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Abstract Background The age distribution of a mosquito population is a major determinant of its vectorial capacity. To contribute to disease transmission, a competent mosquito vector, carrying a pathogen, must live longer than the extrinsic incubation period of that pathogen to enable transmission to a new host. As such, determining the age of female mosquitoes is of significant interest for vector-borne diseases surveillance and control programs. Methods In this contribution, an automated age-grading system was developed to classify the age of female Culex pipiens, which is the primary vector of West Nile virus and other pathogens of medical and veterinary importance in northern latitudes. The system comprises an optical wingbeat sensor coupled to the entrance of a mosquito trap and a machine learning model. Three age classes were used in the study: young (2–4 days), middle (7–9 days) and old (14–16 days). From a balanced dataset of flight data, four features were extracted: wingbeat fundamental frequency, spectrogram, power spectral density and Mel frequency cepstral coefficients. The features were used for training with the XGBoost algorithm to generate a model for age classification. Results The best performing model was trained with the power spectral density feature on two age classes, ≤ 4 days old and ≥ 7 days old, and had an accuracy of 71.8%. Conclusions An automated mosquito age-grading system was applied for the first time to our knowledge for automated age classification in mosquitoes; and complements the mosquito genus and sex classification capability of the system reported in our previous work. The system may find use in mosquito-borne disease surveillance and control to help to discriminate young mosquitoes (≤ 4 days old) from older mosquitoes, which may act as vectors of arboviruses. Graphical Abstract

Malaria remains one of the most important infectious diseases globally due to its high incidence and mortality rates. The influx of infected cases from endemic to non-endemic malaria regions like Europe has resulted in a public health concern over sporadic local outbreaks. This is facilitated by the continued presence of competent Anopheles vectors in non-endemic countries.We modelled the potential distribution of the main malaria vector across Spain using the ensemble of eight modelling techniques based on environmental parameters and the Anopheles maculipennis s.l. presence/absence data collected from 2000 to 2020. We then combined this map with the number of imported malaria cases in each municipality to detect the geographic hot spots with a higher risk of local malaria transmission.The malaria vector occurred preferentially in irrigated lands characterized by warm climate conditions and moderate annual precipitation. Some areas surrounding irrigated lands in northern Spain (e.g. Zaragoza, Logroño), mainland areas (e.g. Madrid, Toledo) and in the South (e.g. Huelva), presented a significant likelihood of A. maculipennis s.l. occurrence, with a large overlap with the presence of imported cases of malaria.While the risk of malaria re-emergence in Spain is low, it is not evenly distributed throughout the country. The four recorded local cases of mosquito-borne transmission occurred in areas with a high overlap of imported cases and mosquito presence. Integrating mosquito distribution with human incidence cases provides an effective tool for the quantification of large-scale geographic variation in transmission risk and pinpointing priority areas for targeted surveillance and prevention.

Abstract Background Mosquito-borne diseases are a major concern for public and veterinary health authorities, highlighting the importance of effective vector surveillance and control programs. Traditional surveillance methods are labor-intensive and do not provide high temporal resolution, which may hinder a full assessment of the risk of mosquito-borne pathogen transmission. Emerging technologies for automated remote mosquito monitoring have the potential to address these limitations; however, few studies have tested the performance of such systems in the field. Methods In the present work, an optical sensor coupled to the entrance of a standard mosquito suction trap was used to record 14,067 mosquito flights of Aedes and Culex genera at four temperature regimes in the laboratory, and the resulting dataset was used to train a machine learning (ML) model. The trap, sensor, and ML model, which form the core of an automated mosquito surveillance system, were tested in the field for two classification purposes: to discriminate Aedes and Culex mosquitoes from other insects that enter the trap and to classify the target mosquitoes by genus and sex. The field performance of the system was assessed using balanced accuracy and regression metrics by comparing the classifications made by the system with those made by the manual inspection of the trap. Results The field system discriminated the target mosquitoes (Aedes and Culex genera) with a balanced accuracy of 95.5% and classified the genus and sex of those mosquitoes with a balanced accuracy of 88.8%. An analysis of the daily and seasonal temporal dynamics of Aedes and Culex mosquito populations was also performed using the time-stamped classifications from the system. Conclusions This study reports results for automated mosquito genus and sex classification using an optical sensor coupled to a mosquito trap in the field with highly balanced accuracy. The compatibility of the sensor with commercial mosquito traps enables the sensor to be integrated into conventional mosquito surveillance methods to provide accurate automatic monitoring with high temporal resolution of Aedes and Culex mosquitoes, two of the most concerning genera in terms of arbovirus transmission. Graphical Abstract

Abstract 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 abstract

Abstract Background Historically, Anopheles atroparvus has 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 indigenous Anopheles populations to imported Plasmodium species. In order to do this, as a first step, an adequate laboratory colony of An. atroparvus is needed. Methods Anopheles atroparvus mosquitoes 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 solution ad libitum. Results An An. atroparvus population 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 for An. atroparvus and a publicly available reference mosquito strain within the INFRAVEC2 project for further research studies involving vector-parasite interactions.

ABSTRACTAedes albopictus mosquitoes have been experimentally demonstrated to be a competent vector for Zika virus (ZIKV) in different countries, but there are still some gaps related to the importance of Ae. albopictus in ZIKV transmission. Recent studies on Spanish Ae. albopictus populations showed controversial results for ZIKV transmission and no studies have been performed yet to detect infectious ZIKV in saliva of progeny of infected female mosquitoes. Herein, the horizontal transmission (HT) and vertical transmission (VT) of ZIKV in field-collected Ae. albopictus mosquitoes from Spain were evaluated for ZIKV strains (African I and Asian lineages) to better estimate the risk of ZIKV transmission by Ae. albopictus. The two field-collected Ae. albopictus populations assayed were infected by all tested ZIKV strains, however differences in terms of vector competence were detected depending on strain-population combination. Moreover, a higher susceptibility to the African I lineage strain than to the Asian lineage strain was observed in both mosquito populations. On the other hand, VT was demonstrated for both ZIKV lineages, detecting the virus in both males and females of the progeny of infected females, although importantly ZIKV dissemination and transmission were not detected in the infected females from the offspring. The results of the present study demonstrate that Spanish Ae. albopictus populations could sustain virus transmission in case of ZIKV introduction, but VT would play a poor role in the ZIKV epidemiology. Overall, our results provide helpful information to health authorities to establish efficient surveillance and vector control programmes for ZIKV.

Abstract Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype–environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat‐shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental‐induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.

Abstract 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-collected Ae. vexans population. Methods Aedes vexans field-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 autochthonous Ae. vexans population was experimentally confirmed as a competent vector for RVFV for the first time. This virus was capable of infecting and disseminating within the studied Ae. vexans mosquitoes. 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-collected Ae. vexans, but further studies are needed to confirm its potential interference in RVFV transmission. Conclusions Our results show that field-collected European Ae. vexans would 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.

Abstract Background Aedes caspius (Pallas, 1771) is a floodwater mosquito species widely distributed in the Western Palaearctic. As an anthropophilic species, its role as an arbovirus vector may be the key for understanding the transmission cycle of certain diseases in Europe such as Zika virus (ZIKV). Concerning vector competence for ZIKV, studies related to Ae. caspius are still scarce. ZIKV is an arbovirus that has provoked a widespread epidemic in the Pacific region (2007–2013) and in the Americas (2015–2016). ZIKV is associated with serious neurological injuries (e.g. microcephaly) and Guillain-Barré syndrome. Due to the ZIKV epidemics in the American continent, some viraemic travellers coming from endemic countries have been reported in Europe. More knowledge is therefore required to define the susceptibility of autochthonous mosquito species such as Ae. caspius for ZIKV in order to improve arbovirus surveillance and control programmes. In the present study, the vector competence of a European population of Ae. caspius was evaluated for two ZIKV lineages, the Suriname ZIKV strain (Asian lineage) and the MR766 ZIKV strain (African I lineage). Females were tested at 7, 14 and 21 days post-exposure (dpe) to infectious blood meals. An Ae. aegypti PAEA strain was used as a positive control. Results Aedes caspius presented low susceptibility to ZIKV infection and the virus was only detected by RT-qPCR in body samples. Low viral loads were detected for the MR766 strain at 7 dpe and for the Suriname strain at 14 and 21 dpe. Aedes caspius was unable to produce a disseminated infection and virus transmission at any of the tested time points. Using Ae. aegypti PAEA strain, infection, dissemination and transmission rates were calculated for the Suriname ZIKV strain (Asian lineage) at each time point. For the MR766 ZIKV strain (African I lineage), while only infection rates were estimated at each time point, no dissemination or transmission were detected in either species. Conclusions The results of the present study reveal that the tested Ae. caspius population has a strong midgut escape barrier that limits the dissemination or transmission of the virus. As such, it seems unlikely that European Ae. caspius mosquitoes could be involved in ZIKV transmission if ZIKV was introduced into Europe. This information may help in designing a better strategy to European surveillance and control programmes for ZIKV.

[This corrects the article DOI: 10.1371/journal.pone.0217860.].

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.

A range of mosquito species that belong to the Culicidae family are responsible for the worldwide transmission of infectious arboviral diseases such as dengue fever, Zika, West Nile fever and Chikungunya fever. Spain is at risk of arbovirus outbreaks, as various arboviral diseases are frequently introduced and it has established competent vector populations. Autochthonous human cases of West Nile virus have been reported infrequently since 2004, and since October 2018 three autochthonous human case of dengue fever have been confirmed. In response to an outbreak of any arboviral disease, space spraying or fogging will be implemented to control adult mosquito populations. To ensure adulticiding is cost-effective, the insecticide susceptibility status of vectors throughout Catalonia, an autonomous region in north-eastern Spain, was assessed through standardized WHO tube and CDC bottle bioassays. All Culex pipiens populations tested were resistant to at least one of the pyrethroids tested, whereas Aedes albopictus populations were susceptible to all pyrethroids tested. More detailed studies on the Cx. pipiens populations from the Barcelona area (the capital and largest city of Catalonia) revealed resistance to all four classes of public health insecticides available (pyrethroids, carbamates, organophosphates and organochlorides). All Ae. albopictus populations were susceptible to those classes, except for one of the tests performed with pirimiphos-methyl (an organophosphate). Pyrethroids are currently the first line chemical class to be used in space spray operations in response to an outbreak of an arboviral disease. While pyrethroids can be effective in reducing Ae. albopictus populations, this class may not be effective to control Cx. pipiens populations.

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.

Eutrophication is one of the major threats to freshwater biodiversity with important ramifications for ecosystem functions and the benefits they provide to society. One of the first visible effects of eutrophication is elevated water turbidity, which reduces the aesthetic appeal of water bodies. Also, turbidity limits organisms’ response to visual cues, which can alter species interactions including prey-consumer relationships. For visual predators, such as most fish, turbidity acts as anti-predation refugee for their prey. This loss of the top-down control can trigger multi-trophic impacts with potential collateral effects such as the proliferation of pests. The mosquito Culex pipiens is one of the most common mosquito species in eutrophic waters where its larvae are favoured by the organic matter enrichment (e.g. microalgae, bacteria and other fine particles). Since mosquitoes are annoying insects for public, and some species are vectors of diseases, mosquito control is a major interest for administration. In many temperate countries, including Spain, this led to the introduction of the eastern mosquitofish (Gambusia holbrooki), one of the world’s 100 worst invasive species. However, this practice is now illegal after the enforcement of new legislation. In an attempt to demonstrate that native species can be as effective as G. holbrooki for mosquito control, this study examined the efficacy of G. holbrooki and the endangered Spanish toothcarp (Aphanius iberus). Specifically, we compared the voracity and total biomass of larvae consumed by the two fish species along a turbidity gradient, simulating phytoplankton and fine sediment levels observed in eutrophic waters. Our results support the replacement of G. holbrooki with A. iberus for mosquito larval control despite indicate the major voracity of the former in all treatments. In conclusion, this study suggests that the introduction of G. holbrooki was perhaps unnecessary for mosquito biocontrol in areas inhabited by A. iberus.