Dengue virus (DENV) is an arbovirus transmitted to humans byAedes mosquitoes 1 . In the insect vector, the small interfering RNA (siRNA) pathway is an important antiviral mechanism against DENV 2-5 . However, it remains unclear when and where the siRNA pathway acts during the virus cycle. Here, we show that the siRNA pathway fails to efficiently silence DENV in the midgut of Aedes aegypti although it is essential to restrict systemic replication. Accumulation of DENV-derived siRNAs in the midgut reveals that impaired silencing results from a defect downstream of small RNA biogenesis. Notably, silencing triggered by endogenous and exogenous dsRNAs remained effective in the midgut where known components of the siRNA pathway, including the dsRNA binding proteins Loquacious and r2d2, had normal expression levels. We identified an Aedes specific paralog of loquacious and r2d2, hereafter named loqs2, which is not expressed in the midgut. Loqs2 interacts with Loquacious and r2d2 and is required to control systemic replication of DENV and also Zika virus (ZIKV). Furthermore, ectopic expression of Loqs2 in the midgut of transgenic mosquitoes is sufficient to restrict DENV replication and dissemination. Together our data reveal a mechanism of tissue specific regulation of the mosquito siRNA pathway controlled by Loqs2.Previous studies observed production of DENV-derived siRNAs and piwi-interacting RNAs (piRNAs) in infected mosquitoes and suggested that different RNA interference (RNAi) mechanisms were important to control virus replication 2,3,6,7 . In order to investigate the activation of RNAi by DENV infection in mosquitoes, we first analyzed different concentrations of the virus in the blood meal (Supplementary Fig. 1a,b). Using 10 7 PFU/mL of virus, we observed that 100% of individual mosquitoes had detectable virus at 1 and 2 days post feeding (dpf) (Fig. 1a). This likely corresponds to the virus inoculum in the blood meal since the amount of viral RNA decreased until 4 dpf when mosquitoes have completed blood digestion 8 . At this point, viral RNA could not be detected in some mosquitoes, suggesting that they had cleared the virus (defined

Wolbachia are intracellular, maternally transmitted bacteria considered the most abundant endosymbionts found in arthropods. They reproductively manipulate their host in order to increase their chances of being transmitted to the offspring, and currently are being used as a tool to control vector-borne diseases. Studies on distribution of Wolbachia among its arthropod hosts are important both for better understanding why this bacterium is so common, as well as for its potential use as a biological control agent. Here, we studied the incidence of Wolbachia in a broad range of insect species, collected from different regions of Brazil, using three genetic markers (16S rRNA, wsp and ftsZ), which varied in terms of their sensitivity to detect this bacterium. The overall incidence of Wolbachia among species belonging to 58 families and 14 orders was 61.9%. The most common positive insect orders were Coleoptera, Diptera, Hemiptera and Hymenoptera, with Diptera and Hemiptera having the highest numbers of Wolbachia-positive families. They included potential human disease vectors whose infection status has never been reported before. Our study further shows the importance of using quantitative polymerase chain reaction for high-throughput and sensitive Wolbachia screening.

The microbial flora associated with Anopheles darlingi Root (Diptera: Culicidae), a major Neotropical malaria vector, was investigated for the development of a paratransgenesis-based approach to control malaria transmission in Brazil. Female mosquitoes were collected using human land catches and captured insects provided a bloodmeal. The controlled blood feeding resulted in increased detection of mosquito bacterial population because it was possible to retrieve bacterial DNA from all blood-fed mosquitoes. The 16S sequences of bacteria recovered, include some closely related to those found in other vector mosquitoes, including Aeromonas, Pantoea and Pseudomonas species.

BackgroundThe bacterium Wolbachia is a promising agent for the biological control of vector-borne diseases as some strains have the ability to block the transmission of key human disease-causing pathogens. Fast, accurate and inexpensive methods of differentiating between infected and uninfected insects will be of critical importance as field-based trials of Wolbachia-based bio-control become increasingly common.FindingsWe have developed a specific and sensitive method of detecting Wolbachia based on the isothermal DNA amplification. This technique can be performed in an ordinary heat block without the need for gel-based visualisation, and is effective for a wide variety of insect hosts.ConclusionHere we present the development of a rapid, highly sensitive and inexpensive method to detect Wolbachia in a variety of insect hosts, including key mosquito disease vectors.

Anopheles darlingi is an important vector of human malaria in the Amazon. Adult females of this mosquito species require a blood meal to develop eggs, preferring humans to other blood sources. Although gonotrophic concordance has been described as the norm for An. darlingi, here we report An. darlingi female mosquitoes taking two or more blood meals within their first gonotrophic cycle. Only half of field-captured adult females fed one blood meal developed follicles to Christophers' stage V. This outcome is dependent on larval nutrition, as 88% of laboratory-raised well-nourished females completed the first gonotrophic cycle with only one blood meal, while less nourished females needed additional blood meals. Half of the fieldcaptured blood-seeking An. darlingi females had follicles in intermediate (IIIa and IIIb) and final (V) stages of the gonotrophic cycle, supporting the conclusion that An. darlingi blood feed more than once during a gonotrophic cycle. Additionally, we observed females attempting to blood feed a second time during the same day. Additional studies of An. darlingi biting behavior are necessary to accurately estimate Plasmodium sp. entomologic inoculation rates throughout the An. darlingi vast geographical distribution. Journal of Vector Ecology 37 (2): 351-358. 2012.

BackgroundMosquito-borne diseases are rapidly spreading to vast territories, putting at risk most of the world’s population. A key player in this scenario is Aedes aegypti, a hematophagous species which hosts and transmits viruses causing dengue and other serious illnesses. Since vector control strategies relying only on insecticides have proven unsustainable, an alternative method involving the release of Wolbachia-harboring individuals has emerged. Its successful implementation vastly depends on how fit the released individuals are in the natural habitat, being able to mate with wild populations and to spread Wolbachia to subsequent generations. In mosquitoes, an important aspect of reproductive fitness is the acoustic communication between males and females, which translates to interactions between harmonic frequencies in close proximity flight. This study aimed to characterize the flight tone produced by individuals harboring Wolbachia, also evaluating their ability to establish stable acoustic interactions.MethodsWild-type (WT) and Wolbachia-harboring specimens (wMelBr) were thorax-tethered to blunt copper wires and placed at close proximity to sensitive microphones. Wing-beat frequencies (WBFs) were characterized at fundamental and harmonic levels, for both single individuals and couples. Harmonic interactions in homogeneous and heterogeneous couples of WT and wMelBr variants were identified, categorized and quantified accordingly.ResultsIn tethered ‘solo’ flights, individuals harboring Wolbachia developed WBFs, differing slightly, in a sex-dependent way, from those of the WT strain. To test the ability to form harmonic ‘duets’, tethered couples of wMelBr and WT individuals were shuffled in different sex pairs and had their flight tones analyzed. All couple types, with WT and/or wMelBr individuals, were able to interact acoustically in the frequency range of 1300–1500 Hz, which translates to the convergence between male’s second harmonic and female’s third. No significant differences were found in the proportions of interacting couples between the pair types. Surprisingly, spectrograms also revealed the convergence between alternative harmonic frequencies, inside and outside the species putative hearing threshold.ConclusionsWolbachia infection leads to small sex-dependent changes on the flight tones of Ae. aegypti, but it does not seem to prevent the stereotyped harmonic interaction between males and females. Therefore, when released in the natural habitat to breed with native individuals, Wolbachia-harboring individuals shall be fit enough to meet the criteria of acoustically-related mating behavior and promote bacteria dispersion effectively.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2695-x) contains supplementary material, which is available to authorized users.

Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

The microbial flora associated with Anopheles darlingi Root (Diptera: Culicidae), a major Neotropical malaria vector, was investigated for the development of a paratransgenesis-based approach to control malaria transmission in Brazil. Female mosquitoes were collected using human land catches and captured insects provided a bloodmeal. The controlled blood feeding resulted in increased detection of mosquito bacterial population because it was possible to retrieve bacterial DNA from all blood-fed mosquitoes. The 16S sequences of bacteria recovered, include some closely related to those found in other vector mosquitoes, including Aeromonas, Pantoea and Pseudomonas species.