rights reserved.

The effects of CpG-C oligodeoxynucleotides on innate immune responses in Eriocheir sinensis (H. Milne-Edwards, 1853)

CpG oligodeoxynucleotides (ODNs) can stimulate the immune system, and therefore are widely used as a therapeutic vaccination and immune adjuvant in human. In the present study, CpG-C, a combination of A- and B-class ODN, was injected into Chinese mitten crab Eriocheir sinensis at three doses (0.1, 1 and 10 mu g crab-1), and the reactive oxygen species (ROS) levels, activities of total intracellular phenoloxidase (PO) and lysozyme-like activities, the mRNA transcripts of EsproPO, EsCrustin and EsALF were assayed to evaluate its modulating effects on the immune system of crab. The ROS levels in all treated and control groups were significantly increased from 6 to 24 h, except that ROS in 0.1 mu g CpG-C-treated crabs was comparable to that of the blank at 6 h. The PO activity was significantly enhanced and EsproPO transcripts were down-regulated (P < 0.01) at 6 h after the injection of 0.1 mu g CpG-C, with no significant changes in the other dosage treatments. The lysozyme-like activities and EsCrustin transcripts in the CpG-C-treatment groups were significantly higher than those of controls. The mRNA expression of EsALF remained almost constant in all the groups during the treatment. These results collectively suggested that CpG-C could activate the immune responses of E. sinensis, and might be used as a novel immunostimulant for disease control in crabs.

The effects of CpG-C oligodeoxynucleotides on innate immune responses in Eriocheir sinensis (H. Milne-Edwards, 1853)

CpG oligodeoxynucleotides (ODNs) can stimulate the immune system, and therefore are widely used as a therapeutic vaccination and immune adjuvant in human. In the present study, CpG-C, a combination of A- and B-class ODN, was injected into Chinese mitten crab Eriocheir sinensis at three doses (0.1, 1 and 10 mu g crab-1), and the reactive oxygen species (ROS) levels, activities of total intracellular phenoloxidase (PO) and lysozyme-like activities, the mRNA transcripts of EsproPO, EsCrustin and EsALF were assayed to evaluate its modulating effects on the immune system of crab. The ROS levels in all treated and control groups were significantly increased from 6 to 24 h, except that ROS in 0.1 mu g CpG-C-treated crabs was comparable to that of the blank at 6 h. The PO activity was significantly enhanced and EsproPO transcripts were down-regulated (P < 0.01) at 6 h after the injection of 0.1 mu g CpG-C, with no significant changes in the other dosage treatments. The lysozyme-like activities and EsCrustin transcripts in the CpG-C-treatment groups were significantly higher than those of controls. The mRNA expression of EsALF remained almost constant in all the groups during the treatment. These results collectively suggested that CpG-C could activate the immune responses of E. sinensis, and might be used as a novel immunostimulant for disease control in crabs.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

The enhanced immune protection of Zhikong scallop Chlamys farreri on the secondary encounter with Listonella anguillarum

It is well known that invertebrates are devoid of adaptive immune components and rely primarily on innate immunity to defend against pathogens, but recent studies have demonstrated the existence of enhanced secondary immune protection in some invertebrates. in the present study, the cumulative mortality of scallops received two successive Listonella anguillarum stimulations was recorded, and variations of immune parameters including phagocytosis (phagocytic rate and phagocytic index), phenoloxidase-like enzyme, acid phosphatase and superoxide dismutase activities were also examined. The scallops received a previous short-term L anguillarum stimulation were protected against a long-term stimulation of L. anguillarum. Significantly higher level of phagocytic activities and acid phosphatase activity were observed in the scallops received twice stimulations compared with those only received the secondary stimulation. These results indicated that a short-term immersion with L. anguillarum modulated the scallops' immune system and endowed the scallops with enhanced resistance to the secondary bacterial stimulation: phagocytosis and acid phosphatase were suspected to be involved in the protection. (C) 2008 Elsevier Inc. All rights reserved.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. (C) 2010 Elsevier Ltd. All rights reserved.

中国明对虾病原检测新方法及酚氧化酶通路相关基因的研究

对虾病害在世界范围内肆虐，给水产养殖和沿海农村经济造成了重大损失。在水产养殖的实践中快速检测水产动物的病害并及时采取隔离等措施对于控制病害尤为重要，其中关键的环节就是快速检测出病害，并在对虾免疫机制上寻找对虾疾病防治的有效方法。研究表明当对虾等甲壳动物受到外界病原刺激时，极微量的微生物多糖就可以激活proPO系统。激活过程中涉及和产生一系列活性物质，如黑色素、酚氧化酶原激活因子（PPA）、模式识别蛋白（BGBP、PGBP、LGBP、LBP）及其膜上受体和A2巨球蛋白等，它们可通过多种方式参与防御反应，包括提供调理素，促进血细胞吞噬作用，形成结节或包囊以及介导凝集和凝固，产生杀菌物质并且黑色素化。黑色素常常在节肢动物的体表形成黑色斑点，形成的色素沉着对机体起到保护作用。所以，酚氧化酶原激活的级联反应是节肢动物免疫的关键因素。本论文研究开发了以环等温介导技术（LAMP）为基础的检测对虾白斑病毒（WSSV）和鳗弧菌（V. anguillarum）的快速检测方法。并从对虾对病害的免疫机制为切入点，从中国明对虾体内克隆了酚氧化酶原（PrpPO）和丝氨酸蛋白酶FcSP3这两个免疫系统中重要的基因，分析了它们的分子结构特征，组织分布及应答鳗弧菌病原刺激的表达变化模式。 建立的对虾常见病害对虾白班病毒（WSSV）和鳗弧菌（V. anguillarum）的LAMP检测方法，经过实验比对和Blast检索，发现本研究中使用的引物，比已经报导的LAMP方法或者PCR方法具有更宽的检测范围（更低的假阴性）。检测WSSV的LAMP方法使用病毒的VP28基因设计引物，而鳗弧菌的检测方法使用empA基因设计引物。在方法中，首次提出加入UNG酶和dUTP的措施来预防污染，在实际检测中非常有效。LAMP方法与PCR检测方法的灵敏性比较也进行了研究，二者灵敏性相当。 依据中国明对虾血液cDNA文库提供的部分片段信息，结合SMART-RACE技术，克隆了酚氧化酶原（PrpPO）基因，通过序列比对分析发现，PrpPO基因cDNA全长为3040 bp，其中开放阅读框2061 bp，编码686个氨基酸，其中推测的信号肽为12个氨基酸。推测的序列与斑节对虾（P. monodon）同源性为93%,与短钩对虾（P. semisulcatus.）同源性为92%。real time RT-PCR实验结果表明， ProPO在血细胞中的相对表达量最高，肝胰脏中表达量最低。弧菌刺激实验中注射弧菌，刺激了血细胞和淋巴器官中的ProPO mRNA显著增加，说明在血细胞和淋巴器官中存在快速反应的ProPO通路。而ProPO mRNA量在淋巴器官中在时间上早于血液中升至最高，说明该动物在在病原刚开始入侵的时候先有淋巴器官发挥主要的免疫作用，随着时间推移血细胞便变成主要的免疫器官。 根据中国明对虾肝胰脏cDNA文库提供EST信息，经过SMART-RACE克隆了一个丝氨酸蛋白酶FcSP3基因，通过序列比对分析发现，该丝氨酸蛋白酶基因cDNA全长为1622 bp，其中开放阅读框1431 bp，编码477个氨基酸，其中推测的信号肽为22个氨基酸。推测的序列与疟蚊的丝氨酸蛋白酶（A. gambiae）同源性为33%,与丽蝇蛹集金小蜂的酚氧化酶原激活因子（N. vitripennis）同源性为32%，与东北大黑鳃金龟的酚氧化酶原激活因子（H. diomphalia）同源性为34%。淋巴器官中PPAⅡ表达量约为血液中表达量的47560倍，肝胰脏中的FCSP3表达量为血细胞表达量的6226倍。鳗弧菌注射对虾后，淋巴器官中刺激组和对照组FcSP3的mRNA量在刺激后6小时显著降低，但是刺激组的表达量明显高于对照组。刺激组的血细胞与肝胰脏中FcSP3 mRNA的相对表达量增高。而病原刺激后的血液与肝胰脏中的FcSP3 mRNA的增长趋势也在时间上先与ProPO mRNA。这说明FcSP3对ProPO有正调控的作用，但这个调控有一个时间差，并且在不同组织中有不同的调控效率。

中国明对虾（Fenneropenaeus chinensis）几种免疫相关因子的分离纯化、基因克隆及相互关系研究

中国明对虾（Fenneropenaeus chinensis）是我国重要的海水养殖动物，但是近年来在其养殖过程中深受病害的影响，细菌性和病毒性疾病给对虾养殖业造成了巨大的经济损失，因此对中国明对虾免疫机理的研究对于对虾的疾病控制以及海水养殖业的持续发展具有重要意义。甲壳动物只具有天然免疫系统。天然免疫系统对于异己物质的识别是通过有限数量的模式识别蛋白（PRPs）来实现的。C型凝集素（C-type lectin）作为宿主防御系统中的一种模式识别受体，在病原识别和细胞间的相互作用上具有重要作用，此外它们还参与止血、凝固、物质运输和创伤修复等一系列作用；酚氧化酶（phenoloxidase）又称为酪氨酸酶(tyrosinase,EC1.14.18.1)。它是一种含铜的酶,能够催化单酚羟化成二酚(如多巴)，把二酚氧化成醌；醌在非酶促条件下形成最终的反应产物黑色素。血蓝蛋白(hemocyanins，Hc)是在软体动物和节肢动物血淋巴中发现的存在于细胞外、负责运输氧的一类巨大蛋白质。本论文从中国明对虾血清中纯化了利用亲和层析纯化了一种凝集素FC-L，并对FC-L的理化性质、多糖结合和细菌结合特性进行了研究。从中国明对虾血细胞中克隆了一种酚氧化酶原(prophenoloxidase,proPO)基因FCproPO，从肝胰腺中克隆了一种血蓝蛋白基因FCHc，分析了它们的分子结构特征，预测了其可能的作用，并对它们的组织分布、应答不同病原感染的表达变化模式以及相互关系进行了研究。

The effect of acute salinity change on white spot syndrome (WSS) outbreaks in Fenneropenaeus chinensis

In order to observe the effect of salinity on disease resistance and white spot syndrome virus (WSSV) proliferation in Fenneropenaeus chinensis, shrimps with latent WSSV were subjected to two acute salinity changes from the original salinity of 22 ppt to 18 and 14 ppt in an hour, respectively. The total haemocyte count (THC) of the challenged group showed no evident change under salinity adjustments, but the phenoloxidase (PO) index declined significantly (P<0.05) corresponding to continuing acute salinity changes from the 24th to the 72nd hour. According to the WSSV load detected by quantitative real-time PCR method, it was found that WSSV carried by the challenged group and control group were significantly different (P<0.05); acute salinity change from 22 to 14 ppt led to the WSSV carried in the challenged group being significantly higher (P<0.05) than that of those surviving in 22 ppt, but salinity change from 22 to 18 ppt had no such effect. At the end of the 72-h experiment, the challenged group subjected to salinity change from 22 to 14 ppt had nearly 3 times the WSSV load as the control group with no salinity change. Therefore, salinity changes over a particular range could result in a decrease of immunocompetence and obvious WSSV proliferation in the shrimps, leading to white spot syndrome developing from a latent infection to an acute outbreak. (C) 2005 Elsevier B.V All rights reserved.

Melanism and disease resistance in insects

There is growing evidence that insects in high-density populations invest relatively more in pathogen resistance than those in low-density populations (i.e. density-dependent prophylaxis). Such increases in resistance are often accompanied by cuticular melanism, which is characteristic of the high-density form of many phase polyphenic insects. Both melanism and pathogen resistance involve the prophenoloxidase enzyme system. In this paper the link between resistance, melanism and phenoloxidase activity is examined in Spodoptera lanae. In S. exempta, cuticular melanism was positively correlated with phenoloxidase activity in the cuticle, haemolymph and midgut. Melanic S. exempta larvae were found to melanize a greater proportion of eggs of the ectoparasitoid Euplectrus laphygmae than non-melanic larvae, and melanic S. littoralis were more resistant to the entomopathogenic fungus Beauveria bassiana (in S. exempta the association between melanism and fungal resistance was non-signficant). These results strengthen the link between melanism and disease resistance and implicate the involvement of phenoloxidase.