Background: Every year, more than 700,000 people die from vector-borne diseases, mainly transmitted by mosquitoes. Vector surveillance plays a major role in the control of these diseases and requires accurate and rapid taxonomical identification. New approaches to mosquito surveillance include the use of acoustic and optical sensors in combination with machine learning techniques to provide an automatic classification of mosquitoes based on their flight characteristics, including wingbeat frequency. The development and application of these methods could enable the remote monitoring of mosquito populations in the field, which could lead to significant improvements in vector surveillance. Methods: A novel optical sensor prototype coupled to a commercial mosquito trap was tested in laboratory conditions for the automatic classification of mosquitoes by genus and sex. Recordings of > 4300 laboratory-reared mosquitoes of Aedes and Culex genera were made using the sensor. The chosen genera include mosquito species that have a major impact on public health in many parts of the world. Five features were extracted from each recording to form balanced datasets and used for the training and evaluation of five different machine learning algorithms to achieve the best model for mosquito classification. Results: The best accuracy results achieved using machine learning were: 94.2% for genus classification, 99.4% for sex classification of Aedes, and 100% for sex classification of Culex. The best algorithms and features were deep neural network with spectrogram for genus classification and gradient boosting with Mel Frequency Cepstrum Coefficients among others for sex classification of either genus. Conclusions: To our knowledge, this is the first time that a sensor coupled to a standard mosquito suction trap has provided automatic classification of mosquito genus and sex with high accuracy using a large number of unique samples with class balance. This system represents an improvement of the state of the art in mosquito surveillance and encourages future use of the sensor for remote, real-time characterization of mosquito populations. Graphical

Background: Aedes vexans(Meigen) is considered a nuisance species in central Europe and the Mediterranean region. It is an anthropophilic and mammalophilic floodwater mosquito involved in the transmission of several arboviruses. Rift Valley fever (RVF) is a relevant mosquito-borne zoonosis, affecting mainly humans and ruminants, that causes severe impact in public health and economic loses. Due to globalization and climate change, the European continent is threatened by its introduction. The main purpose of the present study was to evaluate the vector competence of a European field-collectedAe. vexanspopulation.Methods: Aedes vexansfield-collected larvae were reared in the laboratory under field-simulated conditions. To assess the vector competence for Rift Valley fever phlebovirus (RVFV) transmission, adult F0 females were exposed to infectious blood meals containing the 56/74 RVFV strain. Additionally, intrathoracic inoculations with the same virus strain were performed to evaluate the relevance of the salivary gland barriers. Natural circulation of alphavirus, flavivirus and phlebovirus was also tested.Results: To our knowledge, an autochthonousAe. vexanspopulation was experimentally confirmed as a competent vector for RVFV for the first time. This virus was capable of infecting and disseminating within the studiedAe. vexansmosquitoes. Moreover, infectious virus was isolated from the saliva of disseminated specimens, showing their capacity to transmit the virus. Additionally, a natural infection with a circulating Mosquito flavivirus was detected. The co-infection with the Mosquito flavivirus seemed to modulate RVFV infection susceptibility in field-collectedAe. vexans, but further studies are needed to confirm its potential interference in RVFV transmission.Conclusions: Our results show that field-collected EuropeanAe. vexanswould be able to transmit RVFV in case of introduction into the continent. This should be taken into consideration in the design of surveillance and control programmes.

Worldwide, emerging and re-emerging infectious diseases (EIDs) are a major burden on public and animal health. Arthropod vectors, with mosquitoes being the main contributors of global disease, transmit more than 70% of the recognized EIDs. To assess new alternatives for arthropod-borne viral diseases surveillance, and for the detection of new viruses, honey-baited Flinders Technology Associates (FTA) cards were used as sugar bait in mosquito traps during entomological surveys at the Llobregat River Delta (Catalonia, Spain). Next generation sequencing (NGS) metagenomics analysis was applied on honey-baited FTA cards, which had been exposed to field-captured mosquitoes to characterize their associated virome. Arthropod- and plant-infecting viruses governed the virome profile on FTA cards. Twelve near-complete viral genomes were successfully obtained, suggesting good quality preservation of viral RNAs. Mosquito pools linked to the FTA cards were screened for the detection of mosquito-associated viruses by specific RT-PCRs to confirm the presence of these viruses. The circulation of viruses related to Alphamesonivirus, Quaranjavirus and unclassified Bunyavirales was detected in mosquitoes, and phylogenetic analyses revealed their similarities to viruses previously reported in other continents. To the best our knowledge, our findings constitute the first distribution record of these viruses in European mosquitoes and the first hint of insect-specific viruses in mosquitoes’ saliva in field conditions, demonstrating the feasibility of this approach to monitor the transmissible fraction of the mosquitoes’ virome. In conclusion, this pilot viromics study on honey-baited FTA cards was shown to be a valid approach for the detection of viruses circulating in mosquitoes, thereby setting up an alternative tool for arbovirus surveillance and control programs.

Background: Historically,Anopheles atroparvushas been considered one of the most important malaria vectors in Europe. Since malaria was eradicated from the European continent, the interest in studying its vectors reduced significantly. Currently, to better assess the potential risk of malaria resurgence on the continent, there is a growing need to update the data on susceptibility of indigenousAnophelespopulations to importedPlasmodiumspecies. In order to do this, as a first step, an adequate laboratory colony ofAn. atroparvusis needed.Methods: Anopheles atroparvusmosquitoes were captured in rice fields from the Ebro Delta (Spain). Field-caught specimens were maintained in the laboratory under simulated field-summer conditions. Adult females were artificially blood-fed on fresh whole rabbit blood for oviposition. First- to fourth-instar larvae were fed on pulverized fish and turtle food. Adults were maintained with a 10% sucrose solutionad libitum.Results: AnAn. atroparvuspopulation from the Ebro Delta was successfully established in the laboratory. During the colonization process, feeding and hatching rates increased, while a reduction in larval mortality rate was observed.Conclusions: The present study provides a detailed rearing and maintenance protocol forAn. atroparvusand a publicly available reference mosquito strain within the INFRAVEC2 project for further research studies involving vector-parasite interactions.

Even though malaria was eradicated from Europe after the mid-20th century, in 2017, more than 8000 imported cases were reported in the continent. Due to travel routes to endemic areas, climate change, and the presence of native vector mosquitoes (genus Anopheles), the re-establishment of autochthonous malaria transmission is a current concern. Anopheles atroparvus (Van Thiel, 1972) is one of the 11 sibling species within the Palearctic Anopheles maculipennis complex, which formerly were considered the main vectors of the disease in the European continent. The microbiota (bacteria and viruses) of vector species has been demonstrated to play a significant role in the biology of these organisms, including their infection susceptibility and their capacity to transmit disease-causing agents. Recently, with the improvement of metagenomics techniques, several viruses that naturally infect vector mosquitoes have been identified. The purpose of the present study was to characterize, for the first time, the virome present in An. atroparvus from the Ebro Delta and assess its evolution after ten generations in the laboratory. Small RNA sequencing was used to characterize the virome from wild-caught An. atroparvus females and from the tenth generation produced under controlled laboratory conditions. Through this approach, we were able to identify viral linages previously reported in other invertebrates, such as Chaq virus and several Partiti-like viruses. A reduction in the viral composition was observed during the colonization process. The present study contributes to the understanding of the viral diversity of a medically relevant vector species in its natural setting and under confinement, and sets a baseline for further studies to assess the potential implications of these viruses in the transmission of pathogens.