Wolbachia strain wAlbA blocks Zika virus transmission in Aedes aegypti

Transinfections of the maternally transmitted endosymbiont Wolbachia pipientis can reduce RNA virus replication and prevent transmission by Aedes aegypti, and also have the capacity to invade wild-type populations, potentially reaching and maintaining high infection frequencies. Levels of virus transmission blocking are positively correlated with Wolbachia intracellular density. Despite reaching high densities in Ae. aegypti, transinfections of wAlbA, a strain native to Aedes albopictus, showed no blocking of Semliki Forest Virus in previous intrathoracic injection challenges. To further characterize wAlbA blocking in Ae. aegypti, adult females were intrathoracically challenged with Zika (ZIKV) and dengue viruses, and then fed a ZIKV-containing bloodmeal. No blocking was observed with either virus when challenged by intrathoracic injection. However, when ZIKV was delivered orally, wAlbA-infected females showed a significant reduction in viral replication and dissemination compared with uninfected controls, as well as a complete absence of virus in saliva. Although other Wolbachia strains have been shown to cause more robust viral blocking in Ae. aegypti, these findings demonstrate that, in principle, wAlbA could be used to reduce virus transmission in this species. Moreover, the results highlight the potential for underestimation of the strength of virus-blocking when based on intrathoracic injection compared with more natural oral challenges.     

Wolbachia strain wAlbB maintains high density and dengue inhibition following introduction into a field population of Aedes aegypti

Aedes aegypti mosquitoes carrying the wAlbB Wolbachia strain show a reduced capacity to transmit dengue virus. wAlbB has been introduced into wild Ae. aegypti populations in several field sites in Kuala Lumpur, Malaysia, where it has persisted at high frequency for more than 2 years and significantly reduced dengue incidence. Although these encouraging results indicate that wAlbB releases can be an effective dengue control strategy, the long-term success depends on wAlbB maintaining high population frequencies and virus transmission inhibition, and both could be compromised by Wolbachia–host coevolution in the field. Here, wAlbB-carrying Ae. aegypti collected from the field 20 months after the cessation of releases showed no reduction in Wolbachia density or tissue distribution changes compared to a wAlbB laboratory colony. The wAlbB strain continued to induce complete unidirectional cytoplasmic incompatibility, showed perfect maternal transmission under laboratory conditions, and retained its capacity to inhibit dengue. Additionally, a field-collected wAlbB line was challenged with Malaysian dengue patient blood, and showed significant blocking of virus dissemination to the salivary glands. These results indicate that wAlbB continues to inhibit currently circulating strains of dengue in field populations of Ae. aegypti, and provides additional support for the continued scale-up of Wolbachia wAlbB releases for dengue control.This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases’.     

The use of direct sequencing of dengue virus cDNA from individual field-collected Aedes aegypti for surveillance and epidemiological studies

The relative efficiencies of four methods to extract viral RNA from individual dengue-2 virus (D-2V)-infected mosquitoes, Aedes aegypti (L.) (Diptera: Culicidae), were compared. The most efficient of these methods was then used to extract viral RNA for the preparation of cDNA from the abdomens of six engorged D-2V-infected mosquitoes and sera from three dengue fever (DF) patients collected in an isolated rural town in Colombia. Comparisons of viral envelope (E) gene sequences from each of these strongly suggested that the D-2V population which circulated in this study area was a homogeneous genotype which was unrelated to any of the D-2 viruses isolated from elsewhere in the world. When coupled with our rapid method to identify viruses in individual mosquitoes (Romero-Vivas et al. (1998) Medical and Veterinary Entomology, 12, 101-105), the methodology we describe should be useful for epidemiological and surveillance studies of dengue viruses and other arboviruses.     

Satellite observation to assess dengue risk due to Aedes aegypti and Aedes albopictus in a subtropical city of Argentina

Earth observation environmental features measured through remote sensing and models of vector mosquitoes species Aedes aegypti and Ae. albopictus provide an advancement with regards to dengue risk in urban environments of subtropical areas of Argentina. The authors aim to estimate the effect of landscape coverage and spectral indices (Normalized Difference Vegetation Index [NDVI], Normalized Difference Water Index [NDWI] and Normalized Difference Built-up Index [NDBI]) on the larvae abundance of Ae. aegypti and Ae. albopictus in Eldorado, Misiones, Argentina using remote satellite sensors. Larvae of these species were collected monthly (June 2016 to April 2018), in four environments: tire repair shops, cemeteries, dwellings and an urban natural park. The proportion of landscape coverage (water, urban areas, bare soil, low vegetation and high vegetation) was determined from the supervised classification of Sentinel-2 images and spectral indices, calculated. The authors developed spatial models of both vector species by generalized linear mixed models. The model's results showed that Ae. aegypti larvae abundance was better modelled by NDVI minimum values, NDBI maximum values and the interaction between them. For Ae. albopictus proportion of bare soil, low vegetation and the interaction between both variables explained better the abundance.     

Unusual productivity of Aedes aegypti in septic tanks and its implications for dengue control

Increased DEN-2 virus transmission in Puerto Rico during 2005 prompted the implementation of a rapid intervention programme to suppress Aedes aegypti (L.) (Diptera: Culicidae) emergence, which in turn lead to the discovery of previously unknown breeding sites underground. Initially, the following control measures were applied in Playa/Playita (PP), a town of 1,400 households, to all areas where the number of pupae per person exceeded the expected threshold for dengue transmission; all containers likely to be aquatic habitats were turned over and containers too large to turn were treated with 1 p.p.m. methoprene. The impact of these interventions was evaluated by comparing the number of resting adult mosquitoes (by backpack aspiration and sweepnetting in bedrooms) pre-intervention, with numbers at 3 and 5 weeks post-intervention, and by evaluating pupal density at 4 weeks post-intervention in PP and in a nearby town, Coqui (CO; 1500 households), which was not treated. The pre-intervention and post-intervention densities of resting Ae. aegypti adults were significantly larger in the intervention town, although the density of pupae in surface containers was low and similar in both towns at 4 weeks post-intervention. At 3 weeks post-intervention, the density of resting adults decreased by only 18% of pre-intervention levels, but returned to pre-intervention levels 5 weeks after treatment. By contrast, the density of resting adults in CO steadily decreased to 48% and 61%, at 3 and 5 weeks after the initial surveys, respectively. Geographical Information Systems identified significant clustering of adult mosquitoes, which led to the discovery of underground aquatic habitats (septic tanks) that were producing large numbers of Ae. aegypti and Culex quinquefasciatus (Say) in the treated town. We calculated that septic tanks could produce > 18 000 Ae. aegypti and approximately 170 000 Cx quinquefasciatus adults per day. Septic tanks are likely to be common and widespread in suburban and rural Puerto Rico, where, apparently, they can contribute significantly to the maintenance of island-wide dengue virus endemicity.     

Lessons of Aedes aegypti control in Thailand

Abstract. The incidence of dengue haemorrhagic fever (DHF) in Thailand has increased cyclically since the first recognized outbreak in 1958. Without an effective vaccine against dengue, and considering the clinical difficulty of treating DHF cases, vector control is needed to prevent dengue transmission. Since the establishment of the WHO Aedes Research Unit in 1964, continued since 1973 as the WHO Collaborating Centre at the Department of Medical Research in Bangkok, much operational research has been carried out in Thailand on the bionomics and control of dengue vectors: Aedes aegypti and Ae.albopictus. This review shows that, as in most other countries, dengue vector control programmes in Thailand make little use of the procedures arising from research, nor have they reduced the upward trend of dengue or prevented DHF outbreaks. Implications of the reluctance to use results of operational research on vector control are considered and remedial suggestions made.     

Population structure of the mosquito Aedes aegypti (Stegomyia aegypti) in Pakistan

Eleven microsatellite markers were used to determine the genetic population structure and spread of Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) in Pakistan using mosquitoes collected from 13 different cities. There is a single genetic cluster of Ae. aegypti in Pakistan with a pattern of isolation by distance within the population. The low level of isolation by distance suggests the long‐range passive dispersal of this mosquito, which may be facilitated by the tyre trade in Pakistan. A decrease in genetic diversity from south to north suggests a recent spread of this mosquito from Karachi. A strong negative correlation between genetic distance and the quality of road connections shows that populations in cities connected by better road networks are less differentiated, which suggests the human‐aided passive dispersal of Ae. aegypti in Pakistan. Dispersal on a large spatial scale may facilitate the strategy of introducing transgenic Ae. aegypti or intracellular bacteria such as Wolbachia to control the spread of dengue disease in Pakistan, but it also emphasizes the need for simple measures to control container breeding sites.     

Genetic differentiation of the dengue vector, Aedes aegypti (Ho Chi Minh City, Vietnam) using microsatellite markers

Dengue haemorrhagic fever emerged in the 1950s and has become a major public health concern in most Asian countries. In Vietnam, little is known about the intraspecific variation of the vector and its consequences on vectorial capacity. Here we report the use of microsatellite markers to differentiate Aedes aegypti populations in Ho Chi Minh City, a typical, overcrowded Asian city. Six microsatellite loci, with 5-14 alleles per locus, were scored in 20 mosquito samples collected in 1998 in Ho Chi Minh City. We found substantial differentiation among Ae. aegypti populations from the outskirts, whereas populations from the centre of the city showed less differentiation. These results are consistent with the hypothesis that populations of Ae. aegypti in central Ho Chi Minh City are panmictic because there are abundant larval breeding sites and an abundance of humans for adults to feed upon. In contrast, populations on the outskirts become differentiated largely through the processes of genetic drift because larval breeding sites are not as abundant. These findings implicate human activities associated with urbanization, as factors shaping the genetic structure of Ae. aegypti populations.     

Geographic and ecological distribution of the dengue and chikungunya virus vectors Aedes aegypti and Aedes albopictus in three major Cameroonian towns

Aedes albopictus (Diptera: Culicidae) was first reported in Central Africa in 2000, together with the indigenous mosquito species Aedes aegypti (Diptera: Culicidae). Because Ae. albopictus can also transmit arboviruses, its introduction is a public health concern. We undertook a comparative study in three Cameroonian towns (Sahelian domain: Garoua; equatorial domain: Douala and Yaoundé) in order to document infestation by the two species and their ecological preferences. High and variable levels of pre‐imaginal Ae. aegypti and Ae. albopictus infestation were detected. Only Ae. aegypti was encountered in Garoua, whereas both species were found in Douala and Yaoundé, albeit with significant differences in their relative prevalence. Peridomestic water containers were the most strongly colonized and productive larval habitats for both species. No major differences in types of larval habitat were found, but Ae. albopictus preferentially bred in containers containing plant debris or surrounded by vegetation, whereas Ae. aegypti tended to breed in containers located in environments with a high density of buildings. These findings may have important implications for vector control strategies.     

Sublethal iridovirus disease of the mosquito Aedes aegypti is due to viral replication not cytotoxicity

Invertebrate iridescent viruses (Iridoviridae) possess a highly cytotoxic protein. In mosquitoes (Diptera: Culicidae), invertebrate iridescent virus 6 (IIV-6) usually causes covert (inapparent) infection that reduces fitness. To determine whether sublethal effects of IIV-6 are principally due to cytotoxicity of the viral inoculum (which inhibits macromolecular synthesis in the host), or caused by replication of the virus larvae of the mosquito Aedes aegypti (L) were exposed to untreated IIV-6 virus that had previously been deactivated by heat or ultraviolet light. Control larvae were not exposed to virus. Larval development time was shortest in control larvae and extended in larvae exposed to untreated virus. Covertly infected mosquitoes laid significantly fewer eggs, produced between 20 and 35% fewer progeny and had reduced longevity compared to other treatments. Wing length was shortest in mosquitoes exposed to heat-deactivated virus. Multivariate analysis of the same data identified fecundity and progeny production as the most influential variables in defining differences among treatments. Overall, viral infection resulted in a 34% decrease in the net reproductive rate (R0) of covertly infected mosquitoes, vs. only 5-17% decrease of R0 following treatments with deactivated virus, compared to controls. Sublethal effects of IIV-6 in Ae. aegypti appear to be mainly due to virus replication, rather than cytotoxic effects of the viral inoculum.     

Frequency of pyrethroid resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand

Thirty‐two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda‐cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 – 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) was susceptible (> 98% mortality) to deltamethrin, with incipient resistance (observed 97–82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely susceptible (100% mortality) to the recommended operational dosage of lambda‐cyhalothrin. All five populations of Ae. albopictus were found completely susceptible to both deltamethrin and lambda‐cyhalothrin. Evidence of defined incipient or resistance to synthetic pyrethroids mandates appropriate response and countermeasures to mitigate further development and spread of resistance. In light of these findings, we conclude that routine and comprehensive susceptibility monitoring of dengue mosquito vectors to synthetic pyrethroids should be a required component of resistance management policies and disease control activities.     

Susceptibility and alterations by diflubenzuron in larvae of Aedes aegypti

Benzoylphenyl ureas inhibit chitin synthesis and interfere with the molting process in arthropods. In this study, the effect of diflubenzuron on third‐instar larvae of Aedes aegypti was evaluated. The susceptibility to the product was determined, and the alterations generated were shown through light and electron microscopy. LC₅₀ and LC₉₀ were 0.23 and 0.47 ppm, respectively. The main alterations observed were the incapacity to complete the molt, a reduction of mobility, the fragmentation of the old cuticle, a division of the body segments that was not evident, and the deformation of the caudal structures. Images of the ultrastructure are included, where breaking zones in the cuticle were observed, separation of the cuticle, the epidermis and the muscles, and these latter with a disorganized arrangement. In low concentrations, from 0.15 ppm, diflubenzuron causes alterations in the behavior and morphology of Ae. aegypti.     

The effect of photoperiod on life history and blood‐feeding activity in Aedes albopictus and Aedes aegypti (Diptera: Culicidae)

Several studies have examined how climatic variables such as temperature and precipitation may affect life history traits in mosquitoes that are important to disease transmission. Despite its importance as a seasonal cue in nature, studies investigating the influence of photoperiod on such traits are relatively few. This study aims to investigate how photoperiod alters life history traits, survival, and blood‐feeding activity in Aedes albopictus (Skuse) and Aedes aegypti (Linnaeus). We performed three experiments that tested the effects of day length on female survival, development time, adult size, fecundity, adult life span, and propensity to blood feed in Ae. albopictus and Ae. aegypti. Each experiment had three photoperiod treatments: 1) short‐day (10L:14D), 2) control (12L:12D), and 3) long‐day (14L:10D). Aedes albopictus adult females were consistently larger in size when reared in short‐day conditions. Aedes aegypti adult females from short‐day treatments lived longer and were more likely to take a blood meal compared to other treatments. We discuss how species‐specific responses may reflect alternative strategies evolved to increase survival during unfavorable conditions. We review the potential impacts of these responses on seasonal transmission patterns, such as potentially increasing vectorial capacity of Ae. aegypti during periods of shorter day lengths.     

Polymorphic microsatellite markers for studies of Aedes aegypti (Diptera: Culicidae), the vector of dengue and yellow fever

A significant challenge to population genetic studies of the dengue vector, Aedes aegypti, has been the lack of polymorphic microsatellite loci. In an effort to develop useful markers, we evaluated the genetic variation at 17 microsatellite loci identified in the A. aegypti genome. Nine loci with at least five alleles were identified in field‐collected specimens from Thailand. An additional two loci carried five alleles if samples from an A. aegypti laboratory colony were included. Our results greatly increase the number of highly variable markers available for the study of the genetics and the population structure of this medically important species.     

Prospective field study of transovarial dengue‐virus transmission by two different forms of Aedes aegypti in an urban area of Bangkok,Thailand

A prospective field study was conducted to determine transovarial dengue‐virus transmission in two forms of Aedes aegypti mosquitoes in an urban district of Bangkok, Thailand. Immature Aedes mosquitoes were collected monthly for one year and reared continuously until adulthood in the laboratory. Mosquitoes assayed for dengue virus were processed in pools and their dengue virus infection status was determined by one‐step RT‐PCR and nested‐PCR methods. Of a total 15,457 newly emerged adult Ae. aegypti, 98.2% were dark and 1.8% of the pale form. The results showed that the minimum infection rate (MIR) by transovarial transmission (TOT) of dengue virus during the one‐year study ranged between 0 to 24.4/1,000 mosquitoes. Dengue virus TOT increased gradually during the hot summer months, reaching a peak in April‐June, while dengue cases peaked in September, a rainy month near the end of the rainy season. Therefore, mosquito infections due to TOT were prevalent four months before a high incidence of human infections. TOT dengue virus infections occurred in both forms of Ae. aegypti. All four dengue serotypes were detected, with DEN‐4 predominant, followed by DEN‐3, DEN‐1, and DEN‐2, respectively.     

Natural vertical transmission of dengue viruses by Aedes aegypti in Bolivia

The natural transmission of dengue virus from an infected female mosquito to its progeny, namely the vertical transmission, was researched in wild caught Aedes aegypti during an important outbreak in the town of Santa Cruz de la Sierra, Bolivia. Mosquitoes were collected at the preimaginal stages (eggs, larvae and pupae) then reared up to adult stage for viral detection using molecular methods. Dengue virus serotypes 1 and 3 were found to be co-circulating with significant higher prevalence in male than in female mosquitoes. Of the 97 pools of Ae. aegypti (n = 635 male and 748 female specimens) screened, 14 pools, collected in February-May in 2007, were found positive for dengue virus infection: five DEN-1 and nine DEN-3. The average true infection rate (TIR) and minimum infection rate (MIR) were respectively 1.08% and 1.01%. These observations suggest that vertical transmission of dengue virus may be detected in vectors at the peak of an outbreak as well as several months before an epidemic occurs in human population.     

Proteome of Aedes aegypti in response to infection and coinfection with microsporidian parasites

Hosts are frequently infected with more than one parasite or pathogen at any one time, but little is known as to how they respond to multiple immune challenges compared to those involving single infections. We investigated the proteome of Aedes aegypti larvae following infection with either Edhazardia aedis or Vavraia culicis, and coinfections involving both. They are both obligate intracellular parasites belonging to the phylum microsporidia and infect natural populations of Ae. aegypti. The results found some proteins only showing modified abundance in response to infections involving E. aedis, while others were only differentially abundant when infections involved V. culicis. Some proteins only responded with modified abundance to the coinfection condition, while others were differentially abundant in response to all three types of infection. As time since infection increased, the response to each of the single parasite infections diverged, while the response to the E. aedis and coinfection treatments converged. Some of the proteins differentially abundant in response to infection were identified. They included two vacuolar ATPases, proteins known to have a role in determining the infection success of intracellular parasites. This result suggests microsporidia could influence the infection success of other intracellular pathogens infecting vector species of mosquito, including viruses, Plasmodium and Wolbachia.     

Dengue virus detection in Aedes aegypti larvae from southeastern Brazil

The transmission of dengue, the most important arthropod‐borne viral disease in Brazil, has been intensified over the past decades, along with the accompanying expansion and adaptation of its Aedes vectors. In the present study, we mapped dengue vectors in Ouro Preto and Ouro Branco, Minas Gerais, by installing ovitraps in 32 public schools. The traps were examined monthly between September, 2011 through July, 2012 and November, 2012 to April, 2013. The larvae were reared until the fourth stadium and identified according to species. The presence of dengue virus was detected by real time PCR and agarose gel electrophoresis. A total of 1,945 eggs was collected during the 17 months of the study. The Ovitrap Positivity Index (OPI) ranged from 0 to 28.13% and the Eggs Density Index (EDI) ranged from 0 to 59.9. The predominant species was Aedes aegypti, with 84.9% of the hatched larvae. Although the collection was low when compared to other ovitraps studies, vertical transmission could be detected. Of the 54 pools, dengue virus was detected in four Ae. aegypti pools.