Mosquito Armigeres Subalbatus Research Articles

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.

Population dynamics of Armigeres subalbatus (Diptera: Culicidae) across a temperate altitudinal gradient.

Understanding the impacts of weather fluctuations, and environmental gradients, on the abundance of vectors is fundamental to grasp the dynamic nature of the entomological risk for disease transmission. The mosquito Armigeres subalbatus (Coquillet) is a common vector of filariasis. Nevertheless, its population dynamics have been relatively poorly studied. Here, we present results from a season long study where we studied spatio-temporal abundance patterns of Ar. subalbatus across the altitudinal gradient of Mt. Konpira in Nagasaki, Japan. Spatially, we found that abundance of adult Ar. subalbatus decreased with altitude and increased in areas where the ground was rich in leaf litter. Similarly, adult activity was observed only when relative humidity was over 65%. Temporally, we found that peaks in abundance followed large rainfall events. Nevertheless, this mosquito was under significant density dependence regulation. Our results suggest that Ar. subalbatus population peaks following large rainfall events could reflect the recruitment of individuals that were dormant as dry eggs. We did not find a clear signal of temperature on abundance changes of this mosquito, but only on its phenology. Since ground cover seemed more critical than temperature to its spatial distribution, we propose that this mosquito might have some degree of autonomy to changes in temperature.

Establishment and characterization of two new cell lines from the mosquito Armigeres subalbatus (Coquillett) (Diptera: Culicidae).

Armigeres subalbatus (Coquillett) is a medically important mosquito and a model species for immunology research. We successfully established two cell lines from the neonate larvae of A. subalbatus using two different media. To our knowledge, this is the first report of an established Armigeres mosquito cell line. The cell lines, designated as Ar-3 and Ar-13, consist of adherent and diploid cells with compact colonies. Both these cell lines grow slowly after passage at a split ratio of 1:5 and a population doubling time of 2.7 and 3.0 d, respectively. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm that these lines correspond to the species of origin and are clearly distinct from seven other insect cell lines. Furthermore, reverse-transcription PCR was used to demonstrate that the Ar-3 cell line is susceptible to the Japanese encephalitis virus and two insect flaviviruses associated with Culex and Aedes mosquitoes but relatively insensitive to dengue virus. These data indicate that the newly established cell lines are cellular models of A. subalbatus as well as beneficial tools for the propagation of viruses associated with the Armigeres mosquito.

Functions of Armigeres subalbatus C-type lectins in innate immunity

C-type lectins (CTLs) are a superfamily of calcium-dependent carbohydrate binding proteins containing at least one carbohydrate-recognition domain (CRD) and they are present in almost all metazoans. Insect CTLs may function as pattern-recognition receptors and play important roles in innate immunity. In this study, we selected five AsCTLs from the mosquito Armigeres subalbatus, a natural vector of filarial nematodes, and performed both in vitro and in vivo studies to elucidate their functions in innate immunity. AsCTLMA15, AsCTLGA5 and AsCTL15 were mainly expressed in hemocytes, AsCTL16 was expressed in fat body, while AsCTLMA11 was expressed in both hemocytes and fat body, and only AsCTLMA11 and AsCTL16 were expressed at high levels in adult females. In vitro binding assays showed that all five recombinant AsCTLs could bind to different microbial cell wall components, including lipopolysaccharide (LPS), lipid A, peptidoglycan (PG), lipoteichoic acid (LTA), zymosan and laminarin (beta-1,3-glucan). Recombinant AsCTLs also bound to several Gram-negative and Gram-positive bacteria, and could agglutinate bacterial cells. Injection of double-stranded RNAs (dsRNAs) could significantly reduce expression of the five AsCTL mRNAs, and the survival of mosquitoes treated with dsRNA to AsCTLGA5 was significantly decreased after Escherichia coli infection, but did not change significantly after Micrococcus luteus infection compared to the control groups, suggesting that Ar. subalbatus AsCTLGA5 may participate in innate immunity against E. coli.

Identification and functional analysis of the peptidoglycan recognition protein LD gene in the mosquito, Armigeres subalbatus

Peptidoglycan recognition proteins are important recognition proteins in many organisms ranging from echinoderms to humans. In an attempt to characterize all the PGRPs in the mosquito Armigeres subalbatus, two PGRP-LD isoforms, AsPGRP-LDa and AsPGRP-LDb, which are orthologs of the PGRP-LDs in several other insect species, were identified from this mosquito using homologous cloning. To date the functions of this PGRP gene have not yet been described in detail in other organisms with a known PGRP-LD gene. In the current study, we analyzed the sequences of these AsPGRP-LDs, their evolutionary relationships with their orthologs, their transcriptional expression in various developmental stages and different tissue samples, and their transcriptional responses to different bacterial stimuli. We then knocked down the expression of both AsPGRP-LDs by injection of double-stranded RNAs, and assessed the impact of AsPGRP-LD RNAi on mosquito survival after bacterial challenges and on the transcriptional expression of a number of antimicrobial peptides.

Estimate of survivorship of immature stages of the mosquito Armigeres subalbatus (Coquillett, 1898)

The survivorship of immature stages of the mosquito Armigeres subalbatus (Coquillett, 1898) (Diptera: Culicidae) was evaluated under laboratory and field conditions. In the laboratory, the average time taken by the immature stages to reach the adult stage was less in summer (9.1 days) compared to winter (15.3 days), probably owing to seasonal temperature differences. Survival of eggs to the pupal stage varied between 56% and 75% in the laboratory and 9–60% in field conditions, depending on the season. The instar duration and stage-specific survivorship varied significantly with the season. Field data revealed that cohort survival was highest during the monsoon season and lowest in summer. In a sewage drain habitat, the spatial distribution of larvae and pupae remained highly aggregated as reflected in the coefficient of variation values (consistently > 1). A stable age distribution in terms of constancy in proportions of different immature stages over time was lacking in the sewage drains, possibly due to cannibalism and predation by co-occurring predatory mosquito larvae. The life table parameters of A. subalbatus reveal that the abundance of this mosquito varied with season, though a viable population level may exist throughout the year, posing a public health problem as a vector and nuisance pest.

Insights into the different functions of multiple peptidoglycan recognition proteins in the immune response against bacteria in the mosquito, Armigeres subalbatus

Peptidoglycan recognition proteins (PGRPs) are a group of proteins that recognize and/or bind to peptidoglycan on the surface of a number of pathogens. To understand the roles of multiple PGRPs in the mosquito Armigeres subalbatus (AsPGRPs), we studied the effects of infection of two bacteria, the gram negative Escherichia coli and the gram positive Micrococcus luteus, on the transcriptional expression of AsPGRPs and RNA interference (RNAi) of AsPGRPs on the immune responses of mosquitoes against the two bacteria. Injection of E. coli or M. luteus into adult mosquitoes both significantly increased the transcription of AsPGRP-S1, but not the other AsPGRPs. A mosquito survival assay using injection of E. coli or M. luteus into AsPGRP double-stranded RNA (dsRNA) injected mosquitoes showed that RNAi of AsPGRPs had different impacts on the survival abilities of mosquitoes, and that AsPGRP-LCs seem to be the most critical ones. Real-time Polymerase Chain Reaction (real-time PCR) analysis indicated that the expression of four antimicrobial peptides (AMPs) was dramatically changed after AsPGRP-LB and AsPGRP-LC RNAi, although AsPGRP-S1 and AsPGRP-LE had slight, but significant, effects, suggesting that the changes in survival abilities were potentially due to the changes in AMP expression after AsPGRP RNAi. In addition, bacterial challenges following AsPGRP-LC RNAi did not induce the expression of AMPs to their normal level as in control experiments. An in vivo assay indicated that AsPGRP-LC RNAi had no significant effects on the phagocytic ability of the hemocytes, suggesting that AsPGRP-LC is not a key factor mediating phagocytosis of bacteria in this mosquito.

Cloning and Characterization of the Peptidoglycan Recognition Protein Genes in the Mosquito,Armigeres subalbatus(Diptera: Culicidae)

Peptidoglycan recognition proteins (PGRPs) are a group of proteins that are responsible for the recognition and, in some cases, binding of peptidoglycan (PGN), a unique cell wall component of bacteria, and initiation of immune responses to various types of pathogens. In the current study, full-length cDNA sequences of multiple PGRPs, identified via a database search, were cloned in the mosquito Armigeres subalbatus (Coquillett). During cloning, a novel transcript variant (isoform) of AsPGRP-LC (As: Ar. subalbatus) was also identified that shares a large 5' end fragment with AsPGRP-LC. All four AsPGRP genes (six transcripts) contain a conserved PGRP domain, an ortholog of the amidase-2 domain. Based on predicted functional domain, the six Ar. subalbatus PGRPs resemble both short (AsPGRP-S1) and long (AsPGRP-LBa, AsPGRP-LBb, AsPGRP-LCa, AsPGRP-LCb, and AsPGRP-LE) forms of PGRPs as in other insects. Sequence alignments showed that PGRPs are conserved across Dipterans. Phylogenetic analysis indicated that PGRPs represent an ancient gene family that has primarily diverged through speciation events among these Dipterans, with only a limited number of lineage specific gene duplications. Developmental profiling of the six AsPGRP transcripts using real-time polymerase chain reaction revealed that AsPGRP-LCa and AsPGRP-LCb are constitutively expressed at high levels in all developmental stages, while AsPGRP-S1, AsPGRP-LBa, AsPGRP-LBb, and AsPGRP-LE transcripts have low expression in most of the life stages and are increased only at certain times. Tissue profiling of the six AsPGRP transcripts showed that they are expressed in various patterns, even between the different isoforms of the same PGRP gene, indicating that these AsPGRPs may play different functions.

Hemolytic activity is mediated by the endogenous lectin in the mosquito hemolymph serum

Although cytolysis of invading organisms is an innate form of immunity used by invertebrates, so far the underlying mechanism remains less explored. The pupal hemolymph of the mosquito Armigeres subalbatus induces an activity that causes hemolysis of human red blood cells (HRBC). This hemolytic activity was inhibited by sialic acid (N-acetylneuraminic acid) and serine protease inhibitors. We purified the sialic acid-specific lectin(s) from the pupal hemolymph using formaldehyde-fixed HRBC and determined the sequence of the amino-terminal 19 amino acid residues. A polyclonal antibody produced against this N-terminal peptide clearly inhibited the hemolytic activity of the hemolymph in vitro, thus suggesting that the hemolysis of HRBC is caused by the lectin present in the mosquito hemolymph. We suggest that mosquitoes possess a cytolysis system.

Response of <I>Armigeres subalbatus</I> (Diptera: Culicidae) to Intraperitoneally Isolated <I>Brugia</I> spp. Microfilariae

The relationship between mosquito and parasite involves a delicate balance that is influenced not only by the mosquito but also by parasite determinants. Using the biologically and morphologically similar parasites Brugia malayi and Brugia pahangi and the mosquito Armigeres subalbatus (Coquillett) (Diptera: Culicidae), it should be possible to dissect out the key elements involved in initiating or avoiding an immune response, known as melanotic encapsulation, because in this mosquito B. malayi microfilariae (mf) are melanized and destroyed, but B. pahangi mf develop normally into infective-stage larvae. Because of limitations in isolating sufficient mf from the circulation of an infected mammalian host, Brugia spp. mf that can be obtained in large numbers from the peritoneal cavity of an infected host were tested to ascertain the immune response of Ar. subalbatus to this source of mf. Results indicate that the immune response of Ar. subalbatus against intraperitoneal (i.p.) Brugia spp. mf mimics that which is observed when this mosquito is exposed to mf-infected animals, indicating that i.p. mf are similar to those mf that circulate naturally in the blood of the vertebrate host. Therefore, the i.p. mf should serve as an excellent source of material for genomic and proteomic studies designed to analyze the role of the parasite in influencing the immune response of the mosquito.

Sequence Analysis of Mitochondrial 16S Ribosomal RNA Gene Fragment in the Two Populations of Armigeres (Armigeres) subalbatus (Culcidae: Diptera)

The present research work was aimed at the sequence analysis of mitochondrial 16S rDNA gene fragment in 2 wild populations (A and B) of a mosquito Armigeres subalbatus which is a carrier of filariasis and Japanese encephalitis in humans. The purified samples of mitochondrial DNA were amplified by using 2 specific oligonucleotide primers-5′-CGCCTGTTTATCAAAAACAT-3′ and 5′-CTCCGTTTGAACTCAGATC-3′ which generated a distinct band of 537 base pairs each for the 2 populations. The multiple sequence alignment also revealed indels (insertions deletions) of bases located at positions 1 and 24. As per the incidence of substitutions, the rate of transversions was found to be more than the transitions and the transversion to transition ratio varied from 1.3 to 0.55. This particular gene sequence of Armigeres subalbatus was also found to be A–T rich. In population A the average base frequencies were: A=0.39, C=0.14, G=0.09 and T=0.38 and the A+T content was 78.2% while in population B these frequencies were: A=0.38, C=0.15, G=0.08 and T=0.39 and the A+T content was 77.8%. Since a close relationship exists between the rates of nucleotide substitution and “form and function” of genes, a single gene seems to be functional at different allelic levels and the 2 populations were genomically distinct.

Role of dopachrome conversion enzyme in the melanization of filarial worms in mosquitoes

Melanization is an effective defence reaction of mosquito hosts against invading parasites. In mosquitoes, the biosynthesis of melanin is initiated by the hydroxylation of tyrosine to DOPA by phenoloxidase (PO). DOPA is a branch point of the melanization reaction; it may be oxidized to dopaquinone by PO or be decarboxylated to dopamine by dopa decarboxylase. Further oxidation of dopaquinone by PO produces dopachrome. Dopachrome is then converted to 5, 6-dihydroxyindole by dopachrome conversion enzyme (DCE) to produce melanin. The conversion of dopachrome is a rate-limiting step of the melanization reaction, and the presence of PO and DCE significantly accelerates melanization reactions. In this study, a cDNA encoding DCE was cloned from the mosquito Armigeres subalbatus. Real-time PCR analysis revealed increased transcripts from haemocytes in microfilariae (mf)-inoculated mosquitoes. Gene silencing using double-stranded RNA was used to elucidate the role of DCE in the melanization reaction of parasites in Ar. subalbatus. The levels of both DCE transcripts and protein in gene knockdown mosquitoes were dramatically reduced. Compared with controls, the degree of melanization of mf in DCE-knockdown mosquitoes was significantly decreased. These results suggest that DCE is a critical enzyme that is required for effective melanization immune responses.

The use of a double subgenomic Sindbis virus expression system to study mosquito gene function: effects of antisense nucleotide number and duration of viral infection on gene silencing efficiency

Recently we established a simple, effective antisense strategy using a double subgenomic Sindbis (dsSIN) virus expression system to study gene function in mosquitoes. In this study, we further elucidate the effects of antisense nucleotide number and duration of viral infection on mosquito gene silencing efficiency by the dsSIN virus expression system. Over 15 days post virus infection, the degree of parasite melanization was progressively reduced by more than 95%, 75% and 55% in the mosquito Armigeres subalbatus transduced with 600, 147 or 36 bases antisense RNA, targeted to the highly conserved copper binding region of the Ar. subalbatus prophenoloxidase I gene (As-pro-POI), respectively. As the duration of viral infection increased from day 3-15, the degree of parasite melanization progressively decreased in all mosquitoes transduced with antisense RNA, irrespective of the lengths of antisense RNA. Progressive loss of parasite melanization function was found to correlate with down regulation of As-pro-PO expression at both the mRNA and protein activity levels, and reductions in virus titres in mosquitoes transduced with antisense RNA. A small pro-PO RNA (c. twenty-five nucleotides) was identified in mosquitoes transduced with antisense RNA. These data suggest that As-pro-POI gene expression is knocked down by degrading the As-pro-POI mRNA through the RNAi pathway. In conclusion, our study demonstrates that even a short antisense RNA (thirty-six bases) can cause silencing of the As-pro-POI gene, and the effects of endogenous gene silencing by dsSIN expression system on mosquito gene functions can be accumulative.

Hemocyte-mediated phagocytosis and melanization in the mosquito Armigeres subalbatus following immune challenge by bacteria.

Mosquitoes are important vectors of disease. These insects respond to invading organisms with strong cellular and humoral immune responses that share many similarities with vertebrate immune systems. The strength and specificity of these responses are directly correlated to a mosquito's ability to transmit disease. In the current study, we characterized the hemocytes (blood cells) of Armigeres subalbatus by morphology (ultrastructure), lectin binding, enzyme activity, immunocytochemistry, and function. We found four hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. Granulocytes contained acid phosphatase activity and bound the exogenous lectins Helix pomatia agglutinin, Galanthus nivalis lectin, and wheat germ agglutinin. Following bacteria inoculation, granulocytes mounted a strong phagocytic response as early as 5 min postexposure. Bacteria also elicited a hemocyte-mediated melanization response. Phenoloxidase, the rate-limiting enzyme in the melanization pathway, was present exclusively in oenocytoids and in many of the melanotic capsules enveloping bacteria. The immune responses mounted against different bacteria were not identical; gram(-) Escherichia coli were predominantly phagocytosed and gram(+) Micrococcus luteus were melanized. These studies implicate hemocytes as the primary line of defense against bacteria.

Electron microscopic observations on wound-healing in larvae of the mosquito Armigeres subalbatus (Diptera: Culicidae).

The wound-healing processes in the mosquito Armigeres subalbatus (Coquillett) were observed with electron microscopy. The initial reaction involved wound contractions and aggregation of injured surface tissues, cell debris and movement of granulocytes toward the wound. Granulocytes first aggregated around the surface of the wound and many filamentous filopodia protruded to connect with cytoplasmic strands. These strands were then interconnected to form a network coagulum resulting in wound closure to prevent body fluid loss. Granulocytes lysed on the wound-site and released granular materials around the wound, inducing localized clot formation. These results suggested that wound-healing in this mosquito species involved both humoral and cellular reactions. The latter reaction involved the movement of plasmatocytes to the basement membrane of the epidermis beneath the wound-site and epithelial cells regeneration. Our observations revealed that wound-healing in A. subalbatus involves the wound contraction, formation of a temporary cellular clump, scar formation, basement membrane formation, and reepithelialization. The larvae neither discarded the wound scar nor secreted a new cuticle until the next molting. Based on the ultrastructural observations, it is suggested that the wound-healing reaction in A. subalbatus was probably a typical response employed by other members of the family Culicidae.

Molecular cloning, characterization and tissue expression of prophenoloxidase cDNA from the mosquito Armigeres subalbatus inoculated with Dirofilaria immitis microfilariae.

A cDNA encoding mosquito Armigeres subalbatus prophenol oxidase (As-pro-PO) was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) after Dirofilaria immitis inoculation. The 2205 bp As-pro-PO cDNA contains a 32 bp 5'-noncoding region, a 2055 bp open reading frame (685 amino acids), and a 118 bp 3'-noncoding region. Hydrophobic signal peptide for the endoplasmic reticulum targeting is not found in the NH2-terminal region. Two potential copper-binding domains, amino acids 197-245 and 345-412, are highly homologous to those of the other insect pro-POs. A 2.2 kb As-pro-PO transcript was identified by Northern blot analysis using D. immitis microfilariae-inoculated A. subalbatus. Both in situ hybridization and Northern blot analysis demonstrated that As-pro-PO mRNA was synthesized in mosquito haemocytes but not in other tissues, i.e. fat bodies, midguts and ovaries, etc.

Hemocyte Alterations during Melanotic Encapsulation of Brugia malayi in the Mosquito Armigeres subalbatus

The involvement of hemocytes in melanotic encapsulation reactions against Brugia malayi was assessed in Armigeres subalbatus. Hemocyte populations, epitope changes, phenol oxidase (PO) activity, and the presence of an 84-kDa polypeptide were investigated in mosquitoes exposed to a B. malayi-infective bloodmeal (= immune-activated), in mosquitoes given a noninfective bloodmeal (= controls), in nonbloodfed mosquitoes (= naive), or in some combination of these. Total hemocyte populations in immune-activated mosquitoes significantly decreased at 24 hr postbloodmeal (PB) as compared with controls. At 48 and 72 hr PB, hemocyte population levels in immune-activated mosquitoes increased to control levels. Epitope changes, as indicated by wheat germ agglutinin (WGA) binding, also were observed. There was a significant increase in the percentage of hemocytes binding WGA in immune-activated mosquitoes at 24 hr PB as compared with controls. Furthermore, the activity of hemocyte PO, an enzyme involved in the melanotic encapsulation pathway, was significantly elevated at 12 hr PB in immune-activated mosquitoes as compared with controls. Analysis for the presence of an 84-kDa polypeptide in A. subalbatus indicates that a 2.0-kb message in total RNA hybridized to D6.12, an Aedes aegypti cDNA encoding an 84-kDa polypeptide that is associated with melanotic encapsulation responses. The hybridization of D6.12 to RNA was not greater in immune-activated as compared to control A. subalbatus, as has been observed in A. aegypti. Results indicate that these hemocyte changes correspond in time with the melanotic encapsulation reactions of A. subalbatus against filarial worms.

Reproductive Costs Associated with Resistance in a Mosquito-Filarial Worm System

The mosquito Armigeres subalbatus can encapsulate and kill > 80% of Brugia malayi microfilariae (mf) within 36 hr following ingestion. The cascade of biochemical events constituting this melanotic encapsulation response is also important in other mosquito biological events, including egg-chorion tanning. Certain biochemical entities, including a tyrosine precursor, are thought to be shared among these biological activities. Because of this purported tyrosine link, and because the blood meal both initiates egg development and is the source of mf, we evaluated the possibility that reproductive cost is incurred by the resistant host when undergoing a response to mf acquired in an infected blood meal. Mean time to oviposition was significantly longer for mosquitoes responding to parasites than for controls (77.7 versus 66.5 hr). Tyrosine levels in ovaries from infected mosquitoes were less than half those of controls at 24 and 48 hr, and were still significantly reduced at 72 hr following blood feeding. Ovary development, assessed via measurements and total protein content, also was delayed significantly in the experimental group, with ovary width and protein content never attaining levels found in control mosquitoes. Sections from 24-hr post-blood meal ovaries demonstrated that the normal processes of egg development, including vitelline accumulation, was drastically altered as well. The biological implications of these results are considered.

Double infection in a mosquito Armigeres subalbatus (Coquillett) (A. obturbans auct.) with two different species of filaria.

Étude de l'infection simultanée du moustiqueArmigeres subalbatus par deux espèces de filaires,Brugia pahangi etBreinlia sergenti. Les stades de developpement de ces filaires peuvent être facilement differenciés du fait que leB. pahangi se tient exclusivement dans la musculature thoracique et leB. sergenti dans le tissu adipeux.

Developmental Changes in the Protein Constitution of a Mosquito (Armigeres subalbatus) as Revealed by Disc Electrophoresis

Developmental Changes in the Protein Constitution of a Mosquito (Armigeres subalbatus) as Revealed by Disc Electrophoresis