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Cloning and characterization of cDNAs encoding the antibacterial peptide, defensin A, from the mosquito, Aedes aegypti

Insect defensins are cationic, inducible antibacterial peptides. Four full-length cDNAs encoding defensin A from the mosquito Aedes aegypti were cloned using polymerase chain reaction (PCR) and sequenced. All four cDNAs are 473 base pairs long, bearing an open reading frame of 98 amino acids with a few substitutions in the signal peptide domain. The deduced amino acid sequence of Aedes aegypti defensin (AaDef) contains a signal peptide sequence of 18 amino acids followed by a 40-amino acid putative propeptide domain and a 40-amino acid mature peptide domain. The mature peptide, with a predicted M(r) of 4148, shows 80% identity and 93% similarity to Phormia defensin A, and is identical to the peptide sequencing data for mosquito defensin A of Lowenberger et al. (1995) and B of Chalk et al. (1995). There are three potential phosphorylation sites but no glycosylation sites detected in AaDef. Three putative disulfide linkages between cysteines, characteristic of insect defensins, are conserved in AaDef. Aedes aegypti defensin mRNA is produced in response to a bacterial challenge; it is dramatically enhanced 6 h after bacterial injection, continues to increase through 24 h, and is maintained at high levels until at least 30 h post-bacterial injection.

A potential role for phenylalanine hydroxylase in mosquito immune responses.

In mosquitoes the melanotic encapsulation immune response is an important resistance mechanism against filarial worms and malaria parasites. The rate limiting substrate for melanin production is tyrosine that is hydroxylated by phenoloxidase (PO) to produce 3, 4-dihydroxyphenylalanine. The single pathway for endogenous production of tyrosine is by hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH). In this study we describe a potential role for PAH in melanotic immune responses in the yellow fever mosquito, Aedes aegypti. A 1.6 kb A. aegypti PAH cDNA, encoding a 51 kDa protein, was isolated and subsequently expressed in an Escherichia coli expression system. In developing mosquitoes, PAH transcript is present in all stages and it is differentially expressed in adult tissues. Following an immune-challenge with Dirofilaria immitis microfilariae (mf) or bacteria, PAH transcript is up-regulated in hemocytes. Likewise, western analysis of hemocytes collected from immune-activated mosquitoes show an increase in gene product over control samples. Like PO, ultrastructure observations provide verification that PAH is located in oenocytoid and granulocyte hemocytes. Our results offer the first data that suggest PAH is used in mosquito melanin synthesis and defense responses.

Molecular cloning of a second prophenoloxidase cDNA from the mosquito Armigeres subalbatus: prophenoloxidase expression in blood-fed and microfilariae-inoculated mosquitoes

Melanization constitutes an important component in various aspects of insect life, including cuticular sclerotization, egg‐shell tanning, melanization of parasites and wound healing. Recently, a cDNA encoding prophenoloxidase (pro‐PO), a key enzyme in the biosynthesis of melanotic material in insects, was cloned from microfilariae (mf)‐inoculated mosquitoes, Armigeres subalbatus. However, results of Northern blot analyses indicated that two pro‐POs might be present in Ar. subalbatus and these pro‐POs might be responsible for two distinct physiological functions, egg‐shell tanning and melanization of parasites. Subsequently, the second pro‐PO cDNA (As‐pro‐PO II) was cloned from blood‐fed Ar. subalbatus by rapid amplification of cDNA ends polymerase chain reaction. The 2210 bp As‐pro‐PO II cDNA contains a 41 bp 5′‐non‐coding region, a 2064 bp open reading frame and a 105 bp 3′‐non‐coding region. A hydrophobic signal peptide for endoplasmic reticulum targeting is not found in the N‐terminal region. The deduced amino acid sequence of As‐pro‐PO II shares a high degree of identity (81.5%) with that of the As‐pro‐PO I obtained from mf‐inoculated Ar. subalbatus. Both Northern blot and reverse transcription polymerase chain reaction analysis demonstrated that these two mosquito pro‐POs are persistently expressed in mosquito haemocytes and not in fat body, midgut, or ovaries. The expression of As‐pro‐PO I and As‐pro‐PO II in mosquitoes is associated with melanization of mf and blood feeding, respectively.

Molecular characterization of a defensin gene from the mosquito, Aedes aegypti

Insect immune proteins, defensins, are inducible anti-Gram-positive bacterial peptides. We report here the identification of two defensin genes from the mosquito, Aedes aegypti, which encode a large 541 bp transcript (AaDef Ala) and a small 473 bp transcript (AaDef Asm). The cDNA corresponding to AaDef Ala was cloned, sequenced, and compared with the previously reported AaDef Asm cDNA. The AaDef Ala gene was isolated through genomic library screening and characterized. It putative regulatory region contains a 64 bp intron, a TATA box and a putative arthropod initiator. Two 150 bp long direct and several palindromic repeats are present in this sequence. Similar to other insect immune peptide genes, the AaDef Ala gene contains numerous putative regulatory motifs with impressive similarity to elements of vertebrate acute phase response protein genes.

The use of gene silencing to study the role of dopa decarboxylase in mosquito melanization reactions

Mosquito melanization involves hydroxylation of tyrosine to dopa, which then is oxidized to dopaquinone by phenoloxidase, or decarboxylated to dopamine by dopa decarboxlase (DDC). An Armigeres subalbatus cDNA encoding DDC was cloned and real‐time PCR analysis revealed increased transcripts in blood‐fed and microfilariae (mf)‐inoculated mosquitoes. A double subgenomic Sindbis virus was used to silence DDC and assess its role in melanization of mf. DDC transcription and activity were significantly decreased in silenced mosquitoes, as was the degree of mf melanization 48 h postinoculation; however, melanization increased after 72 and 96 h, demonstrating that DDC influences the rate of melanization. DDC‐silenced mosquitoes also exhibit high mortality, over‐feeding and abnormal movement, consistent with an involvement of DDC in neurotransmission.

Mosquito transcriptome changes and filarial worm resistance in Armigeres subalbatus

BackgroundArmigeres subalbatus is a natural vector of the filarial worm Brugia pahangi, but it rapidly and proficiently kills Brugia malayi microfilariae by melanotic encapsulation. Because B. malayi and B. pahangi are morphologically and biologically similar, the Armigeres-Brugia system serves as a valuable model for studying the resistance mechanisms in mosquito vectors. We have initiated transcriptome profiling studies in Ar. subalbatus to identify molecular components involved in B. malayi refractoriness.ResultsThese initial studies assessed the transcriptional response of Ar. subalbatus to B. malayi at 1, 3, 6, 12, 24, 48, and 72 hrs after an infective blood feed. In this investigation, we initiated the first holistic study conducted on the anti-filarial worm immune response in order to effectively explore the functional roles of immune-response genes following a natural exposure to the parasite. Studies assessing the transcriptional response revealed the involvement of unknown and conserved unknowns, cytoskeletal and structural components, and stress and immune responsive factors. The data show that the anti-filarial worm immune response by Ar. subalbatus to be a highly complex, tissue-specific process involving varied effector responses working in concert with blood cell-mediated melanization.ConclusionThis initial study provides a foundation and direction for future studies, which will more fully dissect the nature of the anti-filarial worm immune response in this mosquito-parasite system. The study also argues for continued studies with RNA generated from both hemocytes and whole bodies to fully expound the nature of the anti-filarial worm immune response.

Filarial worms reduce Plasmodium infectivity in mosquitoes.

Background Co-occurrence of malaria and filarial worm parasites has been reported, but little is known about the interaction between filarial worm and malaria parasites with the same Anopheles vector. Herein, we present data evaluating the interaction between Wuchereria bancrofti and Anopheles punctulatus in Papua New Guinea (PNG). Our field studies in PNG demonstrated that An. punctulatus utilizes the melanization immune response as a natural mechanism of filarial worm resistance against invading W. bancrofti microfilariae. We then conducted laboratory studies utilizing the mosquitoes Armigeres subalbatus and Aedes aegypti and the parasites Brugia malayi, Brugia pahangi, Dirofilaria immitis, and Plasmodium gallinaceum to evaluate the hypothesis that immune activation and/or development by filarial worms negatively impact Plasmodium development in co-infected mosquitoes. Ar. subalbatus used in this study are natural vectors of P. gallinaceum and B. pahangi and they are naturally refractory to B. malayi (melanization-based refractoriness). Methodology/Principal Findings Mosquitoes were dissected and Plasmodium development was analyzed six days after blood feeding on either P. gallinaceum alone or after taking a bloodmeal containing both P. gallinaceum and B. malayi or a bloodmeal containing both P. gallinaceum and B. pahangi. There was a significant reduction in the prevalence and mean intensity of Plasmodium infections in two species of mosquito that had dual infections as compared to those mosquitoes that were infected with Plasmodium alone, and was independent of whether the mosquito had a melanization immune response to the filarial worm or not. However, there was no reduction in Plasmodium development when filarial worms were present in the bloodmeal (D. immitis) but midgut penetration was absent, suggesting that factors associated with penetration of the midgut by filarial worms likely are responsible for the observed reduction in malaria parasite infections. Conclusions/Significance These results could have an impact on vector infection and transmission dynamics in areas where Anopheles transmit both parasites, i.e., the elimination of filarial worms in a co-endemic locale could enhance malaria transmission.

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.

Construction and characterization of an expressed sequenced tag library for the mosquito vector Armigeres subalbatus.

BackgroundThe mosquito, Armigeres subalbatus, mounts a distinctively robust innate immune response when infected with the nematode Brugia malayi, a causative agent of lymphatic filariasis. In order to mine the transcriptome for new insight into the cascade of events that takes place in response to infection in this mosquito, 6 cDNA libraries were generated from tissues of adult female mosquitoes subjected to immune-response activation treatments that lead to well-characterized responses, and from aging, naïve mosquitoes. Expressed sequence tags (ESTs) from each library were produced, annotated, and subjected to comparative analyses.ResultsSix libraries were constructed and used to generate 44,940 expressed sequence tags, of which 38,079 passed quality filters to be included in the annotation project and subsequent analyses. All of these sequences were collapsed into clusters resulting in 8,020 unique sequence clusters or singletons. EST clusters were annotated and curated manually within ASAP (A Systematic Annotation Package for Community Analysis of Genomes) web portal according to BLAST results from comparisons to Genbank, and the Anopheles gambiae and Drosophila melanogaster genome projects.ConclusionThe resulting dataset is the first of its kind for this mosquito vector and provides a basis for future studies of mosquito vectors regarding the cascade of events that occurs in response to infection, and thereby providing insight into vector competence and innate immunity.

Existence of Hepatitis C Virus in Culex quinquefasciatus after Ingestion of Infected Blood: Experimental Approach to Evaluating Transmission by Mosquitoes

ABSTRACT We used PCR to detect hepatitis C virus (HCV) RNA among supernatants of ground Culex quinquefasciatus mosquitoes that (i) had been fed HCV-positive blood, (ii) had been intrathoracically inoculated with HCV-positive blood, or (iii) were from homes of hepatitis C patients. HCV RNA was detectable under all three conditions, but it did not replicate in mosquitoes and was not detectably transmitted during feeding.