Cloning and Sequence Analysis of Prophenoloxidase from Haemocytes of the Red Swamp Crayfish,Procambarus clarkii

The full-length cDNA sequence of prophenoloxidase was obtained through RACE technology. The complete cDNA sequence is 3 721-bp long, containing an open reading frame （ORF） of 1 881 bp, a 154-bp 5′-untranslated region, and a 1 686- bp 3′-untranslated region with three potential functional poly（A） signals （AATAAA）. The molecular mass of the deduced amino acid sequence （627 aa） was 72.3 kDa with an estimatedpI of 5.88. It contained putative copper-binding sites （copper A： 131, 135, 167 and copper B： 301,305, 341）, and a tentative complement-like motif （GCGWPDHL）. Eight potential N-linked glycosylation sites were predicted to be present in P. clarkii prophenoloxidase. Similar to those in other arthropod prophenoloxidases reported so far, no signal peptide was detected in the crayfish prophenoloxidase. The phylogenetic trees confirmed that P. clarkii prophenoloxidase was most closely related to that of freshwater crayfish P. leniusculus and more closely related to other crustacean prophenoloxidases from shrimp, prawn, and lobster than to the insect prophenoloxidases. Besides, two putative introns were found in this sequence of genomic DNA.

Modulation by Biogenic Amines for the Hemocyte Count and Prophenoloxidase Exocytosis via Receptors in Litopenaeus vannamei

Hemocyte counts and phenoloxidase(PO)activity were examined after hemolymph being incubated in dopamine(DA),noradrenaline(NE)and serotonin(5-HT).Results showed that all the three biogenic amines(BAs)had a significant impact on total hemocyte count(THC),differential hemocyte count(DHC),and intracelluar and extracelluar phenoloxidase(PO)activity.Among these Bas,DA had the strongest effect on the above parameters,whereas 5-HT had the least effect.Preincubation with D1 receptor antagonist SCH23390,D2 receptor antagonist Sulpiride and 1:1 admixture of the two could significantly inhibit the effect of DA on these parameters.SCH23390 showed a stronger inhibitory effect than Sulpiride,and the admixture exhibited the strongest effect.These results suggested that the change of hemocyte count and activation of prophenoloxidase(proPO)system in Litopenaeusvan-namei hemocyte can be regulated by BAs,and DA modulates the two parameters via its receptors.

Effects of acute salinity stress on the survival and prophenoloxidase system of Exopalaemon carinicauda

The ridgetail white prawn Exopalaemon carinicauda is a euryhaline shrimp species in the estuarine and coastal areas of China.In this study,survival rates,transcription levels of two prophenoloxidase system-related genes(Ec LGBP and Ecpro PO)and PO activity were determined quantitatively in juvenile and adult E.carinicauda under different salinity levels.The results showed that E.carinicauda juveniles could survive in a wider range of salinity conditions than adults.For juvenile E.carinicauda,the expression levels of Ec LGBP and Ec Pro PO were upregulated in low salinities and showed no significant difference at 20–40,while PO activities in low salinities were higher compared to those in high salinities.For adult E.carinicauda,the expression profiles of Ec LGBP and Ecpro PO had a different trend of up-regulation in salinity stress treatments and no obvious difference was observed in the gene expression levels and PO activity between 30 and 40.The salinity tolerance range of immunity for juvenile and adult E.carinicauda is 20–40 and 30–40,respectively.

Prophenoloxidase from Pieris rapae:gene cloning,activity,and transcription in response to venom/calyx fluid from the endoparasitoid wasp Cotesia glomerata

Prophenoloxidase (PPO) plays an important role in melanization, necessary for defense against intruding parasitoids. Parasitoids have evolved to inject maternal virulence factors into the host hemocoel to suppress hemolymph melanization for the successful development of their progeny. In this study, the full-length complementary DNA (cDNA) of a Pieris rapae PPO was cloned. Its cDNA contained a 2 076-base pair (bp) open reading frame (ORF) encoding 691 amino acids (aa). Two putative copper-binding sites, a proteolytic activation site, three conserved hemocyanin domains, and a thiol ester motif were found in the deduced amino acid sequence. According to both multiple alignment and phylogenetic analysis, P. rapae PPO gene cloned here is a member of the lepidopteran PPO-2 family. Injection of Cotesia glomerata venom or calyx fluid resulted in reduction of P. rapae hemolymph phenoloxidase activity, demonstrating the ability to inhibit the host's melanization. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of P. rapae PPO-2 in the haemocytes from larvae had not significantly changed following venom injection, suggesting that the regulation of PPO messenger RNA (mRNA) expression by venom was not employed by C. glomerata to cause failure of melanization in parasitized host. While decreased P. rapae PPO-2 gene expression was observed in the haemocytes after calyx fluid injection, no detectable transcriptional change was induced by parasitization, indicating that transcriptional down-regulation of PPO by calyx fluid might play a minor role involved in inhibiting the host's melanization.

中华绒螯蟹酚氧化酶原系统与颤抖病的关系(英文)

近年来,颤抖病给中华绒螯蟹养殖业造成了巨大的经济损失.酚氧化酶原系统是甲壳动物最主要的体液免疫系统,当外源微生物侵入体内时,它发挥最基本的免疫防御作用来抵抗感染.酚氧化酶原系统的变化是蟹类健康状况的重要指标.prophenoloxidase和peroxinectin是酚氧化酶原系统中两个重要的体液免疫分子.实验从分子和蛋白两个水平研究了中华绒螯蟹酚氧化酶原系统与颤抖病的关系,建立了一种新的通过测定酚氧化酶原系统活性来判断蟹体质状况的方法.在分子水平上,中华绒螯蟹体液免疫分子prophenoloxidase和peroxinectin的部分基因序列被克隆和测序,并检测出这两种分子mR-NA水平的表达情况与颤抖病的感染程度呈正相关,患病组蟹mRNA的表达水平远高于正常组;在蛋白水平上,酚氧化酶的活性被测定,在正常情况下,蟹体内酚氧化酶活性处于低水平状态,当被感染颤抖病后,酚氧化酶活性有一定程度的升高.这些结果提示了酚氧化酶原系统在抗颤抖病感染时起重要作用,为进一步研究预防和治疗颤抖病的新途径提供了理论依据.

Effect of the endoparasitoid Campoletis chlorideae on phenoloxidase activity in Helicoverpa armigera hemo-lymph

Endoparasitoid wasps can develop inside permissive host due to their ability to overcome or to evade the host’s cellular and humoral immune response. Oviposition of Campoletis chlorideae (Hymenoptera: Ichneumonidae) in larvae of Helicoverpa armigera (Lepidoptera) was accompanied by inhibition of phenoloxidase (PO) activity and melanization reaction in host hemolymph in vitro. PO activity in host plasma was decreased about 83% 48 h post para-sitization. A similar result was found when the host insect was injected with 0.5 wasp equivalent calyx fluid. This indicated that the calyx fluid was concerned with suppression of PO activity after parasitization. Furthermore, the prophen-oloxidase (proPO) in host haemocytes could be activated by bovine trypsin in unparasitized insects, while it could not be activated in parasitized or calyx fluid-injected host. The results suggested that inhibition of PO activity by parasitization was related to the calyx fluid of Campoletis chlorideae, and the components of calyx

Identification of two clip domain serine proteases involved in the pea aphid's defense against bacterial and fungal infection

Phenoloxidases(POs)are required for the pea aphid s defense against bacterial and fungal infection.Prophenoloxidases(PPOs)are protcolytically converted to its active form PO through a clip domain serine protease cascade.In this study,we identified five clip domain serine proteases in the pea aphids.The messenger RNA levels of two of them,Ap.SPLP and Ap_VP,were upregulated by Gram-positive bacterium Staphylococcus aureus and fungus Beauveha bassiana infections.Double-stranded RNA-based expression knockdown of these two genes resulted in reduced PO activity of the aphid hemolymph,higher loads of S.aureus and B.bassiana in the aphids,and lower survival rates of the aphids after infections.Our data suggest that Ap.SPLP and Ap_VP are involved in PPO activation pathway in the pea aphid.

Growth and immune response of pond-reared giant freshwater prawn Macrobrachium rosenbergii post larvae fed diets containing Chlorella vulgaris

A 70-day feeding trial was conducted to evaluate the efficacy of Chlorella vulgaris supplementation in diets of giant freshwater prawn(Macrobrachium rosenbergii)post larvae.Resistance of the prawns against Aeromonas hydrophila and several immune parameters(total hemocyte count and prophenoloxidase activity)were also assessed in the study.Iso-nitrogenous,iso-lipidic,and iso-caloric prawn feeds were prepared utilizing a fishmeal based positive control diet(F0)and four dietary treatments with C.vulgaris inclusion levels of 2(F2),4(F4),6(F6),and 8(F8)%.Post larvae of M.rosenbergii were stocked randomly(mean initial body weight of 0.39�0.38 g)in fifteen net cages(8.1 m�8.9 m with an average depth of 1 m)in an earthen pond for the assessment of growth parameters.Overall growth indices for prawn fed Chlorella containing diets were enhanced in comparison to the control treatment.Prawn fed diets with Chlorella showed significantly higher(P<0.05)prophenol oxidase activity,total hemocyte counts,and survival rates post bacterial infection.These findings demonstrated that inclusion of 4%,6%,and 8%C.vulgaris in prawn diets provided optimal growth rates and improved immunity of the post larvae.