Knock-down of REL2, but not defensin A, augments Aedes aegypti susceptibility to Bacillus subtilis and Escherichia coli

Some components of the Toll and Imd immune signaling pathways have remained conserved between Drosophila and mosquitoes, however, important differences in the way invading microorganisms activate these pathways in these organisms have started to be revealed. In the present study, we have attempted to silence the Aedes aegypti NF-κB-like factor REL2, which is analogous to Drosophila Relish, and analyze the effects on mosquito mortality upon infection with a Gram-negative and a Gram-positive bacterium, both containing a DAP-type peptidoglycan, and effects on embryo development. Moreover, we have silenced one of the antimicrobial peptides (AMPs) controlled by REL2, defensin A, a major AMP in A. aegypti, and compared the results on mosquito mortality upon bacterial infection to those obtained with REL2 silencing. Results show that REL2 is crucial for A. aegypti immunity upon infection with Escherichia coli and Bacillus subtilis, corroborating with previous studies on that REL2/Imd in mosquitoes is involved in a generalized antibacterial defense, differently than its analogous in Drosophila. However, defensin A silencing did not cause a significant increase in mortality in infected mosquitoes, indicating that this peptide is not essential in mosquito protection against the two bacteria and that other immune factors controlled by REL2 are playing this role. In regard to embryo development, REL2 knock-down did not cause any significant effect on: the number of laid eggs, number of developed pupae, percent of emerged adults, and ratio between emerged females versus males. A slight decrease in the number of hatched eggs (percent eclosion) was observed in REL2 knock-down mosquitoes, but these observations were inconclusive.     

Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti

Mosquitoes (Aedes aegypti) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The A. aegypti carboxypeptidase A promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs' white eye) strain by using the nonautonomous mariner MosI transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission in vitro of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic A. aegypti that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.     

Relish-mediated immune deficiency in the transgenic mosquito Aedes aegypti

The lack of genetic means has been a serious limitation in studying mosquito immunity. We generated Relish-mediated immune deficiency (RMID) by transforming Aedes aegypti with the Delta Rel transgene driven by the vitellogenin (Vg) promoter using the pBac[3xP3-EGFP, afm] vector. A stable transformed line had a single copy of the Vg-Delta Rel transgene. The Vg-Delta Rel transgene expression was highly activated by blood feeding, and transgenic mosquitoes were extremely susceptible to the infection by Gram-negative bacteria. This RMID phenotype was characterized by severely reduced postinfection levels of antimicrobial peptides genes, defensin and cecropin. Crossing the RMID line with the wild-type strain produced the same RMID phenotype, indicating its dominant nature, whereas crossing with the Vg-def transgenic line, in which Defensin A was activated by blood feeding, restored the immunity to Enterobacter cloacae.     

Toll and Toll-9 in Drosophila innate immune response

In both insects and mammals, members of the Toll receptor family play important roles in the initial events leading to the activation of immunity genes. The prototypic Toll in Drosophila appears to be activated by a host protein ligand after microbial stimulation. The cellular events and the biological response after Toll activation, however, require further investigation. We used transgenic Drosophila strains expressing NF-kappaB and Toll proteins to investigate innate immune response in whole larvae and dissected larval fat bodies. Substantial activation of antimicrobial peptide genes was observed after septic injury. To circumvent the contribution of injury-induced response, we used dissected larval fat bodies to show that commercially available microbial compounds were able to alter the cellular distribution of Toll. The results also demonstrate that complex cellular events, including receptor trafficking, likely take place after stimulation of the larval immune tissue. By genome-wide expression analysis, we further show that Toll and Toll-9 may utilize the same signaling pathway in activating many immunity genes. Thus, the innate immune response in Drosophila is regulated by complex mechanisms, which involve Toll and other Toll-related proteins.     

An evolutionary conserved function of the JAK-STAT pathway in anti-dengue defense

Here, we show that the major mosquito vector for dengue virus uses the JAK-STAT pathway to control virus infection. Dengue virus infection in Aedes aegypti mosquitoes activates the JAK-STAT immune signaling pathway. The mosquito''s susceptibility to dengue virus infection increases when the JAK-STAT pathway is suppressed through RNAi depletion of its receptor Domeless (Dome) and the Janus kinase (Hop), whereas mosquitoes become more resistant to the virus when the negative regulator of the JAK-STAT pathway, PIAS, is silenced. The JAK-STAT pathway exerts its anti-dengue activity presumably through one or several STAT-regulated effectors. We have identified, and partially characterized, two JAK-STAT pathway-regulated and infection-responsive dengue virus restriction factors (DVRFs) that contain putative STAT-binding sites in their promoter regions. Our data suggest that the JAK-STAT pathway is part of the A. aegypti mosquito''s anti-dengue defense and may act independently of the Toll pathway and the RNAi-mediated antiviral defenses.     

Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti

Progress in molecular genetics makes possible the development of alternative disease control strategies that target the competence of mosquitoes to transmit pathogens. We tested the regulatory region of the vitellogenin (Vg) gene of Aedes aegypti for its ability to express potential antipathogen factors in transgenic mosquitoes. Hermes-mediated transformation was used to integrate a 2.1-kb Vg-promoter fragment driving the expression of the Defensin A (DefA) coding region, one of the major insect immune factors. PCR amplification of genomic DNA and Southern blot analyses, carried out through the ninth generation, showed that the Vg-DefA transgene insertion was stable. The Vg-DefA transgene was strongly activated in the fat body by a blood meal. The mRNA levels reached a maximum at 24-h postblood meal, corresponding to the peak expression time of the endogenous Vg gene. High levels of transgenic defensin were accumulated in the hemolymph of bloodfed female mosquitoes, persisting for 20-22 days after a single blood feeding. Purified transgenic defensin showed antibacterial activity comparable to that of defensin isolated from bacterially challenged control mosquitoes. Thus, we have been able to engineer the genetically stable transgenic mosquito with an element of systemic immunity, which is activated through the blood meal-triggered cascade rather than by infection. This work represents a significant step toward the development of molecular genetic approaches to the control of vector competence in pathogen transmission.     

Robust gut-specific gene expression in transgenic Aedes aegypti mosquitoes

Genetic modification of the vectorial capacity of mosquito vectors of human disease requires promoters capable of driving gene expression with appropriate tissue and stage specificity. We report on the characterization in transgenic Aedes aegypti of two mosquito gut-specific promoters. A 1.4-kb DNA fragment adjacent to the 5' end of the coding region of the Ae. aegypti carboxypeptidase (AeCP) gene and a corresponding 3.4-kb DNA fragment at the 5' end of the Anopheles gambiae carboxypeptidase (AgCP) gene were linked to a firefly luciferase reporter gene and introduced into the Ae. aegypti germ line by using Hermes and mariner (Mos1) transposons. Six independent transgenic lines were obtained with the AeCP construct and one with the AgCP construct. Luciferase mRNA and protein were abundantly expressed in the guts of transgenic mosquitoes in four of the six AeCP lines and in the AgCP line. Expression of the reporter gene was gut-specific and reached peak levels at about 24 h post-blood ingestion. The AeCP and AgCP promoters can be used to drive the expression of genes that hinder parasite development in the mosquito gut.     

Vector competence of mosquitoes as a marker to distinguish Central American and Mexican epizootic from enzootic strains of Venezuelan encephalitis virus.

Two epizootic strains of Venezuelan encephalitis (VE) virus from Central America and Mexico were transmitted by a colonized epizootic vector mosquito, Aedes taeniorhynchus, at higher rates than were two enzootic strains when the mosquitoes were infected by intrathroacic inoculation or feeding of virus. Differences in transmission rates also occurred with colonized Aedes aegypti, but were less marked. Following intrathoracic inoculation of A. taeniorhynchus or A. aegypti, epizootic strains grew to slightly higher concentrations in the mosquitoes than did enzootic strains. Intestinal thresholds of infection for A. taeniorhynchus were slightly lower for epizootic than for enzootic virus strains, but were essentially equal for A. aegypti. Only a small percentage of individual Culex pipiens quinquefasciatus mosquitoes supported growth of epizootic VE virus, and only 1 of 6 tested C. p. quinquefasciatus transmitted virus by bite. Thus, transmission and growth of virus in these Aedes mosquitoes distinguished between these epizootic and enzootic strains of VE virus.     

Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing

Homology-dependent RNA silencing occurs in many eukaryotic cells. We reported recently that nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila, which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Here, we present further evidence to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway, as the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. We demonstrate that RNAi also functions as a natural antiviral immunity in mosquito cells. We further show that vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila. The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. We found that the double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells.     

RNA interference-mediated knockdown of a GATA factor reveals a link to anautogeny in the mosquito Aedes aegypti

Blood feeding tightly regulates the reproductive cycle in anautogenous mosquitoes. Vitellogenesis (the synthesis of yolk protein precursors) is a key event in the mosquito reproductive cycle and is activated in response to a blood meal. Before blood feeding, Aedes aegypti is in a state of reproductive arrest during which the yolk protein precursor genes (YPPs) are repressed. The regulatory region of the major YPP gene vitellogenin (Vg) has multiple GATA-binding sites required for the high expression level of this gene. However, a GATA factor (AaGATAr) likely acts as a repressor, preventing activation of this gene before a blood meal. Here we report in vivo data confirming the role of AaGATAr as a repressor of the Vg gene at the state of previtellogenic arrest. Using an RNA interference (RNAi)-mediated technique in conjunction with the Sindbis viral expression system, we show that knockdown of the AaGATAr gene results in an increased basal level of expression of the Vg gene and an elevated response to the steroid hormone 20-hydroxyecdysone in mosquitoes in a state of arrest. These experiments have revealed a component in the molecular mechanism by which anautogeny is maintained in A. aegypti.     

Small RNA Response to Infection of the Insect-Specific Lammi Virus and Hanko Virus in an Aedes albopictus Cell Line

RNA interference (RNAi)-mediated antiviral immunity is believed to be the primary defense against viral infection in mosquitoes. The production of virus-specific small RNA has been demonstrated in mosquitoes and mosquito-derived cell lines for viruses in all of the major arbovirus families. However, many if not all mosquitoes are infected with a group of viruses known as insect-specific viruses (ISVs), and little is known about the mosquito immune response to this group of viruses. Therefore, in this study, we sequenced small RNA from an Aedes albopictus-derived cell line infected with either Lammi virus (LamV) or Hanko virus (HakV). These viruses belong to two distinct phylogenetic groups of insect-specific flaviviruses (ISFVs). The results revealed that both viruses elicited a strong virus-derived small interfering RNA (vsiRNA) response that increased over time and that targeted the whole viral genome, with a few predominant hotspots observed. Furthermore, only the LamV-infected cells produced virus-derived Piwi-like RNAs (vpiRNAs); however, they were mainly derived from the antisense genome and did not show the typical ping-pong signatures. HakV, which is more distantly related to the dual-host flaviviruses than LamV, may lack certain unknown sequence elements or structures required for vpiRNA production. Our findings increase the understanding of mosquito innate immunity and ISFVs’ effects on their host.     

Assessing fitness costs for transgenic Aedes aegypti expressing the GFP marker and transposase genes

The development of transgenic mosquitoes that are refractory to the transmission of human diseases such as malaria, dengue, and yellow fever has received much interest due to the ability to transform a number of vector mosquito species with transposable elements. Transgenic strains of mosquitoes have been generated with molecular techniques that exhibit a reduced capacity to transmit pathogens. These advancements have led to questions regarding the fitness of transgenic mosquitoes and the ability of transformed mosquitoes to compete and effectively spread beneficial genes through nontransformed field populations, the core requirement of a genetically based control strategy aimed at reducing the spread of mosquito-borne human disease. Here we examine the impact of transgenesis on the fitness of Aedes aegypti, a mosquito that transmits yellow fever. Mosquitoes were altered with two types of transgene, the enhanced GFP gene and two transposase genes from the Hermes and MOS1 transposable elements. We examined the effects of these elements on the survivorship, longevity, fecundity, sex ratio, and sterility of transformed mosquitoes and compared results to the nontransformed laboratory strain. We show that demographic parameters are significantly diminished in transgenic mosquitoes relative to the untransformed laboratory strain. Reduced fitness in transgenic mosquitoes has important implications for the development and utilization of this technology for control programs based on manipulative molecular modification.     

Entomologic investigations of a chikungunya virus epidemic in the Union of the Comoros, 2005

From January to April 2005, an epidemic of chikungunya virus (CHIKV) illness occurred in the Union of Comoros. Entomological studies were undertaken during the peak of the outbreak, from March 11 to March 31, aimed at identifying the primary vector(s) involved in transmission so that appropriate public health measures could be implemented. Adult mosquitoes were collected by backpack aspiration and human landing collection in homes and neighborhoods of clinically ill patients. Water-holding containers were inspected for presence of mosquito larvae. Adult mosquitoes were analyzed by RT-PCR and cultivation in cells for the presence of CHIK virus and/or nucleic acid. A total of 2,326 mosquitoes were collected and processed in 199 pools. The collection consisted of 62.8% Aedes aegypti, 25.5% Culex species, and 10.7% Aedes simpsoni complex, Eretmapodites spp and Anopheles spp. Seven mosquito pools were found to be positive for CHIKV RNA and 1 isolate was obtained. The single CHIKV mosquito isolate was from a pool of Aedes aegypti and the minimum infection rate (MIR) for this species was 4.0, suggesting that Ae. aegypti was the principal vector responsible for the outbreak. This was supported by high container (31.1%), household (68%), and Breteau (126) indices, with discarded tires (58.8%) and small cooking and water storage vessels (31.1%) registering the highest container indices.     

The effectiveness of mosquito coils containing esbiothrin under laboratory and field conditions

Tests were made to correlate the chemical content of mosquito coils with the knockdown and bite-inhibitory action of the smoke in the laboratory and with the protective effect in field use. Smoke from a blank coil, containing no pyrethroid, gave no knockdown, 10% inhibition of biting in the laboratory using Aedes aegypti, and 39% protection in field tests in village huts on the Kenyan coast with a mixed population of mosquitoes including 71% Anopheles gambiae. Coils containing low experimental contents of 0.044% and 0.099% Esbiothrin, an isomer blend rich in the D-allethrolone ester of D-trans-chrysanthemic acid, gave rapid knockdown in both small chamber and 25 m3 room tests, and 71% and 94% inhibition of biting respectively in the laboratory. In the field they gave 74% and 84% protection from mosquitoes alighting. Chemical content was therefore a good guide to knockdown and bite inhibition in the laboratory and also to the considerable protection found in the field.     

Utility of comparative anchor-tagged sequences as physical anchors for comparative genome analysis among the Culicidae

The development of comparative genetic maps in multiple species of mosquitoes could prove extremely useful in the search for those genes that contribute to mosquito vector competence or genes associated with other phenotypes of interest. To effectively compare these gene maps, markers must be developed that are based on chromosomal regions conserved throughout the Culicidae. We designed 35 polymerase chain reaction (PCR) primer pairs based upon orthlogous exons in Aedes aegypti and Drosophila melanogaster or Anopheles gambiae. Twenty-three of the primers yielded a single PCR product in at least one dipteran, in addition to Ae. aegypti, when screened with genomic DNA from seven dipterans, including five mosquito species. Eight of the primers amplified a single PCR product in only Ae. aegypti, while four primer pairs gave no PCR product in any species. The 23 successful comparative anchor-tagged sequence primer pairs give broad genome coverage in Ae. aegypti, and more importantly demonstrate an efficient strategy for developing comparative anchor marker loci for any species of Culicidae.     

Experimental studies on the transmission of hepatitis B by mosquitoes.

Culex tarsalis and Aedes aegypti mosquitoes were fed on chimpanzees carrying hepatitis B surface antigen (HBS Ag) of known infectivity and pools were tested by radioimmunoassay daily for the presence of HBS Ag. HBS Ag continued to be detected at low levels in mosquito tissue after digestion of the blood meal. Inoculation of susceptible chimpanzees with macerated pools of A. aegypti mosquitoes at two intervals after digestion of the blood meal did not produce hepatitis or serologic evidence of hepatitis B virus infection. Mechanical transmission studies by interrupting feeding of A aegypti from HBS Ag-carrier chimpanzees and transferring them to susceptible chimpanzees did not produce hepatitis. These findings do not support the hypothesis that mosquitoes are involved in either biological or mechanical transmission of hepatitis B.     

Discovery of an alternate metabolic pathway for urea synthesis in adult Aedes aegypti mosquitoes

We demonstrate the presence of an alternate metabolic pathway for urea synthesis in Aedes aegypti mosquitoes that converts uric acid to urea via an amphibian-like uricolytic pathway. For these studies, female mosquitoes were fed a sucrose solution containing (15)NH4Cl, [5-(15)N]-glutamine, [(15)N]-proline, allantoin, or allantoic acid. At 24 h after feeding, the feces were collected and analyzed in a mass spectrometer. Specific enzyme inhibitors confirmed that mosquitoes incorporate (15)N from (15)NH4Cl into [5-(15)N]-glutamine and use the (15)N of the amide group of glutamine to produce labeled uric acid. More importantly, we found that [(15)N2]-uric acid can be metabolized to [(15)N]-urea and be excreted as nitrogenous waste through an uricolytic pathway. Ae. aegypti express all three genes in this pathway, namely, urate oxidase, allantoinase, and allantoicase. The functional relevance of these genes in mosquitoes was shown by feeding allantoin or allantoic acid, which significantly increased unlabeled urea levels in the feces. Moreover, knockdown of urate oxidase expression by RNA interference demonstrated that this pathway is active in females fed blood or (15)NH4Cl based on a significant increase in uric acid levels in whole-body extracts and a reduction in [(15)N]-urea excretion, respectively. These unexpected findings could lead to the development of metabolism-based strategies for mosquito control.