The dual roles of Armigeres subalbatus prophenoloxidase V in parasite melanization and egg chorion melanization in the mosquito Ar. subalbatus

Phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning, cuticle formation and melanotic encapsulaction of pathogens. Previously, we identified five POs, designated As-pro-PO I–V, from the mosquito Armigeres subalbatus and demonstrated that the functions of As-pro-PO I, II and III, were associated with filarial parasite melanization, blood feeding and cuticle formation, respectively. In the present study, we delineate the dual functions of As-pro-PO V. We found that the level of As-pro-PO V mRNA in mosquitoes was significantly increased after microfilaria challenge or blood feeding, and decreased to normal level after oviposition. Knockdown of As-pro-PO V by dsRNA resulted in significant decreases in the degree of microfilaria melanization, egg chronic melanization rates and egg hatching rates in Ar. subalbatus. Further transfection and electrophoretic mobility-shift assays verified the As-pro-PO V gene might regulated by both AP-1, a putative immune-related regulatory element and CdxA, a developmental regulatory element. The binding of AP-1 and CdxA motif with mosquito nuclear extracts was significantly enhanced after microfilaria challenge and blood-feeding in Ar. subalbatus, respectively. These results indicate that As-pro-PO V is a critical enzyme that is required for both an effective melanization immune response and egg chorion melanization in this mosquito.     

Rhythms in the biting behaviour of a mosquito Armigeres subalbatus

Summary The biting cycle of Armigeres subalbatus is distinctly crepuscular, exhibiting two peaks of activity, a smaller one at dawn and a larger one at dusk. The biting cycle is entrained to natural light-dark cycles and the time interval from dawn to dawn or dusk to dusk peaks is exactly 24 h and from dawn to dusk or dusk to dawn is about 12 h measured at 50% level. This rhythm manifests itself day after day without any marked qualitative change.The rate of change of light intensity may determine the onset of crepuscular biting. The sudden increase (up to ca. 17 lx) or decrease (down to ca. 4 lx) in the intensity of ambient light at the time of sunrise or sunset coincides with the peak of the biting activity.The density of the population of the host-seeking females fluctuates in relation to the phases of the moon, increasing with the full moon phase and decreasing with the new moon phase.Even though the density of the population is greater outdoors than indoors both at ground levels and in the first floor, the peak of activity occurs at the same time in all the places. A vertical stratification of biting activity was also noticed.     

Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus,Anopheles sinensis,Armigeres subalbatus,Culex pipiens and Cx. tritaeniorhynchus in China

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.     

Haemolymph monophenol oxidase activity in Armigeres subalbatus infected with Brugia pahangi.

The activity of monophenol oxidase can be elicited in the haemolymph of Armigeres subalbatus by both blood and filaria-infected blood feeding. Haemolymph collected from both blood-fed and filaria-infected mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of L-[3,5-3H]tyrosine to dopa. Enzyme activity in filaria-infected mosquitoes was found to be significantly lower than that found in the blood-fed mosquitoes within 3 days post-ingestion, but still remained measurable 72 h post-ingestion. The decreased enzyme activity coincided in time with the development of capsules around the microfilariae. The consumption of monophenol oxidase by the melanization of migrating microfilariae in the haemocoel of filaria-infected mosquitoes and the effects of excretory and secretory products of developing larvae on monophenol oxidase activity are suggested.     

Induction of Fas mediated caspase-8 independent apoptosis in immune cells by Armigeres subalbatus saliva

Background

It is widely recognized that the introduction of saliva of bloodsucking arthropods at the site of pathogen transmission might play a central role in vector-borne infections. However, how the interaction between salivary components and the host immune system takes place and which physiological processes this leads to has yet to be investigated. Armigeres subalbatus is one of the prominent types of mosquitoes involved in the transmission of parasitic and viral diseases in humans and animals.

Methodology/Principal Findings

Using murine peritoneal macrophages and lymphocytes, and human peripheral mononuclear cells (PBMCs), this study shows that saliva of the female Ar. subalbatus induces apoptosis via interaction with the Fas receptor within a few hours but without activating caspase-8. The process further activates downstream p38 MAPK signaling, a cascade that leads to the induction of apoptosis in capase-3 dependent manner. We further illustrate that Ar. subalbatus saliva suppresses proinflammatory cytokines without changing IL-10 levels, which might happen as a result of apoptosis.

Conclusions

Our study shows for the first time that saliva-induced apoptosis is the leading phenomenon exerted by Ar. subalbatus that impede immune cells leading to the suppression of their effecter mechanism.     

Brugia malayi and Brugia pahangi: inherent difference in immune activation in the mosquitoes Armigeres subalbatus and Aedes aegypti

The inherent ability of Brugia malayi and Brugia pahangi (Nematoda) to establish successful relationships with the mosquitoes Armigeres subalbatus and Aedes aegypti Liverpool strain was evaluated. Brugia pahangi microfilariae (mff) avoided the immune response and developed normally in A. subalbatus exposed to the parasite by an infective bloodmeal, whereas nearly 85% of B. malayi were destroyed by the immune response. Because A. aegypti supports the development of both filarial worm species but destroys intrathoracically inoculated B. pahangi isolated from jird blood, blood-isolated B. malayi were inoculated into A. aegypti, and the immune response was compared with that observed against B. pahangi. The response against B. malayi was significantly more rapid and effective than the response against B. pahangi. Similar results were obtained when blood-isolated B. pahangi or B. malayi were inoculated into A. subalbatus. Microfilariae of both species were able to avoid immune destruction in A. aegypti if they were allowed to penetrate the Liverpool midgut in vitro prior to inoculation. Most B. pahangi that had first penetrated an Armigeres midgut prior to inoculation into A. subalbatus were able to avoid the immune response, but by day 3 postinoculation, less than 40% of the B. malayi, treated in the same manner, were able to escape the immune response. Genetic susceptibility of mosquitoes to infection by filarial worms and potential mechanisms of immune evasion/suppression are discussed regarding B. malayi and B. pahangi.     

Cloning and characterization of prophenoloxidase A3 (proPOA3) from Culex pipiens pallens

The prophenoloxidase subunit A3 (proPOA3) gene was cloned from Culex pipiens pallens, which had an open reading frame of 2061 bp encoding a putative 686 amino acid protein. The deduced amino acid sequence shares 98% with proPOA3 from Culex quinquefasciatus. ProPOA3 is expressed at all developmental stages of C. pipiens pallens. Significant negative correlation was observed between proPOA3 expression and deltamethrin resistance in resistant C. pipiens pallens. Furthermore, proPOA3 expression levels were significantly lower in deltamethrin-resistant mosquitoes than in susceptible mosquitoes collected at four locations in Eastern China. However, we did not find any substantial change in proPOA3 expression in field-collected resistant Anopheles mosquitoes. Moreover, overexpressing proPOA3 in C6/36 cells led to more sensitivity to deltamethrin treatment. In laboratory and field-collected resistant C. pipiens pallens, a valine to isoleucine mutation (769G>A) and two synonymous mutations (1116G>C and 1116G>A) were identified in proPOA3. In addition, the mutation frequency of 769G>A and 1116G>C increased gradually, which corresponded with raised deltamethrin resistance levels. Taken together, our study provides the first evidence that proPOA3 may play a role in the regulation of deltamethrin-resistance in C. pipiens pallens.     

Beta 1, 3-glucan recognition protein from the mosquito, Armigeres subalbatus, is involved in the recognition of distinct types of bacteria in innate immune responses

The activation of an immune response to invading microorganisms generally requires recognition by pattern recognition receptors. Beta 1, 3-glucan recognition proteins (GRPs) have specific affinity for beta 1, 3-glucan, a component on the surface of fungi and bacteria. In this study, we show that GRP from Armigeres subalbatus mosquitoes (AsGRP) is able to bind different bacterial species, and that this binding varies from species to species and is independent of Gram type. AsGRP knockdown with double-stranded RNA increases the mortality of mosquitoes to those bacteria that strongly bind AsGRP, but not to bacteria that do not detectably bind AsGRP. This increase in susceptibility is partially evidenced by decreased melanization in Salmonella typhimurium. Furthermore, AsGRP expression is differentially affected by the presence of different species of bacteria. These results demonstrate that AsGRP is selective in its affinity to different bacteria and; therefore, plays a role in the antibacterial immune response of mosquitoes.     

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 (Hübner) 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 SDS–PAGE, MALDI–TOF MS and LC–ESI 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.     

Cloning, overexpression, and characterization of recombinant heparinase III from Bacteroides stercoris HJ-15

Recombinant heparinase III (rHepIII) from Bacteroides stercoris HJ-15 was cloned, expressed, and characterized. The full-length heparinase III gene from B. stercoris HJ-15 was identified by Southern blotting, and the sequence was deposited in GenBank. The heparinase III gene, which is 2,001-bp long, was cloned and overexpressed in Escherichia coli; highly active rHepIII was easily purified using only one step of immobilized Ni²⁺ affinity column chromatography. Enzymatic properties and substrate specificities of rHepIII were assessed, and its kinetic constants were calculated. rHepIII was most active in 50 mM sodium phosphate buffer with 350 mM NaCl (pH 6.6) at 45°C. Through amino acid modification studies and site-directed mutagenesis assay, cysteines and histidines were identified as crucial residues for enzymatic activity. Moreover, this enzyme digested not only heparan sulfate but also heparin and hyaluronic acid, and their degradation products were verified by strong anion exchange/high-performance liquid chromatography. These characteristics, including active residues and substrate specificities were interesting compared with those of existing heparinase III from other species. We anticipate that the convenience of purification and the characteristics of this enzyme will make it a powerful tool for studies of glycosaminoglycans and their lyases.     

红螯螯虾酚氧化酶原及其特性研究

采用同源克隆策略和RACE技术, 从红螯螯虾Cherax quadricarinatus血细胞中克隆得到酚氧化酶原基因的全长cDNA序列, 共2951 bp, 开放读码框为1995 bp, 编码665个氨基酸. 预测的分子量和等电点分别为75.7 kD和6.23. 酚氧化酶原含有两个推测的tyrosinase copper-binding motifs (带有六个组氨酸残基)和一个thiol-ester-like motif, 这些特征和其他甲壳动物的酚氧化酶原特征相同. 红螯螯虾酚氧化酶原氨基酸序列与通讯螯虾Pacifastacus leniusculus、欧洲龙虾Homarus gammarus、美洲龙虾Homarus americanus 和克氏原螯虾Procambarus clarkii 酚氧化酶原的相似率分别为68%、63%、63%和59%. 酚氧化酶原基因双酶切后连接入pET-28a原核表达载体, 转化到大肠杆菌BL21后重组表达酚氧化酶原蛋白. 在重组蛋白纯化后, 免疫新西兰大耳兔制备得到的酚氧化酶原多克隆抗体, 其效价大于1:12800. 红螯螯虾血淋巴、肝和鳃组织中的酚氧化酶原mRNA表达和酚氧化酶活性较高, 而神经、心、肠和肌肉中较低. 中华绒螯蟹螺原体和嗜水气单胞菌免疫红螯螯虾后, 血淋巴细胞、肝和鳃组织中的酚氧化酶原和酚氧化酶活性在免疫后的不同时间均出现了显著性的增加, 此结果表明酚氧化酶原和酚氧化酶在红螯螯虾对抗细菌感染的过程中起到重要的免疫作用. 此结果为进一步深入研究酚氧化酶原基因和酚氧化酶的功能及其调控机理奠定基础.       

柑橘全爪螨酚氧化酶原激活酶基因cDNA克隆及生物信息学分析

酚氧化酶原激活酶(prophenoloxidase activating enzyme,PPAE)是酚氧化酶激活系统(prophenoloxidase activating system,PPO-AS)的组成部分,是无脊椎动物抵御入侵微生物的关键酶。本研究利用RACE技术从柑橘全爪螨Panonychus citri(McGergor)体内获得一条PPAE基因全长cDNA,命名为PcPPAE(GenBank:KC136292),属于丝氨酸蛋白酶家族。该基因cDNA全长1 676 bp,开放阅读框1 377 bp,编码458个氨基酸。该基因编码蛋白预测分子量为49.9 ku,理论等电点(pI值)为5.68,分子式为C2201H3467N607O678S22,不稳定系数为41.51,总亲水性系数为-0.268。经序列比对发现该基因具有发夹结构域,丝氨酸蛋白酶结构域,以及丝氨酸蛋白酶结构域中保守的催化三联体。系统发育分析表明该基因与二斑叶螨发夹结构丝氨酸蛋白酶基因的亲缘关系最近。本研究首次从柑橘全爪螨体内克隆获得酚氧化酶原激活酶,为后期进行柑橘全爪螨抵御微生物侵染机制研究奠定了基础。     

Dopaminergic systems in the European eel: characterization,brain distribution,and potential role in migration and reproduction

In fish like in mammals, dopamine (DA) is a major catecholaminergic neurotransmitter that contributes to many functions of the nervous system like sensory perception, tuning of sensori-motor cues, and hypothalamic and pituitary functions. In the eel, DA inhibits gonadal development, and juvenile silver eels remain blocked at a prepubertal stage if their reproductive migration does not occur. From data in other teleosts and vertebrates, it is suggested that DA would be involved also in the last steps of eel reproduction (oocyte maturation, ovulation, and spermiation) as well as in eel reproductive migration (locomotion and olfaction). Investigating dopaminergic systems in the eel may help in understanding the mechanisms of its complex life cycle and provide new data for its conservation and reproduction. In this article we review the biosynthesis and catabolism of catecholamines and discuss available methods to investigate brain dopaminergic systems in vertebrates and their application to the eel. Immunocytochemistry, in situ hybridization, and different tracing methods are used to map dopaminergic neurons and projections in the brain and pituitary and infer their potential functions. Moreover, variations in dopaminergic activity may be approached by means of quantitative methods like quantitative real-time RT-PCR and HPLC. These tools are currently used to study dopaminergic systems in the eel brain, their anatomy, regulation, and potential roles with special emphasis on the regulation of reproduction and reproductive migration. Guest editors: S. Dufour, E. Prévost, E. Rochard & P. Williot Fish and diadromy in Europe (ecology, management, conservation)     

Cloning,characterization, and functional studies of a 47-kDa platelet receptor for type III collagen

A 1.2-kb cDNA fragment encoding a platelet 47-kDa protein has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide of the sequenced amino terminus of the purified platelet protein with a poly(dT)(12).(dG) by polymerase chain reaction. A computer search revealed that the cDNA represents the coding sequence of a protein with a fragmentary homology to several proteins. Using a prokaryotic expression system, pBad TOPO-47 cDNA, a 47-kDa recombinant protein was obtained and purified to apparent homogeneity by nickel-nitrilotriacetic acid resin and collagen affinity column. The recombinant protein binds to type III but not type I collagen-Sepharose 2B affinity columns. Anti-47-kDa but not anti-65-kDa antibody inhibits the binding of the recombinant protein to the type III collagen-coated micro titer wells in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type III collagen-induced platelet aggregation also in a dose-dependent manner. We have defined two active peptides from the cloned deduced amino acid sequence. Both peptides inhibit type III but not type I collagen-induced platelet aggregation in a dose-dependent fashion. These results suggest that the active binding site of the platelet receptor to type III collagen resides in these portions of the protein.     

Cloning and characterization of a type III polyketide synthase from Aspergillus niger

Type III polyketide synthases (PKSs) are the condensing enzymes that catalyze the formation of a myriad of aromatic polyketides in plant, bacteria, and fungi. Here we report the cloning and characterization of a putative type III PKS from Aspergillusniger, AnPKS. This enzyme catalyzes the synthesis of alkyl pyrones from C2 to C18 starter CoA thioesters with malonyl-CoA as an extender CoA through decaboxylative condensation and cyclization. It displays broad substrate specificity toward fatty acyl-CoA starters to yield triketide and tetraketide pyrones, with benzoyl-CoA as the most preferred starter. The optimal temperature and pH of AnPKS are 50°C and 8, respectively. Under optimal conditions, the enzyme shows the highest catalytic efficiency (k(cat)/K(m)) of 7.4×10(5)s(-1)M(-1) toward benzoyl-CoA. Homology modeling and site-directed mutagenesis were used to probe the molecular basis of its substrate specificity. This study should open doors for further engineering of AnPKS as a biocatalyst for synthesis of value-added polyketides.