Aedes aegypti lachesin protein binds to the domain III of envelop protein of Dengue virus‐2 and inhibits viral replication

Dengue virus (DENV) comprises of four serotypes (DENV‐1 to ‐4) and is medically one of the most important arboviruses (arthropod‐borne virus). DENV infection is a major human health burden and is transmitted between humans by the insect vector, Aedes aegypti. Ae. aegypti ingests DENV while feeding on infected humans, which traverses through its gut, haemolymph and salivary glands of the mosquito before being injected into a healthy human. During this process of transmission, DENV must interact with many proteins of the insect vector, which are important for its successful transmission. Our study focused on the identification and characterisation of interacting protein partners in Ae. aegypti to DENV. Since domain III (DIII) of envelope protein (E) is exposed on the virion surface and is involved in virus entry into various cells, we performed phage display library screening against domain III of the envelope protein (EDIII) of DENV‐2. A peptide sequence showing similarity to lachesin protein was found interacting with EDIII. The lachesin protein was cloned, heterologously expressed, purified and used for in vitro interaction studies. Lachesin protein interacted with EDIII and also with DENV. Further, lachesin protein was localised in neuronal cells of different organs of Ae. aegypti by confocal microscopy. Blocking of lachesin protein in Ae. aegypti with anti‐lachesin antibody resulted in a significant reduction in DENV replication.     

Wolbachia pipientis occurs in Aedes aegypti populations in New Mexico and Florida,USA

The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two‐step PCR assay to detect Wolbachia in wild‐collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop‐mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia‐positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.     

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.     

Susceptibility of Florida Aedes aegypti and Aedes albopictus to dengue viruses from Puerto Rico

Locally acquired dengue cases in the continental U.S. are rare. However, outbreaks of dengue‐1 during 2009, 2010, and 2013 in Florida and dengue‐1 and −2 in Texas suggest vulnerability to transmission. Travel and commerce between Puerto Rico and the U.S. mainland is common, which may pose a risk for traveler‐imported dengue cases. Mosquitoes were collected in Florida and used to evaluate their susceptibility to dengue viruses (DENV) from Puerto Rico. Aedes aegypti and Ae. albopictus were susceptible to virus infection with DENV‐1 and −2. No significant differences were observed in rates of midgut infection or dissemination between Ae. aegypti or Ae. albopictus for DENV‐1 (6–14%). Aedes aegypti was significantly more susceptible to midgut infection with DENV‐2 than Ae. albopictus (Ae. aegypti, ∼28%; Ae. albopictus, ∼9%). The dissemination rate with dengue‐2 virus for Ae. aegypti (23%) was greater than Ae. albopictus (0%), suggesting that Ae. albopictus is not likely to be an important transmitter of the DENV‐2 isolate from Puerto Rico. These results are discussed in light of Florida's vulnerability to DENV transmission.     

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.     

A dengue receptor as possible genetic marker of vector competence in <Emphasis Type="Italic">Aedes aegypti</Emphasis>

Background  

Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. Notwithstanding studies of Quantitative Trait Loci (QTL) that influence the ability of Aedes aegypti midgut (MG) to become infected with dengue virus (DENV), no study to date has been undertaken to identify genetic markers of vector competence. Furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. Differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. The first interaction of DENV with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. The susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. To study this interaction in Ae. aegypti strains that differ in their vector competence for DENV, we investigated the DS3 strain (susceptible to DENV), the IBO-11 strain (refractory to infection) and the membrane escape barrier strain, DMEB, which is infected exclusively in the midgut epithelial cells.     

The challenge of invasive mosquito vectors in the U.K. during 2016–2018: a summary of the surveillance and control of Aedes albopictus

Mosquito‐borne diseases resulting from the expansion of two key vectors, Aedes aegypti and Aedes albopictus (Diptera: Culicidae), continue to challenge whole regions and continents around the globe. In recent years there have been human cases of disease associated with Chikungunya, dengue and Zika viruses. In Europe, the expansion of Ae. albopictus has resulted in local transmission of Chikungunya and dengue viruses. This paper considers the risk that Ae. aegypti and Ae. albopictus represent for the U.K. and details the results of mosquito surveillance activities. Surveillance was conducted at 34 points of entry, 12 sites serving vehicular traffic and two sites of used tyre importers. The most common native mosquito recorded was Culex pipiens s.l. (Diptera: Culicidae). The invasive mosquito Ae. albopictus was detected on three occasions in southern England (September 2016, July 2017 and July 2018) and subsequent control strategies were conducted. These latest surveillance results demonstrate ongoing incursions of Ae. albopictus into the U.K. via ground vehicular traffic, which can be expected to continue and increase as populations in nearby countries expand, particularly in France, which is the main source of ex‐continental traffic.     

Electrophysiological and behavioural response of Aedes albopictus to n‐heinecosane,an ovipositional pheromone of Aedes aegypti

Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae) are highly anthropophilic mosquito species and potential vectors of dengue and yellow fever. The location of suitable sites for oviposition requires a set of visual, tactile, and olfactory cues that influence females before they lay their eggs. In this study, the effect of n‐heneicosane, a recognized oviposition pheromone of Ae. aegypti, on the olfactory receptors of the antennae of Ae. aegypti and Ae. albopictus was studied using electroantennographic detection coupled to gas chromatography (GC‐EAD). A significant electroantennographic response to n‐heneicosane in adult females of both mosquito species was observed. In addition, gravid Ae. albopictus females laid more eggs in substrate treated with n‐heneicosane at 0.1, 1, or 10 p.p.m. than in the control, denoting oviposition attractancy. Conversely, at 30, 50, 100, and 200 p.p.m., more eggs were laid in the control substrate, indicating oviposition repellency. Analysis of the larval cuticle by GC and mass spectrometry confirmed the presence of n‐heneicosane in the cuticles of Ae. albopictus larvae. The species‐specific role of n‐heneicosane as an oviposition pheromone in Ae. aegypti and its significance as a behaviour modifier of Ae. albopictus in breeding sites is discussed.     

Wolbachia infection alters the relative abundance of resident bacteria in adult Aedes aegypti mosquitoes,but not larvae

Insect–symbiont interactions are known to play key roles in host functions and fitness. The common insect endosymbiont Wolbachia can reduce the ability of several human pathogens, including arboviruses and the malaria parasite, to replicate in insect hosts. Wolbachia does not naturally infect Aedes aegypti, the primary vector of dengue virus, but transinfected Ae. aegypti have antidengue virus properties and are currently being trialled as a dengue biocontrol strategy. Here, we assess the impact of Wolbachia infection of Ae. aegypti on the microbiome of wild mosquito populations (adults and larvae) collected from release sites in Cairns, Australia, by profiling the 16S rRNA gene using next‐generation sequencing. Our data indicate that Wolbachia reduces the relative abundance of a large proportion of bacterial taxa in Ae. aegypti adults, that is in accordance with the known pathogen‐blocking effects of Wolbachia on a variety of bacteria and viruses. In adults, several of the most abundant bacterial genera were found to undergo significant shifts in relative abundance. However, the genera showing the greatest changes in relative abundance in Wolbachia‐infected adults represented a low proportion of the total microbiome. In addition, there was little effect of Wolbachia infection on the relative abundance of bacterial taxa in larvae, or on species diversity (accounting for species richness and evenness together) detected in adults or larvae. These results offer insight into the effects of Wolbachia on the Ae. aegypti microbiome in a native setting, an important consideration for field releases of Wolbachia into the population.     

Heterogeneous genetic invasions of three insecticide resistance mutations in Indo‐Pacific populations of Aedes aegypti (L.)

Nations throughout the Indo‐Pacific region use pyrethroid insecticides to control Aedes aegypti, the mosquito vector of dengue, often without knowledge of pyrethroid resistance status of the pest or origin of resistance. Two mutations (V1016G + F1534C) in the sodium channel gene (Vssc) of Ae. aegypti modify ion channel function and cause target‐site resistance to pyrethroid insecticides, with a third mutation (S989P) having a potential additive effect. Of 27 possible genotypes involving these mutations, some allelic combinations are never seen whereas others predominate. Here, five allelic combinations common in Ae. aegypti from the Indo‐Pacific region are described and their geographical distributions investigated using genome‐wide SNP markers. We tested the hypothesis that resistance allele combinations evolved de novo in populations versus the alternative that dispersal of Ae. aegypti between populations facilitated genetic invasions of allele combinations. We used latent factor mixed‐models to detect SNPs throughout the genome that showed structuring in line with resistance allele combinations and compared variation at SNPs within the Vssc gene with genome‐wide variation. Mixed‐models detected an array of SNPs linked to resistance allele combinations, all located within or in close proximity to the Vssc gene. Variation at SNPs within the Vssc gene was structured by resistance profile, whereas genome‐wide SNPs were structured by population. These results demonstrate that alleles near to resistance mutations have been transferred between populations via linked selection. This indicates that genetic invasions have contributed to the widespread occurrence of Vssc allele combinations in Ae. aegypti in the Indo‐Pacific region, pointing to undocumented mosquito invasions between countries.     

Natural vertical transmission of dengue viruses in Aedes aegypti in selected sites in Cebu City,Philippines

We attempted to determine the vertical transmission of dengue virus (DENV) in Aedes aegypti in selected sites in Cebu City, Philippines. Mosquito sub‐adults were collected monthly from households and the field during the wet‐dry‐wet season from November, 2011 to July, 2012 and were laboratory‐reared to adults. Viral RNA extracts in mosquitoes were assayed by hemi‐nested RT‐PCR. Results showed that 62 (36.26%; n=679) out of 171 mosquito pools (n=2,871) were DENV+. The minimum infection rate (MIR) of DENV ranged from 0 in wet months to 48.22/1,000 mosquitoes in April, 2012 (mid‐dry). DENVs were detected in larvae, pupae, and male and female adults, with DENV‐4, DENV‐3, and DENV‐1, in that rank of prevalence. DENV‐1 co‐infected with either DENV‐3 or ?4 or with both in April, 2012; DENV‐3 and ?4 were present in both seasons. More DENV+ mosquitoes were collected from households than in field premises (p<0.001) and in the dry than in the wet season (p<0.05), with significant interaction (p<0.05) between sites and premises but no interaction between sites and seasons (p>0.05). By Generalized Linear Mixed models, the type of premises nested in sites and monthly total rainfall were significant predictors of monthly dengue cases (p<0.05) and not MIR, season, temperature, and relative humidity. Surveillance of DENV prevalence in Ae. aegypti and detecting their natural foci in the dry season provide an early warning signal of dengue outbreak.     

A lethal ovitrap‐based mass trapping scheme for dengue control in Australia: II. Impact on populations of the mosquito Aedes aegypti

In Cairns, Australia, the impacts on Aedes aegypti L. (Diptera: Culicidae) populations of two types of ‘lure & kill’ (L&K) lethal ovitraps (LOs), the standard lethal ovitrap (SLO) and the biodegradable lethal ovitrap (BLO) were measured during three mass‐trapping interventions. To assess the efficacy of the SLO, two interventions (one dry season and one wet season) were conducted in three discrete areas, each lasting 4 weeks, with the following treatments: (i) SLOs (>200 traps, ∼4/premise), BG‐sentinel traps (BGSs; ∼15, 1/premise) and larval control (container reduction and methoprene treatment) and (ii) larval control alone, and (iii) untreated control. Female Ae. aegypti populations were monitored for 4 weeks pre‐ and post‐treatment in all three areas using BGSs and sticky ovitraps (SOs) or non‐lethal regular ovitraps (ROs). In the dry season, 206 SLOs and 15 BGSs set at 54 and 15 houses, respectively, caught and killed an estimated 419 and 73 female Ae. aegypti, respectively. No significant decrease in collection size of female Ae. aegypti could be attributed to the treatments. In the wet season, 243 SLOs and 15 BGSs killed ∼993 and 119 female Ae. aegypti, respectively. The mean number of female Ae. aegypti collected after 4 weeks with SOs and BGSs was significantly less than the control (LSD post‐hoc test). The third mass‐trapping intervention was conducted using the BLO during the wet season in Cairns. For this trial, three treatment areas were each provided with BLOs (>500, ∼4/premise) plus larval control, and an untreated control area was designated. Adult female Ae. aegypti were collected for 4 weeks pre‐ and post‐treatment using 15 BGSs and 20 SOs. During this period, 53.2% of BLOs contained a total of 6654 Ae. aegypti eggs. Over the intervention period, collections of Ae. aegypti in the treatment areas were significantly less than in the control area for BGSs but not SOs. An influx of relatively large numbers of young females may have confounded the measurement of changes in populations of older females in these studies. This is an important issue, with implications for assessing delayed action control measures, such as LOs and parasites/pathogens that aim to change mosquito age structure. Finally, the high public acceptability of SLOs and BLOs, coupled with significant impacts on female Ae. aegypti populations in two of the three interventions reported here, suggest that mass trapping with SLOs and BLOs can be an effective component of a dengue control strategy.     

Elimination of Aedes aegypti in northern Australia, 2004–2006

The Northern Territory (NT) of Australia is currently free of the dengue mosquito Aedes (Stegomyia) aegypti (L). However, on 17 February 2004, two Ae. aegypti adults were captured in two routine CO₂‐baited encephalitis virus surveillance traps in Tennant Creek, located 990 km south of Darwin in the NT. The detection triggered an immediate survey and control response undertaken by the NT Department of Health and Community Services, followed by a Commonwealth of Australia‐funded Ae. aegypti elimination program. This report details the methods and results of the detection and subsequent elimination activities that were carried out between 2004 and 2006, returning the NT to its dengue vector‐free status. There have been very few successful Ae. aegypti elimination programs in the world. This purposeful mosquito elimination for Australia was officially declared on 5 April 2006.     

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.     

The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in <Emphasis Type="Italic">Aedes aegypti</Emphasis>

Background  

The RNA interference (RNAi) pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus) is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.     

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.