Changes in the haemocyte population of the mosquito, Culex quinquefasciatus, following infection with the filarial parasite, Wuchereria bancrofti

The mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is the vector of the filarial parasite Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae), which causes human bancroftian filariasis. Information on the mosquito humoral response against the filarial parasite during the process of its infection and development is important, as it decides the vector competence of the mosquito. Visible changes in the haemocyte population of mosquito, if any, will be an indicator of the possible humoral factors. The present study was aimed at investigating changes in the populations of various types of haemocytes of Cx. quinquefasciatus following infection with W. bancrofti. On day 2 post-feeding on microfilaraemic blood, the haemolymph perfusate of infected mosquitoes with L1 stage of the parasite showed 44.1% granulocytes, 42% prohaemocytes and 13.9% plasmatocytes, whereas that of the control mosquitoes fed on amicrofilaraemic blood showed 63.4% plasmatocytes, 22.2% prohaemocytes and 14.4% granulocytes. Differences in the population numbers of haemocyte types between the infected and control were significant (P > 0.05). However, the mosquitoes examined on day 6 post-feeding, when the parasite was in L2 stage, did not show any such changes. But, similar changes reappeared on day 12 in mosquitoes with L3 stage of the parasite. The observed haemocyte population changes indicate the possibility of some amount of humoral immune response, through the production of certain immune molecules, in Cx. quinquefasciatus infected with W. bancrofti. The nature and exact role of such a response on the filarial parasite development need further investigation.     

Reproductive aspects of the mosquito Culex quinquefasciatus (Diptera:Culicidae) infected with Wuchereria bancrofti (Spirurida: Onchocercidae)

This study reports on the relationship between Wuchereria bancrofti infection and female body size, intake of blood and fecundity in the mosquito Culex quinquefasciatus, vector of this filarial parasite in Recife (Brazil). Adults from field collected larvae were infected via a membrane feeding procedure, using blood with parasitaemia ranging from 724-6,000 mf/ml. A positive correlation was observed between mosquito size (measured by wing length) and egg production in uninfected females. However, this relationship did not exist in W. bancrofti infected mosquitoes. This change is unlikely to be the result of changes in blood ingestion as no significant difference was found when infected and uninfected females were compared. Variation in egg production observed between trials could not be associated with parasite density in the blood. These results suggest infection with W. bancrofti may disrupt the relationship between mosquito size and egg production during the first gonotrophic cycle of C. quinquefasciatus such that fecundity is sometimes reduced. However, this overall affect is variable and many groups of mosquitoes do not respond in this way.     

Actin protein up-regulated upon infection and development of the filarial parasite, Wuchereria bancrofti (Spirurida: Onchocercidae), in the vector mosquito, Culex quinquefasciatus (Diptera: Culicidae)

Detection and identification of humoral proteins, which are up-regulated in Culex quinquefasciatus upon infection by Wuchereria bancrofti, is important in tracing out the biochemical consequences of the filarial parasite development in the vector mosquito. Analysis of the haemolymph of infected mosquitoes through SDS-PAGE and RP-HPLC showed up-regulation of five proteins of molecular weights 40, 66, 22, 14, and 7-kDa. Among these, only the 40-kDa was unknown and the others were comparable with those already reported as transferrin, attacin, lysozyme, and defensin, respectively. In the present study, the 40-kDa protein up-regulated upon infection was identified as actin through nano-LC-MS/MS analysis. Actin is known to be one of the cytoskeletal proteins up-regulated in the haemolymph, as part of the innate immune system, of Escherichia coli challenged Drosophila melanogaster larvae. For the first time, we have observed an increased level of actin in the haemolymph of W. bancrofti-infected Cx. quinquefasciatus. However, the exact mechanism of actin involvement in the immune system of this mosquito is yet to be studied.     

A polymerase chain reaction based method for the detection of Culex quinquefasciatus (Diptera: culicidae)

Culex quinquefasciatus Say is the major vector of the filarial parasite Wuchereria bancrofti (Cobbold) which causes lymphatic filariasis in humans. A repetitive DNA sequence from the genome of C. quinquefasciatus has been cloned and completely sequenced. The 693 bp cloned fragment had an A+T content of 72%. Dot matrix analysis of the fragment did not reveal any direct or inverted repeats within it. Southern blot analysis using a variety of restriction enzymes appeared to indicate that the cloned fragment was interspersed within the genome with a copy number of approximately 30,000. A search of the GenBank database did not reveal significant homologies to any previously cloned sequences. Although the probe was sensitive enough to detect picogram quantities of DNA, it was not specific for C. quinquefasciatus, as it hybridized with DNA from other mosquito species, Culex pseudovishnui Colless, Culex gelidus Theobald, Culex tritaeniorhynchus Giles, Anopheles vagus D?nitz and Mansonia uniformis (Theobald). However PCR primers derived from the cloned sequence, IpC, were found to be specific and amplified only C. quinquefasciatus DNA. The optimized PCR assay was found to be very sensitive and was capable of detecting DNA from all stages of C. quinquefasciatus thus making it an ideal diagnostic tool.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

PCR and Mosquito dissection as tools to monitor filarial infection levels following mass treatment

BACKGROUND: Entomological methods may provide important tools for monitoring the progress of lymphatic filariasis elimination programs. In this study, we compared dissection of the vector, Culex quinquefasciatus, with the polymerase chain reaction (PCR) to assess filarial infection levels in mosquitoes in the context of a lymphatic filariasis elimination program in Leogane, Haiti. METHODS: Mosquitoes were collected using gravid traps located in 4 sentinel communities with Wuchereria bancrofti microfilaria prevalence that ranged from 0.8% to 15.9%. Captured mosquitoes were divided between dissection, to enumerate W. bancrofti larvae (L1, L2, L3) and desiccation for later analysis by PCR. PCR was conducted on DNA extracts from pooled mosquitoes (1-15 pooled females) utilizing a competitive PCR system with primers specific for the Ssp I repeat. PCR products were analyzed with a hybridization ELISA using probes specific for a control sequence and the Ssp I repeat. RESULTS: The prevalence of mosquito infection with W. bancrofti ranged from 0%-3.66% by dissection (L1-L3) and point estimates of infection prevalence, as assayed by PCR, ranged from 0.25% - 9.16%. Following mass treatment, W. bancrofti infection prevalence dropped significantly as determined by PCR and dissection in 2 of the 4 sentinel sites (Leogane and Barrier Jeudi, P = 0.04 and P = 0.005, respectively). Although transmission declined in the other two sites, larval recoveries were low and these changes were not statistically significant. DISCUSSION: Our results suggest that a single round of mass treatment can have an impact on transmission of lymphatic filariasis. The use of entomologic methods as a tool to monitor filariasis programs and the statistical limitations of mosquito trapping are discussed.     

Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene

Anopheles gambiae, the major vector of human malaria parasite, is an important insect model to study vector–parasite interactions. Here, we developed a simple in vivo double-stranded RNA (dsRNA) knockout approach to determine the function of the mosquito antimicrobial peptide gene Defensin. We injected dsRNA into adults and observed efficient and reproducible silencing of Defensin. Analysis of the knockdown phenotype revealed that this peptide is required for the mosquito antimicrobial defense against Gram-positive bacteria. In contrast, in mosquitoes infected by Plasmodium berghei, no loss of mosquito viability and no significant effect on the development and morphology of the parasite midgut stages were observed in the absence of Defensin. We conclude that this peptide is not a major antiparasitic factor in A. gambiae in vivo. Our results open new perspectives for the study of mosquito gene function in vivo and provide a basis for genome-scale systematic functional screens by targeted gene silencing.     

Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum

Deciphering molecular interactions between the malaria parasite and its mosquito vector is an emerging area of research that will be greatly facilitated by the recent sequencing of the genomes of Anopheles gambiae mosquito and of various Plasmodium species. So far, most such studies have focused on Plasmodium berghei, a parasite species that infects rodents and is more amenable to studies. Here, we analysed the expression pattern of nine An.gambiae genes involved in immune surveillance during development of the human malaria parasite P.falciparum in mosquitoes fed on parasite-containing blood from patients in Cameroon. We found that P.falciparum ingestion triggers a midgut-associated, as well as a systemic, response in the mosquito, with three genes, NOS, defensin and GNBP, being regulated by ingestion of gametocytes, the infectious stage of the parasite. Surprisingly, we found a different pattern of expression of these genes in the An.gambiae-P.berghei model. Therefore, differences in mosquito reaction against various Plasmodium species may exist, which stresses the need to validate the main conclusions suggested by the P.berghei-An.gambiae model in the P.falciparum-An.gambiae system.     

Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized alpha/beta motifs

Insect defensins containing cysteine-stabilized alpha/beta motifs (Cs-alpha/beta defensin) are cationic, inducible antibacterial peptides involved in humoral defence against pathogens. To examine trends in molecular evolution of these antimicrobial peptides, sequences similar to the well-characterized Cs-alpha/beta defensin peptide of Anopheles gambiae, using six cysteine residues as landmarks, were retrieved from genomic and protein databases. These sequences were derived from different orders of insects. Genes of insect Cs-alpha/beta defensin appear to constitute a multigene family in which the copy number varies between insect species. Phylogenetic analysis of these sequences revealed two main lineages, one group comprising mainly lepidopteran insects and a second, comprising Hemiptera, Coleoptera, Diptera and Hymenoptera insects. Moreover, the topology of the phylogram indicated dipteran Cs-alpha/beta defensins are diverse, suggesting diversity in immune mechanisms in this order of insects. Overall evolutionary analysis indicated marked diversification and expansion of mature defensin isoforms within the species of mosquitoes relative to non-mosquito defensins, implying the presence of finely tuned immune responses to counter pathogens. The observed higher synonymous substitution rate relative to the nonsynonymous rate in almost all the regions of Cs-alpha/beta defensin of mosquitoes suggests that these peptides are predominately under purifying selection. The maximum-likelihood models of codon substitution indicated selective pressure at different amino acid sites in mosquito mature Cs-alpha/beta defensins is differ and are undergoing adaptive evolution in comparison to non-mosquito Cs-alpha/beta defensins, for which such selection was inconspicuous; this suggests the acquisition of selective advantage of the Cs-alpha/beta defensins in the former group. Finally, this study represents the most detailed report on the evolutionary strategies of Cs-alpha/beta defensins of mosquitoes in particular and insects in general, and indicates that insect Cs-alpha/beta defensins have evolved by duplication followed by divergence, to produce a diverse set of paralogues.     

斯氏按蚊泛素羧端水解酶对约氏疟原虫感染应答性表达增高(英文)

分离和研究疟疾感染蚊的差异表达基因 ,对阐明媒介与疟原虫之间相互作用及其分子机制尤为重要。利用已建立的斯氏按蚊感染约氏疟原虫的差减cDNA库的进行表达筛选 ,发现表达增高基因中有一个编码与黑腹果蝇泛素羧端水解酶高度同源蛋白的序列。相似性比较显示该编码序列在氨基酸水平与已知的冈比亚按蚊EST序列对应部位的同源性为 89% ,与果蝇和人类的同源性均为 63%。模拟Northern印迹的表达动态分析提示 ,感染后至少 1～ 7天内该基因在蚊体内的表达显著增高 ,与疟原虫发育动合子穿越蚊中肠壁和子孢子从卵囊向蚊眼涎腺移行等关键阶段相一致。目前对有关蚊天然免疫系统激活的泛素途径所知甚少 ,现有结果提示该基因与疟原虫感染相关 ,它的克隆和表达分析有可能推测其在疟原虫感染中所起的作用     

Identification and partial characterization of a parasite antigen in sera from humans infected with Wuchereria bancrofti

The purpose of this study was to identify and characterize soluble parasite antigens present in sera from humans infected with the filarial nematode Wuchereria bancrofti. Affinity chromatography and immunoblot methods were used to demonstrate a 200,000 m.w. circulating parasite antigen in sera from infected humans which corresponded to an antigen released by adult W. bancrofti during in vitro culture. Two monoclonal antibodies were produced to this antigen by immunizing mice with antigens from Dirofilaria immitis, a filarial parasite that is closely related to W. bancrofti, and screening cell fusion supernatants by enzyme immunoassay and counterimmunoelectrophoresis inhibition. The antibodies bound to a single repeated epitope (not phosphorylcholine) that was resistant to heat, acid, and protease treatments but sensitive to periodate oxidation. Immunoperoxidase studies showed that the epitope was concentrated in the cuticle and reproductive organs in D. immitis, and it was released in relatively large amounts by adult female D. immitis in vitro. The epitope is also present in antigens of other species of filarial and nonfilarial nematodes, but on the basis of preliminary studies, its presence in human serum appears to be specific for W. bancrofti infection.     

Anopheles and Plasmodium: from laboratory models to natural systems in the field

Parasites that cause malaria must complete a complex life cycle in Anopheles vector mosquitoes in order to be transmitted from human to human. Previous gene-silencing studies have shown the influence of mosquito immunity in controlling the development of Plasmodium. Thus, parasite survival to the oocyst stage increased when the parasite antagonist gene LRIM1 (leucine-rich repeat immune protein 1) of the mosquito was silenced, but decreased when the C-type lectin agonist gene CTL4 or CTLMA2 (CTL mannose binding 2) was silenced. However, such effects were shown for infections of the human mosquito vector Anopheles gambiae with the rodent parasite Plasmodium berghei. Here, we report the first results of A. gambiae gene silencing on infection by sympatric field isolates of the principal human pathogen P. falciparum. In contrast with the results obtained with the rodent parasite, silencing of the same three genes had no effect on human parasite development. These results highlight the importance of following up discoveries in laboratory model systems with studies on natural parasite-mosquito interactions.     

Brugia pahangi: effects upon the flight capability of Aedes aegypti.

Aedes aegypti mosquitoes were adversely affected by infections of the filarial worm Brugia pahangi. Infected mosquitoes flew significantly shorter distances and showed marked reductions in total flight time during 24-hr flight mill tests compared to uninfected controls. Total flight range and duration flown by infected mosquitoes remained relatively constant throughout the infection process, while control mosquitoes flew further and longer with increasing time after their blood meal. Furthermore, a significantly greater number of infected mosquitoes either died or were rendered incapable of flight. Of flying and nonflying mosquitoes with 6-day-old or older infections dissected for parasite burdens, the nonflying group contained significantly more worms. Results of this study indicate that developing filarial larvae within this mosquito vector reduce its ability to survive and to transmit its infection by reducing its flight capabilities. Conclusions from this study relate only to A. aegypti homozygous for the gene fm which is fully susceptible to this filarial parasite.     

Anopheles gambiae SRPN2 facilitates midgut invasion by the malaria parasite Plasmodium berghei

We report on a phylogenetic and functional analysis of genes encoding three mosquito serpins (SRPN1, SRPN2 and SRPN3), which resemble known inhibitors of prophenoloxidase-activating enzymes in other insects. Following RNA interference induction by double-stranded RNA injection, knockdown of SRPN2 in adult Anopheles gambiae produced a notable phenotype: the appearance of melanotic pseudotumours, which increased in size and number with time, indicating spontaneous melanization and association with an observed lifespan reduction. Furthermore, knockdown of SRPN2 strongly interfered with the invasion of A. gambiae midguts by the rodent malaria parasite Plasmodium berghei. It did not affect ookinete formation, but markedly reduced oocyst numbers, by 97%, as a result of increased ookinete lysis and melanization.     

Dirofilaria immitis: effect on the longevity of Aedes trivittatus

Laboratory studies on vector mortality as related to parasite burden revealed that the mosquito Aedes trivittatus showed considerable tolerance to infection with the filarial nematode Dirofilaria immitis. Although mosquitoes exposed to a dog with a high microfilaremia (347 microfilariae/20 mm³) had a significant increase in mortality during the first 16 days postexposure, 66% of the mosquitoes lived long enough for complete parasite development to occur. Those mosquitoes exposed to a dog with a low microfilaremia (62 microfilariae/20 mm³) had no significant increase in mortality. There was a strong negative correlation between parasite burden and mosquito survival, but only mosquitoes harboring more than 15 juveniles had an increased chance of dying before D. immitis could develop to the infective stage. The retention of microfilariae within the blood clot and peritrophic membrane of A. trivittatus seems beneficial to this vector-parasite system by reducing the parasite burden and increasing mosquito longevity.     

UP REGULATION OF UBIQUITIN C-TERMINAL HYDROLASE IN THE RESPONSE OF THE MOSQUITO ANOPHELES STEPHENS TO PLASMODIUM YOELII INFECTION

Abstract The isolation and study of genes that are differentially expressed in malaria-infected mosquitoes is important for the elucidation of basic molecular mechanisms underlying vector-parasite interactions. When screening against a previously established cDNAs pool representing specifically expressed genes in the mosquito Anopheles stephemi infected by Plasnwdium γoelii , it was found that one of these encodes a protein with extensive sequence similarity to the Drosophila melarwgaster ubiquitin C-terminal hydrolase (UCTH). Similarity alignment showed that the fragment is 89% identical at amino acid level to the corresponding region of the known An. gambiae EST sequence, as well as 63 % identical to that of both the fruittly and human sequence. Virtual Northern blot expression dynamics of the gene indicated that it was up-regulated significantly in the mosquito at least 1–7 days post-infection, consistent with the critical transition stages of midgut invasion and relocation of sporozoites from the oocysts to the salivary glands during parasite development. Rather little is known about the role of the ubiquitin pathway in the activation of the mosquito innate immune system. The results indicate that the gene is related to malaria infection in mosquito. The cloning and expression profile analysis of AsUCTH enables us to make predictions as to the roles it may play during malaria infection.     

The mosquito genome: perspectives and possibilities

Anopheles gambiae is the mosquito vector responsible for transmitting Plasmodium falciparum, a malaria parasite of humans. With the emergence of genome projects for a variety of prokaryotic and eukaryotic microorganisms, there has been a long-standing interest in sequencing the genomes of the malaria parasite and its insect vector. This tour de force effort has now been completed and reported. The alignment of putative orthologs in An. gambiae with those of Drosophila melanogaster highlights several similarities and differences. These findings could have implications in: (1) identifying new targets for insecticide development; (2) strengthening our understanding of the developmental biology of mosquitoes; and (3) possibly controlling pathogen transmission. A brief overview of these interesting findings and the implications for further studies will be discussed here.