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, an intracellular endosymbiont present in up to 70% of all insect species, has been suggested as a sustainable strategy for the control of arboviruses such as Dengue, Zika and Chikungunya. As Mayaro virus outbreaks have also been reported in Latin American countries, the objective of this study was to evaluate the vector competence of Brazilian field-collected Ae. aegypti and the impact of Wolbachia (wMel strain) upon this virus. Our in vitro studies with Aag2 cells showed that Mayaro virus can rapidly multiply, whereas in wMel-infected Aag2 cells, viral growth was significantly impaired. In addition, C6/36 cells seem to have alterations when infected by Mayaro virus. In vivo experiments showed that field-collected Ae. aegypti mosquitoes are highly permissive to Mayaro virus infection, and high viral prevalence was observed in the saliva. On the other hand, Wolbachia-harboring mosquitoes showed significantly impaired capability to transmit Mayaro virus. Our results suggest that the use of Wolbachia-harboring mosquitoes may represent an effective mechanism for the reduction of Mayaro virus transmission throughout Latin America.

Diseases transmitted by mosquitoes impose enormous burden towards human morbidity and mortality. Over the last three decades, Brazil has suffered from severe Dengue epidemics. In September 2014, this situation is further complicated by the introduction of two other viruses, Zika and Chikungunya, placing Brazil in a triple epidemic. In this article, we discuss the biology of Aedes aegypti Linnaeus, and the principal initiatives currently used to control mosquito populations and the diseases they transmit. Aedes aegypti has broad global distribution and is involved in the transmission of various arboviral diseases such as Dengue, Zika, and Chikungunya. Several factors contribute to the success of the species, particularly behavioral plasticity, rapid development, desiccation-resistant eggs, resistance to the principle insecticide classes currently available on the market, preference for the urban environment, and proximity to humans. Vector control programs are the best way to reduce the burden of mosquito-borne diseases. Chemical control is most commonly used in recent times, and unfortunately, the results have not been satisfactory but instead, there is increased vector dispersal and, subsequently, the spread of disease epidemics. Investigations of alternative control methods such as release of Wolbachia-infected mosquitoes for blocking vector-borne pathogens, release of transgenic mosquitoes carrying a lethal gene for offspring, and the use of insecticide-dispersing mosquitoes are under way in Brazil, and some have shown promising results. Special emphasis should be placed on integrated management of all available tactics, so as to maximize efforts towards mosquito control. Finally, we emphasize that continuous actions and community participation control initiatives are critically important for success.

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.

Newly emerging or re-emerging arthropod-borne viruses (arboviruses) are important causes of human morbidity and mortality worldwide. Arboviruses such as Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), and West Nile virus (WNV) have undergone extensive geographic expansion in the tropical and subtropical regions of the world. In the Americas the main vectors of DENV, ZIKV, and CHIKV are mosquito species adapted to urban environments, namely Aedes aegypti and Aedes albopictus, whereas the main vector of WNV is Culex quinquefasciatus. Given the widespread distribution in the Americas and high permissiveness to arbovirus infection, these mosquito species may play a key role in the epidemiology of other arboviruses normally associated with sylvatic vectors. Here, we test this hypothesis by determining the vector competence of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus to Mayaro (MAYV) virus, a sylvatic arbovirus transmitted mainly by Haemagogus janthinomys that has been causing an increasing number of outbreaks in South America, namely in Brazil. Using field mosquitoes from Brazil, female mosquitoes were experimentally infected, and their competence for infection and transmission rates of MAYV was evaluated. We found consistent infection rate for MAYV in Ae. aegypti (57.5%) and Ae. albopictus (61.6%), whereas very low rates were obtained for Cx. quinquefasciatus (2.5%). Concordantly, we observed high potential transmission ability in Ae. aegypti and Ae. albopictus (69.5% and 71.1% respectively), in contrast to Cx. quinquefasciatus, which could not transmit the MAYV. Notably, we found that very low quantities of virus present in the saliva (undetectable by RT-qPCR) were sufficiently virulent to guarantee transmission. Although Ae. aegypti and Ae. albopictus mosquitoes are not the main vectors for MAYV, our studies suggest that these mosquitoes could play a significant role in the transmission of this arbovirus, since both species showed significant vector competence for MAYV (Genotype D), under laboratory conditions.

Field release of Wolbachia-infected Aedes aegypti has emerged as a promising solution to manage the transmission of dengue, Zika and chikungunya in endemic areas across the globe. Through an efficient self-dispersing mechanism, and the ability to induce virus-blocking properties, Wolbachia offers an unmatched potential to gradually modify wild Ae. aegypti populations turning them unsuitable disease vectors. Here we describe a proof-of-concept field trial carried out in a small community of Niterói, greater Rio de Janeiro, Brazil. Following the release of Wolbachia-infected eggs, we report here a successful invasion and long-term establishment of the bacterium across the territory, as denoted by stable high-infection indexes (> 80%). We have also demonstrated that refractoriness to dengue and Zika viruses, either thorough oral-feeding or intra-thoracic saliva challenging assays, was maintained over the adaptation to the natural environment of Southeastern Brazil. These findings further support Wolbachia’s ability to invade local Ae. aegypti populations and impair disease transmission, and will pave the way for future epidemiological and economic impact assessments.

-The Mediterranean fruit fl y, Ceratitis capitata (Wiedemann), is considered one of the main pests that affect fruit production in the world. This insect spends part of its life cycle in the soil, making it a target for entomopathogenic nematodes. This work aimed at evaluating the infl uence of soil temperature and moisture on the infectivity of Heterorhabditis sp. RSC01 and Steinernema carpocapsae ALL to third-instars of C. capitata, and to compare the effi ciency of these isolates at fi ve different soil temperatures (19, 22, 25, 28, and 31°C) and three levels of relative soil moisture (100, 75, and 50% of fi eld capacity). Ten C. capitata larvae were transferred to plastic jars (12 cm × 6 cm) containing 100 g soil, followed by the application of an aqueous suspension containing 125 infective juveniles (IJ)/cm 2 . In the control treatment, 3 ml of distilled water was applied. Mortality evaluations were made fi ve days later and were confi rmed by observations of the characteristic symptoms and cadaver dissection. The infectivity was directly proportional to temperature increase, with maximum percent mortality of 86.7% and 80.0% for S. carpocapsae and Heterorhabditis sp., respectively, at 31°C. At 25°C, the highest mortality for both species was obtained at 75% of fi eld capacity (96.7% and 26.7% for S. carpocapsae and Heterorhabditis sp., respectively).

Entomopathogenic nematodes are considered excellent biological control agents, with greater potential against soil insect pests and pests of cryptic environments. The fruit fly Ceratitis capitata is considered one of the main fruit crop pests worldwide. This insect stays in the soil during a phase of life, where it becomes a target for entomopathogenic nematodes. The objective of this study was to evaluate the pathogenicity and virulence of entomopathogenic nematodes on C. capitata. The bioassays were organized with four replications, containing 10 individuals; 1 mL of a nematode suspension containing 200 IJ/insect was applied. The most virulent isolates against C. capitata larvae were selected and applied at concentrations of 0, 50, 100, 150, 200, 250, 300, 350 and 400 IJ/insect. All isolates were pathogenic for C. capitata. The S. carpocapsae ALL and Heterorhabditis sp. RSC01 isolates were the most virulent against the larval stage, with mortalities of over 85%. As to the pupal stage, isolates Heterorhabditis sp. PI, Heterorhabditis sp. JPM4, H. bacteriophora HP88, S. feltiae and S. glaseri were the best, with mortalities ranging between 35 and 44%.Key words: biological control, mediterranean fruit fly, pathogenicity Seleção de nematoides entomopatogênicos visando ao controle da mosca das frutas Ceratitis capitata (Diptera: Tephritidae) RESUMONematoides entomopatogênicos são considerados excelentes agentes de controle biológico demonstrando maior potencialidade em insetos-praga de solo e de ambientes crípticos. A mosca das frutas Ceratitis capitata é considerada uma das principais pragas para a fruticultura mundial. Este inseto passa uma fase de sua vida no solo, tornando-se alvo em potencial para esses entomopatógenos. Assim, teve-se como objetivo avaliar a patogenicidade e a virulência de nematoides entomopatogênicos sobre C. capitata. Os bioensaios foram realizados com quatro repetições contendo 10 indivíduos e aplicado 1 mL de suspensão de nematoides com 200 JI/inseto. Os isolados que se apresentaram mais virulentos para larvas de C. capitata foram selecionados e aplicados nas concentrações de 0, 50, 100, 150, 200, 250, 300, 350 e 400 JI/inseto. Verificou-se que todos os isolados foram patogênicos para C. capitata. Os isolados S. carpocapsae ALL e Heterorhabditis sp. RSC01 foram os mais virulentos para a fase larval causando mortalidade superior a 85%. Em relação à fase de pupa os isolados Heterorhabditis sp. PI, Heterorhabditis sp. JPM4, H.