Haemocyte population and ultrastructural changes during the immune response of the mosquito Culex quinquefasciatus to microfilariae of Wuchereria bancrofti

Abstract Haemocytes circulating in the haemolymph protect insects against pathogens that enter the haemocoel. Changes in haemocyte morphology and differences in haemocyte counts during the immune response of Culex quinquefasciatus Say (Diptera: Culicidae) to microfilariae of Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae) were investigated in the present study. The mean number of total haemocytes was significantly elevated in infected mosquitoes (P < 0.001), reaching a peak on the third day post‐infection. Differential counts show that mean numbers of prohaemocytes, plasmatocytes, granular cells and oenocytoids increased significantly after infection with microfilariae granulocytes compared to the control and näive groups of Cx. quinquefasciatus (P < 0.05). Changes in proportional counts of haemocytes were also analysed in haemolymph perfusates of Cx. quinquefasciatus infected with W. bancrofti. On the first day post‐infection, infected mosquitoes showed an increase in the proportion of prohaemocytes (18.8% compared to 9.6% for the control) and of oenocytoids (7.1% compared to 4.7% control); however, they exhibited lower levels of plasmatocytes (36.6% compared to 42.1% control) and granular cells (36.1% compared to 41.4% control). On day 14 post‐infection, similar changes were observed for these haemocyte types, except that the proportion of granular cells was significantly greater than the control (41.2% compared to 31.3% control). Although an enhancement of prohaemocyte numbers was observed, this cellular type did not show any ultrastructural alteration. On the other hand, granular cells, plasmatocytes and oenocytoids presented morphological alterations indicative of innate immunological activation in mosquitoes infected with W. bancrofti.     

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.     

Deletion of the NSm Virulence Gene of Rift Valley Fever Virus Inhibits Virus Replication in and Dissemination from the Midgut of Aedes aegypti Mosquitoes

Background

Previously, we investigated the role of the Rift Valley fever virus (RVFV) virulence genes NSs and NSm in mosquitoes and demonstrated that deletion of NSm significantly reduced the infection, dissemination, and transmission rates of RVFV in Aedes aegypti mosquitoes. The specific aim of this study was to further characterize midgut infection and escape barriers of RVFV in Ae. aegypti infected with reverse genetics-generated wild type RVFV (rRVF-wt) or RVFV lacking the NSm virulence gene (rRVF-ΔNSm) by examining sagittal sections of infected mosquitoes for viral antigen at various time points post-infection.

Methodology and Principal Findings

Ae. aegypti mosquitoes were fed an infectious blood meal containing either rRVF-wt or rRVF-ΔNSm. On days 0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 post-infection, mosquitoes from each experimental group were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and examined for RVFV antigen by immunofluorescence assay. Remaining mosquitoes at day 14 were assayed for infection, dissemination, and transmission. Disseminated infections were observed in mosquitoes as early as three days post infection for both virus strains. However, infection rates for rRVF-ΔNSm were statistically significantly less than for rRVF-wt. Posterior midgut infections in mosquitoes infected with rRVF-wt were extensive, whereas midgut infections of mosquitoes infected with rRVF-ΔNSm were confined to one or a few small foci.

Conclusions/Significance

Deletion of NSm resulted in the reduced ability of RVFV to enter, replicate, and disseminate from the midgut epithelial cells. NSm appears to have a functional role in the vector competence of mosquitoes for RVFV at the level of the midgut barrier.     

Molecular Xenomonitoring Using Mosquitoes to Map Lymphatic Filariasis after Mass Drug Administration in American Samoa

Background

Mass drug administration (MDA) programs have dramatically reduced lymphatic filariasis (LF) incidence in many areas around the globe, including American Samoa. As infection rates decline and MDA programs end, efficient and sensitive methods for detecting infections are needed to monitor for recrudescence. Molecular methods, collectively termed ‘molecular xenomonitoring,’ can identify parasite DNA or RNA in human blood-feeding mosquitoes. We tested mosquitoes trapped throughout the inhabited islands of American Samoa to identify areas of possible continuing LF transmission after completion of MDA.

Methodology/Principle Findings

Mosquitoes were collected using BG Sentinel traps from most of the villages on American Samoa''s largest island, Tutuila, and all major villages on the smaller islands of Aunu''u, Ofu, Olosega, and Ta''u. Real-time PCR was used to detect Wuchereria bancrofti DNA in pools of ≤20 mosquitoes, and PoolScreen software was used to infer territory-wide prevalences of W. bancrofti DNA in the mosquitoes. Wuchereria bancrofti DNA was found in mosquitoes from 16 out of the 27 village areas sampled on Tutuila and Aunu''u islands but none of the five villages on the Manu''a islands of Ofu, Olosega, and Ta''u. The overall 95% confidence interval estimate for W. bancrofti DNA prevalence in the LF vector Ae. polynesiensis was 0.20–0.39%, and parasite DNA was also detected in pools of Culex quinquefasciatus, Aedes aegypti, and Aedes (Finlaya) spp.

Conclusions/Significance

Our results suggest low but widespread prevalence of LF on Tutuila and Aunu''u where 98% of the population resides, but not Ofu, Olosega, and Ta''u islands. Molecular xenomonitoring can help identify areas of possible LF transmission, but its use in the LF elimination program in American Samoa is limited by the need for more efficient mosquito collection methods and a better understanding of the relationship between prevalence of W. bancrofti DNA in mosquitoes and infection and transmission rates in humans.     

Understanding the mechanism of Chikungunya virus vector competence in three species of mosquitoes

Chikungunya virus (CHIKV) is primarily transmitted by Aedes spp. mosquitoes. The present study investigated vector competence for CHIKV in Aedes aegypti and Aedes albopictus mosquitoes found in Madurai, South India. The role of receptor proteins on midguts contributing to permissiveness of CHIKV to Aedes spp. mosquitoes was also undertaken. Mosquitoes were orally infected with CHIKV DRDE‐06. Infection of midguts and dissemination to heads was confirmed by immunofluorescence assay at different time points. A plaque assay was performed from mosquito homogenates at different time points to study CHIKV replication. Presence of putative CHIKV receptor proteins on mosquito midgut epithelial cells was detected by virus overlay protein binding assay (VOPBA). The identity of these proteins was established using mass spectrometry. CHIKV infection of Ae. aegypti and Ae. albopictus midguts and dissemination to heads was observed to be similar. A plaque assay performed with infected mosquito homogenates revealed that CHIKV replication dynamics was similar in Aedes sp. mosquitoes until 28 days post infection. VOPBA performed with mosquito midgut membrane proteins revealed that prohibitin could serve as a putative CHIKV receptor on Aedes mosquito midguts, whereas an absence of CHIKV binding protein/s on Culex quinquefasciatus midguts can partially explain the non‐permissiveness of these mosquitoes to infection.     

Wolbachia enhances insect‐specific flavivirus infection in Aedes aegypti mosquitoes

Mosquitoes transmit a diverse group of human flaviviruses including West Nile, dengue, yellow fever, and Zika viruses. Mosquitoes are also naturally infected with insect‐specific flaviviruses (ISFs), a subgroup of the family not capable of infecting vertebrates. Although ISFs are not medically important, they are capable of altering the mosquito's susceptibility to flaviviruses and may alter host fitness. Wolbachia is an endosymbiotic bacterium of insects that when present in mosquitoes limits the replication of co‐infecting pathogens, including flaviviruses. Artificially created Wolbachia‐infected Aedes aegypti mosquitoes are being released into the wild in a series of trials around the globe with the hope of interrupting dengue and Zika virus transmission from mosquitoes to humans. Our work investigated the effect of Wolbachia on ISF infection in wild‐caught Ae. aegypti mosquitoes from field release zones. All field mosquitoes were screened for the presence of ISFs using general degenerate flavivirus primers and their PCR amplicons sequenced. ISFs were found to be common and widely distributed in Ae. aegypti populations. Field mosquitoes consistently had higher ISF infection rates and viral loads compared to laboratory colony material indicating that environmental conditions may modulate ISF infection in Ae. aegypti. Surprisingly, higher ISF infection rates and loads were found in Wolbachia‐infected mosquitoes compared to the Wolbachia‐free mosquitoes. Our findings demonstrate that the symbiont is capable of manipulating the mosquito virome and that Wolbachia‐mediated viral inhibition is not universal for flaviviruses. This may have implications for the Wolbachia‐based DENV control strategy if ISFs confer fitness effects or alter mosquito susceptibility to other flaviviruses.     

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.     

Impact of untreated bednets on prevalence of Wuchereria bancrofti transmitted by Anopheles farauti in Papua New Guinea

Abstract Despite the growing evidence that insecticide‐treated mosquito nets reduce malaria morbidity and mortality in a variety of epidemiological conditions, their value against lymphatic filariasis infection and disease is yet to be established. The impact of untreated bednets on the prevalence of Wuchereria bancrofti (Cobbold) (Nematoda: Filarioidea) infection and disease was investigated on Bagabag island in Papua New Guinea, where both malaria and filariasis are transmitted by the same vector mosquitoes of the Anopheles punctulatus Dönitz group (Diptera: Culicidae). Community‐wide surveys were conducted recording demographic characteristics including bednet usage. Physical examinations for hydrocoele and lymphoedema were performed and blood samples assessed for filarial and malaria parasites. Mosquitoes were sampled using the all‐night landing catch method and individually dissected to determine W. bancrofti infection and infective rates. Bednet usage among residents was 61% and the mean age of users (25.6 years) was similar to non‐users (22.5 years). Anopheles farauti Laveran was the only species were found to contain filarial larvae: 2.7% infected (all stages), 0.5% infective (L3). The overall W. bancrofti microfilaraemia and antigenaemia rates were 28.5% and 53.1%, respectively. Bednet users had lower prevalence of W. bancrofti microfilaraemia, antigenaemia and hydrocoele rates than non‐users. In comparison, untreated bednets had no effect on the prevalence and intensity of Plasmodium falciparum and P. vivax infections. The impact of bednet usage on rates of microfilaraemia and antigenaemia remained significant even when confounding factors such as age, location and sex were taken into account, suggesting that untreated bednets protect against W. bancrofti infection.     

Adverse ffects of covert iridovirus infection on life history and demographic parameters of Aedes aegypti

Abstract Sublethal viral infections can cause changes in the body size and demography of insect vectors, with important consequences for population dynamics and the probability that individual mosquitoes will transmit disease. This study examined the effects of covert (sublethal) infection by Invertebrate iridescent virus 6 (IIV‐6) on the demography of female Aedes aegypti and the relationship between key life history parameters in covertly infected female insects compared with healthy (control) insects or non‐infected mosquitoes that had survived exposure to virus inoculum without becoming infected. Of the female mosquitoes that emerged following exposure to virus inoculum and were offered blood meals, 29% (43/150) proved positive for covert IIV‐6 infection. The net reproductive rate (R₀) of covertly infected females was 50% lower for infected females compared to control mosquitoes, whereas non‐infected exposed females had an R₀ approximately 15% lower than that of controls. Reproduction caused a significant decrease of about 13 days in mosquito longevity compared to females that did not reproduce (P < 0.001). Infected females lived 5–8 days less than non‐infected exposed females or controls, respectively (P = 0.028). Infected females and non‐infected exposed females both had significantly shorter wings than control insects (P < 0.001). There was a significant positive correlation between wing length and longevity in covertly infected female mosquitoes but not in control or non‐infected exposed mosquitoes. Longer lived females produced more eggs in all treatments. There were no significant correlations between body size and fecundity or the production of offspring. There was also no correlation between fecundity and fertility, suggesting that sperm inactivation was a more likely cause of decreased fertility in older mosquitoes than sperm depletion. We conclude that covert infection by iridescent virus is likely to reduce the vectorial capacity of this mosquito.     

Age-Dependent Effects of Oral Infection with Dengue Virus on Aedes aegypti (Diptera: Culicidae) Feeding Behavior,Survival, Oviposition Success and Fecundity

Background

Aedes aegypti is the main vector of dengue, a disease that is increasing its geographical range as well as incidence rates. Despite its public health importance, the effect of dengue virus (DENV) on some mosquito traits remains unknown. Here, we investigated the impact of DENV-2 infection on the feeding behavior, survival, oviposition success and fecundity of Ae. aegypti females.

Methods/Principal Findings

After orally-challenging Ae. aegypti females with a DENV-2 strain using a membrane feeder, we monitored the feeding behavior, survival, oviposition success and fecundity throughout the mosquito lifespan. We observed an age-dependent cost of DENV infection on mosquito feeding behavior and fecundity. Infected individuals took more time to ingest blood from anesthetized mice in the 2^nd and 3^rd weeks post-infection, and also longer overall blood-feeding times in the 3^rd week post-infection, when females were around 20 days old. Often, infected Ae. aegypti females did not lay eggs and when they were laid, smaller number of eggs were laid compared to uninfected controls. A reduction in the number of eggs laid per female was evident starting on the 3^rd week post-infection. DENV-2 negatively affected mosquito lifespan, since overall the longevity of infected females was halved compared to that of the uninfected control group.

Conclusions

The DENV-2 strain tested significantly affected Ae. aegypti traits directly correlated with vectorial capacity or mosquito population density, such as feeding behavior, survival, fecundity and oviposition success. Infected mosquitoes spent more time ingesting blood, had reduced lifespan, laid eggs less frequently, and when they did lay eggs, the clutches were smaller than uninfected mosquitoes.     

Population genomics of the filarial nematode parasite Wuchereria bancrofti from mosquitoes

Wuchereria bancrofti is a parasitic nematode and the primary cause of lymphatic filariasis – a disease specific to humans. W. bancrofti currently infects over 90 million people throughout the tropics and has been acknowledged by the world health organization as a vulnerable parasite. Current research has focused primarily on the clinical manifestations of disease and little is known about the evolutionary history of W. bancrofti. To improve upon knowledge of the evolutionary history of W. bancrofti, we whole genome sequenced 13 W. bancrofti larvae. We circumvent many of the difficulties of multiple infections by sampling larvae directly from mosquitoes that were experimentally inoculated with infected blood. To begin, we used whole genome data to reconstruct the historical population size. Our results support a history of fluctuating population sizes that can be correlated with human migration and fluctuating mosquito abundances. Next, we reconstructed the putative pedigree of W. bancrofti worms within an infection using the kinship coefficient. We deduced that there are full‐sib and half‐sib relationships residing within the same larval cohort. Through combined analysis of the mitochondrial and nuclear genomes we concluded that this is likely a results of polyandrous mating, the first time reported for W. bancrofti. Lastly, we scanned the genomes for signatures of natural selection. Annotation of putative selected regions identified proteins that may have aided in a parasitic life style or may have evolved to protect against current drug treatments. We discuss our results in the greater context of understanding the biology of an animal with a unique life history and ecology.     

Wolbachia infection does not alter attraction of the mosquito Aedes (Stegomyia) aegypti to human odours

The insect endosymbiont Wolbachia pipientis (Rickettsiales: Rickettsiaceae) is undergoing field trials around the world to determine if it can reduce transmission of dengue virus from the mosquito Stegomyia aegypti to humans. Two different Wolbachia strains have been released to date. The primary effect of the wMel strain is pathogen protection whereby infection with the symbiont limits replication of dengue virus inside the mosquito. A second strain, wMelPop, induces pathogen protection, reduces the adult mosquito lifespan and decreases blood feeding success in mosquitoes after 15 days of age. Here we test whether Wolbachia infection affects mosquito attraction to host odours in adults aged 5 and 15 days. We found no evidence of reduced odour attraction of mosquitoes, even for those infected with the more virulent wMelPop. This bodes well for fitness and competitiveness in the field given that the mosquitoes must find hosts to reproduce for the biocontrol method to succeed.     

Xenomonitoring of Different Filarial Nematodes Using Single and Multiplex PCR in Mosquitoes from Assiut Governorate,Egypt

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.     

Gene silencing in adult Aedes aegypti mosquitoes through oral delivery of double‐stranded RNA

The induction of the naturally occurring phenomenon of RNA interference (RNAi) to study gene function in insects is now common practice. With appropriately chosen targets, the RNAi pathway has also been exploited for insect control, typically through oral delivery of dsRNA. Adapting current methods to deliver foreign compounds, such as amino acids and pesticides, to mosquitoes through sucrose solutions, we tested whether such an approach could be used in the yellow fever mosquito, Aedes aegypti. Using a non‐specific dsRNA construct, we found that adult Ae. aegypti ingested dsRNA through this method and that the ingested dsRNA can be recovered from the mosquitoes post‐feeding. Through the feeding of a species‐specific dsRNA construct against vacuolar ATPase, subunit A, we found that significant gene knockdown could be achieved at 12, 24 and 48 h post‐feeding.     

Susceptibility of adult Aedes aegypti (Diptera: Culicidae) to infection by Metarhizium anisopliae and Beauveria bassiana: prospects for Dengue vector control

Dengue fever vectored by the mosquito Aedes aegypti is one of the most rapidly spreading insect-borne diseases, stimulating the search for alternatives to current control methods. Screening assays using a range of Metarhizium anisopliae and Beauveria bassiana isolates were performed against adult female Ae. aegypti. Four virulent isolates were selected for detailed study. Adult female mosquitoes were exposed to supports previously inoculated with fungal suspensions. Fungal isolates were suspended in Tween 80+8% vegetable oil. The isolates caused between 70 and 89% mortality as a result of fungal infection over the 7-day test period. Mean survival times varied between 3 and 5 days for treated insects, whilst control survival exceeded 40 days. The most promising isolate, M. anisopliae LPP133, based not only on virulence but facility for mass production, was used for lethal exposure time determinations. An exposure time of only 3.5 h was necessary to cause 50% mortality. Large cage trails were also carried out and mean survival time of insects exposed to fungus impregnated black cloths was significantly reduced. These results show that entomopathogenic fungi could be promising biological control agents for use against adult Ae. aegypti, by inoculating fungi onto surfaces on which the mosquitoes tend to rest. The subsequent mortality caused by the fungi could potentially reduce the populations of this insect thus reducing the incidence of Dengue.     

Touchdown-touchup nested PCR for low-copy gene detection of benzimidazole-susceptible Wuchereria bancrofti with a Wolbachia endosymbiont imported by migrant carriers

A novel, sensitive and specific touchdown–touchup nested PCR (TNPCR) technique based on two useful molecular markers, a Wuchereria bancrofti β-tubulin gene involved in benzimidazole susceptibility and a Wolbachia ftsZ gene involved in cell division, was developed to simultaneously detect the parasite W. bancrofti (W1) with its Wolbachia endosymbiont (W2) from both microfilaremic and post-treatment samples of at-risk migrant carriers infected with geographical W. bancrofti isolates. The detection and characterization of authentically low-copy gene-derived amplicons revealed no false positive identifications in amicrofilaremia with or without antigenemia. The W1-TNPCR was 100-fold more sensitive than the W2-TNPCR regardless of the microfilarial DNA isolation method and compared well with the thick blood film and membrane filtration techniques. These locus-specific TNPCRs could also detect Wolbachia-carrying W. bancrofti genotype in addition to a link to benzimidazole sensitivity among those with unknown infection origins that exhibited microfilaremia responsiveness against treatment with diethylcarbamazine plus albendazole. These TNPCR methods can augment the results of microscopic detection of the parasite because these methods enhance DNA isolation and PCR amplification capabilities.