Wolbachia strains wMel and wAlbB differentially affect Aedes aegypti traits related to fecundity.

Two Wolbachia strains, wMel and wAlbB, have been transinfected into Aedes aegypti mosquitoes for population replacement with the aim of reducing dengue transmission. Epidemiological data from various endemic sites suggest a pronounced decrease in dengue transmission after implementing this strategy. In this study, we investigated the impact of the Wolbachia strains wMel and wAlbB on Ae. aegypti fitness in a common genetic background. We found that Ae. aegypti females infected with the wMel strain exhibited several significant differences compared with those infected with the wAlbB strain. Specifically, wMel-infected females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on Ae. aegypti fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations in endemic settings. Further research is needed to better understand the underlying mechanisms responsible for these differences in fitness effects and their potential impact on the long-term efficacy of Wolbachia-based dengue control programs.IMPORTANCEThe transmission of arboviruses such as dengue, Zika, and chikungunya is on the rise globally. Among the most promising strategies to reduce arbovirus burden is the release of one out of two strains of Wolbachia-infected Aedes aegypti: wMel and wAlbB. One critical aspect of whether this approach will succeed involves the fitness cost of either Wolbachia strains on mosquito life history traits. For instance, we found that wMel-infected Ae. aegypti females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on mosquito fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations.

DENV-1 Titer Impacts Viral Blocking in wMel Aedes aegypti with Brazilian Genetic Background.

Several countries have been using Wolbachia deployments to replace highly competent native Aedes aegypti populations with Wolbachia-carrying mosquitoes with lower susceptibility to arboviruses such as dengue, Zika, and chikungunya. In Rio de Janeiro, Wolbachia deployments started in 2015 and still present a moderate introgression with a modest reduction in dengue cases in humans (38%). Here, we evaluated the vector competence of wild-type and wMel-infected Ae. aegypti with a Brazilian genetic background to investigate whether virus leakage could contribute to the observed outcomes in Brazil. We collected the specimens in three areas of Rio de Janeiro with distinct frequencies of mosquitoes with wMel strain and two areas with wild Ae. aegypti. The mosquitoes were orally exposed to two titers of DENV-1 and the saliva of DENV-1-infected Ae. aegypti was microinjected into wMel-free mosquitoes to check their infectivity. When infected with the high DENV-1 titer, the presence of wMel did not avoid viral infection in mosquitoes' bodies and saliva but DENV-1-infected wMel mosquitoes produced lower viral loads than wMel-free mosquitoes. On the other hand, wMel mosquitoes infected with the low DENV-1 titer were less susceptible to virus infection than wMel-free mosquitoes, although once infected, wMel and wMel-free mosquitoes exhibited similar viral loads in the body and the saliva. Our results showed viral leakage in 60% of the saliva of wMel mosquitoes with Brazilian background; thus, sustained surveillance is imperative to monitor the presence of other circulating DENV-1 strains capable of overcoming the Wolbachia blocking phenotype, enabling timely implementation of action plans.

Aedes aegypti oviposition-sites choice under semi-field conditions.

Vector control is still the recommended approach to avoid arbovirus outbreaks. Herein, we investigate oviposition preferences of Aedes aegypti (Diptera: Culicidae) females under a semi-field structure Rio de Janeiro, Brazil. For that, in Experiment 1, we used two settings: 'Single items', which included as containers drain, beer bottle, bucket, car tyre, water tank, and a potted Peace Lily (Spathiphyllum wallisii) in a saucer with water, or 'Multiple containers', as an urban simulation, in which one drain, two additional beer bottles, and an extra plant pot saucer were added. Experiment 2 (sensory cues) used five variations of potted plant, each one varying in the range of sensory cues known to attract gravid females to oviposition containers. Our results indicate that gravid Ae. aegypti prefer to oviposit close to the ground and in open water containers with organic compounds from plant watering. Domestic large artificial containers containing tap water received significantly fewer eggs, except for the car tyre, which exhibited as many eggs as the potted plant. We also show that visual (potted plant shape) and olfactory clues (odour of the plant or from water containing organic matter) were equally attractive separately as were these stimuli together.

Gut Bacterial Diversity of Field and Laboratory-Reared Aedes albopictus Populations of Rio de Janeiro, Brazil.

BACKGROUND: The mosquito microbiota impacts different parameters in host biology, such as development, metabolism, immune response and vector competence to pathogens. As the environment is an important source of acquisition of host associate microbes, we described the microbiota and the vector competence to Zika virus (ZIKV) of Aedes albopictus from three areas with distinct landscapes. METHODS: Adult females were collected during two different seasons, while eggs were used to rear F1 colonies. Midgut bacterial communities were described in field and F1 mosquitoes as well as in insects from a laboratory colony (>30 generations, LAB) using 16S rRNA gene sequencing. F1 mosquitoes were infected with ZIKV to determine virus infection rates (IRs) and dissemination rates (DRs). Collection season significantly affected the bacterial microbiota diversity and composition, e.g., diversity levels decreased from the wet to the dry season. Field-collected and LAB mosquitoes' microbiota had similar diversity levels, which were higher compared to F1 mosquitoes. However, the gut microbiota composition of field mosquitoes was distinct from that of laboratory-reared mosquitoes (LAB and F1), regardless of the collection season and location. A possible negative correlation was detected between Acetobacteraceae and Wolbachia, with the former dominating the gut microbiota of F1 Ae. albopictus, while the latter was absent/undetectable. Furthermore, we detected significant differences in infection and dissemination rates (but not in the viral load) between the mosquito populations, but it does not seem to be related to gut microbiota composition, as it was similar between F1 mosquitoes regardless of their population. CONCLUSIONS: Our results indicate that the environment and the collection season play a significant role in shaping mosquitoes' bacterial microbiota.

Rapid and Non-Invasive Detection of Aedes aegypti Co-Infected with Zika and Dengue Viruses Using Near Infrared Spectroscopy.

The transmission of dengue (DENV) and Zika (ZIKV) has been continuously increasing worldwide. An efficient arbovirus surveillance system is critical to designing early-warning systems to increase preparedness of future outbreaks in endemic countries. The Near Infrared Spectroscopy (NIRS) is a promising high throughput technique to detect arbovirus infection in Ae. aegypti with remarkable advantages such as cost and time effectiveness, reagent-free, and non-invasive nature over existing molecular tools for similar purposes, enabling timely decision making through rapid detection of potential disease. Our aim was to determine whether NIRS can differentiate Ae. aegypti females infected with either ZIKV or DENV single infection, and those coinfected with ZIKV/DENV from uninfected ones. Using 200 Ae. aegypti females reared and infected in laboratory conditions, the training model differentiated mosquitoes into the four treatments with 100% accuracy. DENV-, ZIKV-, and ZIKV/DENV-coinfected mosquitoes that were used to validate the model could be correctly classified into their actual infection group with a predictive accuracy of 100%, 84%, and 80%, respectively. When compared with mosquitoes from the uninfected group, the three infected groups were predicted as belonging to the infected group with 100%, 97%, and 100% accuracy for DENV-infected, ZIKV-infected, and the co-infected group, respectively. Preliminary lab-based results are encouraging and indicate that NIRS should be tested in field settings to evaluate its potential role to monitor natural infection in field-caught mosquitoes.

High throughput estimates of Wolbachia, Zika and chikungunya infection in Aedes aegypti by near-infrared spectroscopy to improve arbovirus surveillance.

Deployment of Wolbachia to mitigate dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) transmission is ongoing in 12 countries. One way to assess the efficacy of Wolbachia releases is to determine invasion rates within the wild population of Aedes aegypti following their release. Herein we evaluated the accuracy, sensitivity and specificity of the Near Infrared Spectroscopy (NIRS) in estimating the time post death, ZIKV-, CHIKV-, and Wolbachia-infection in trapped dead female Ae. aegypti mosquitoes over a period of 7 days. Regardless of the infection type, time post-death of mosquitoes was accurately predicted into four categories (fresh, 1 day old, 2-4 days old and 5-7 days old). Overall accuracies of 93.2, 97 and 90.3% were observed when NIRS was used to detect ZIKV, CHIKV and Wolbachia in dead Ae. aegypti female mosquitoes indicating NIRS could be potentially applied as a rapid and cost-effective arbovirus surveillance tool. However, field data is required to demonstrate the full capacity of NIRS for detecting these infections under field conditions.

Yeast-encapsulated essential oils: a new perspective as an environmentally friendly larvicide.

BACKGROUND: Effective mosquito control approaches incorporate both adult and larval stages. For the latter, physical, biological, and chemical control have been used with varying results. Successful control of larvae has been demonstrated using larvicides including insect growth regulators, e.g. the organophosphate temephos, as well as various entomopathogenic microbial species. However, a variety of health and environmental issues are associated with some of these. Laboratory trials of essential oils (EO) have established the larvicidal activity of these substances, but there are currently no commercially available EO-based larvicides. Here we report on the development of a new approach to mosquito larval control using a novel, yeast-based delivery system for EO. METHODS: Food-grade orange oil (OO) was encapsulated into yeast cells following an established protocol. To prevent environmental contamination, a proprietary washing strategy was developed to remove excess EO that is adsorbed to the cell exterior during the encapsulation process. The OO-loaded yeast particles were then characterized for OO loading, and tested for efficacy against Aedes aegypti larvae. RESULTS: The composition of encapsulated OO extracted from the yeast microparticles was demonstrated not to differ from that of un-encapsulated EO when analyzed by high performance liquid chromatography. After lyophilization, the oil in the larvicide comprised 26-30 percentage weight (wt%), and is consistent with the 60-65% reduction in weight observed after the drying process. Quantitative bioassays carried with Liverpool and Rockefeller Ae. aegypti strains in three different laboratories presented LD50 of 5.1 (95% CI: 4.6-5.6) to 27.6 (95% CI: 26.4-28.8) mg/l, for L1 and L3/L4 mosquito larvae, respectively. LD90 ranged between 18.9 (95% CI: 16.4-21.7) mg/l (L1 larvae) to 76.7 (95% CI: 69.7-84.3) mg/l (L3/L4 larvae). CONCLUSIONS: The larvicide based on OO encapsulated in yeast was shown to be highly active (LD50 < 50 mg/l) against all larval stages of Ae. aegypti. These results demonstrate its potential for incorporation in an integrated approach to larval source management of Ae. aegypti. This novel approach can enable development of affordable control strategies that may have significant impact on global health.

Limited risk of Zika virus transmission by five Aedes albopictus populations from Spain.

BACKGROUND: Aedes albopictus, the Asian tiger mosquito, is an exotic invasive species in Europe. It has substantial public health relevance due to its potential role in transmitting several human pathogens. Out of the European countries, Spain has one of the highest risk levels of autochthonous arbovirus transmission due to both the high density of Ae. albopictus and the extensive tourist influx from vector-endemic areas. This study aims to investigate the susceptibility of five Ae. albopictus populations from mainland Spain and the Balearic Islands to a Brazilian Zika virus (ZIKV) strain. METHODS: The F1 generation of each Ae. albopictus population was orally challenged with a ZIKV-infected blood meal (1.8 × 106 PFU/ml). At 7 and 14 days post-infection (dpi), mosquito bodies (thorax and abdomen) and heads were individually analysed through RT-qPCR to determine the infection rate (IR) and dissemination rate (DR), respectively. The saliva of infected mosquitoes was inoculated in Vero cells and the transmission rate was assessed by plaque assay or RT-qPCR on ~33 individuals per population. RESULTS: The IR and DR ranged between 12-88%, and 0-60%, respectively, suggesting that ZIKV is capable of crossing the midgut barrier. Remarkably, no infectious viral particle was found in saliva samples, indicating a low ability of ZIKV to overcome the salivary gland barrier. A subsequent assay revealed that a second non-infective blood meal 48 h after ZIKV exposure did not influence Ae. albopictus vector competence. CONCLUSIONS: The oral experimental ZIKV infections performed here indicate that Ae. albopictus from Spain become infected and disseminate the virus through the body but has a limited ability to transmit the Brazilian ZIKV strain through biting. Therefore, the results suggest a limited risk of autochthonous ZIKV transmission in Spain by Ae. albopictus.

Rapid, noninvasive detection of Zika virus in Aedes aegypti mosquitoes by near-infrared spectroscopy.

The accelerating global spread of arboviruses, such as Zika virus (ZIKV), highlights the need for more proactive mosquito surveillance. However, a major challenge during arbovirus outbreaks has been the lack of rapid and affordable tests for pathogen detection in mosquitoes. We show for the first time that near-infrared spectroscopy (NIRS) is a rapid, reagent-free, and cost-effective tool that can be used to noninvasively detect ZIKV in heads and thoraces of intact Aedes aegypti mosquitoes with prediction accuracies of 94.2 to 99.3% relative to quantitative reverse transcription polymerase chain reaction (RT-qPCR). NIRS involves simply shining a beam of light on a mosquito to collect a diagnostic spectrum. We estimated in this study that NIRS is 18 times faster and 110 times cheaper than RT-qPCR. We anticipate that NIRS will be expanded upon for identifying potential arbovirus hotspots to guide the spatial prioritization of vector control.

Marking Triatoma brasiliensis, Triatoma pseudomaculata and Rhodnius nasutus Nymphs with Trace Elements: Element Persistence and Effects of Marking on Insect Mortality.

BACKGROUND: Field ecologists often rely on mark-release-recapture (MRR) experiments to estimate population dynamics parameters for a given species. In the case of a medically important taxon, i.e., a disease vector, inferences on species survival and dispersal rates are particularly important as they have the potential to provide insights into disease transmission dynamics in endemic areas. Medical entomologists have traditionally used fluorescent dusts to externally mark the cuticle of insects. However, dust marking is usually restricted to the adult life stage because immature insects lose the mark when they molt. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the efficacy of 13 trace elements in marking nymphs of three native Brazilian Chagas disease vectors: Triatoma brasiliensis, Triatoma pseudomaculata, and Rhodnius nasutus. Cr and Cu were detected in over 97% of T. brasiliensis (34/35 31/31 for Cr and Cu), while Cu and Mn were detected in more than 95% of T. pseudomaculata (29/29 for Cu and 28/29 for Mn) tested 120 days after marking. Only Mn marked over 90% of R. nasutus nymphs (38/41). Overall, trace elements had no negative effects on T. pseudomaculata longevity, but As-marked T. brasiliensis nymphs (p<0.01), and Cd-marked R. nasutus nymphs (p<0.01) had significantly shorter lifespan. CONCLUSIONS/SIGNIFICANCE: Previous evidence shows that there is little or no genetic differentiation between populations at the microgeographic level, which often precludes indirect estimations of dispersal capability based on genetic markers. In such situations, MRR studies are more suitable as they measure insect movement directly from one site to another, instead of effective migration (i.e. gene flow). The determination of a reliable and persistent marking method is the first step towards the development of meaningful ecological estimates through the application of MRR methodology. Here, we have identified trace elements that can be used for mark and recapture studies of three triatomine species in Brazil.