Microbiota Variation Across Life Stages of European Field-Caught Anopheles atroparvus and During Laboratory Colonization: New Insights for Malaria Research

The potential use of bacteria for developing novel vector control approaches has awakened new interests in the study of the microbiota associated with vector species. To set a baseline for future malaria research, a high-throughput sequencing of the bacterial 16S ribosomal gene V3-V4 region was used to profile the microbiota associated with late-instar larvae, newly emerged females, and wild-caught females of a sylvan Anopheles atroparvus population from a former malaria transmission area of Spain. Field-acquired microbiota was then assessed in non-blood-fed laboratory-reared females from the second, sixth, and 10th generations. Diversity analyses revealed that bacterial communities varied and clustered differently according to origin with sylvan larvae and newly emerged females distributing closer to laboratory-reared females than to their field counterparts. Inter-sample variation was mostly observed throughout the different developmental stages in the sylvan population. Larvae harbored the most diverse bacterial communities; wild-caught females, the poorest. In the transition from the sylvan environment to the first time point of laboratory breeding, a significant increase in diversity was observed, although this did decline under laboratory conditions. Despite diversity differences between wild-caught and laboratory-reared females, a substantial fraction of the bacterial communities was transferred through transstadial transmission and these persisted over 10 laboratory generations. Differentially abundant bacteria were mostly identified between breeding water and late-instar larvae, and in the transition from wild-caught to laboratory-reared females from the second generation. Our findings confirmed the key role of the breeding environment in shaping the microbiota of An. atroparvus. Gram-negative bacteria governed the microbiota of An. atroparvus with the prevalence of proteobacteria. Pantoea, Thorsellia, Serratia, Asaia, and Pseudomonas dominating the microbiota associated with wild-caught females, with the latter two governing the communities of laboratory-reared females. A core microbiota was identified with Pseudomonas and Serratia being the most abundant core genera shared by all sylvan and laboratory specimens. Overall, understanding the microbiota composition of An. atroparvus and how this varies throughout the mosquito life cycle and laboratory colonization paves the way when selecting potential bacterial candidates for use in microbiota-based intervention strategies against mosquito vectors, thereby improving our knowledge of laboratory-reared An. atroparvus mosquitoes for research purposes.info:eu-repo/semantics/publishedVersio

Field-captured Aedes vexans (Meigen, 1830) is a competent vector for Rift Valley fever phlebovirus in Europe

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.info:eu-repo/semantics/publishedVersio

Laboratory colonization and maintenance of Anopheles atroparvus from the Ebro Delta, Spain

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 signifcantly. 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 frst step, an adequate laboratory colony of An. atroparvus is needed. Methods: Anopheles atroparvus mosquitoes were captured in rice felds from the Ebro Delta (Spain). Field-caught specimens were maintained in the laboratory under simulated feld-summer conditions. Adult females were artifcially blood-fed on fresh whole rabbit blood for oviposition. First- to fourth-instar larvae were fed on pulverized fsh 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 vectorparasite interactions. Keywords: Anopheles atroparvus, Colonization, Malaria, Europeinfo:eu-repo/semantics/publishedVersio

Viromics on honey-baited FTA cards as a new tool for the detection of circulating viruses in mosquitoes

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.info:eu-repo/semantics/publishedVersio

European Aedes caspius mosquitoes are experimentally unable to transmit Zika virus

Background: Aedes caspius (Pallas, 1771) is a foodwater mosquito species widely distributed in the Western Palae‑ arctic. 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 Pacifc 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 defne 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.info:eu-repo/semantics/publishedVersio

Evidence of Zika virus horizontal and vertical transmission in Aedes albopictus from Spain but not infectious virus in saliva of the progeny

Aedes 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 programs for ZIKV.info:eu-repo/semantics/publishedVersio

The microbiome and vector mosquitoes : New Insights for Malaria and Arbovirus Control and Surveillance

A Europa, l'amenaça del ressorgiment i introducció de malalties transmeses per mosquits, com la malària i la febre de la vall del Rift, ha despertat un nou interès en l'estudi del microbioma associat amb mosquits autòctons per a una millor comprensió de les interaccions mosquit-patogen amb la finalitat de desenvolupar eines de vigilància vectorial i control ecològicament més adequades - En conseqüència, la tesi present es va enfocar en i) explorar la microbiota de l'Anopheles atroparvus, ii) avaluar la influencia de flavivirus insecto-específic en la competència vectorial del Culex pipiens i de l'Aedes vexans d' Europa per a la transmissió de RVFV, i iii) Aplicar metagenòmica a targetes FTA com a nou mètode per a la detecció de virus i vigilància d'arbovirus. Al primer capítol, es va establir una colònia de laboratori d' An. atroparvus del Delta de l' Ebre i es va actualitzar el seu protocol de cria. El seqüènciament del gen bacterià 1S rRNA va mostrar que l'ambient de cria, fisiologia i hàbits de farratge va influir en la microbiota tant de mosquits de camp com de laboratori. Addicionalment, els anàlisis de diversitat van mostrar variació entre els diferents estadis de desenvolupament silvestres i un declivi de la diversitat en el grup de femelles de la desena generació de laboratori F10, tot i això, van conservar una fracció significativa de la microbiota de les femelles silvestres. Finalment, proteobactèries Gram-negatives van predominar en la microbiota de l' An. atroparvus, entre les quals Pseudomonas, Asaia i Serratia van ser identificades com a candidates potencials per al control vectorial local. Per avaluar la influencia de ISFVs en la competència vectorial enfront de la transmissió de RVFV (Capítol 2), primer es va estudiar la infecció de Culex flavivirus (CxFV) a Cx. Pipiens a través d'exposició oral i inoculació intratoràcica. CxFV va infectar a Cx. Pipiens després d'inoculacions intratoràciques però no a través de l'exposició oral. Posteriorment, els assajos de competència vectorial enfront de RVFV es van realitzar en co-infecció amb CxFV i un flavivirus de mosquit de circulació natural, respectivament, en Cx. pipiens i Ae. vexans. Ambdues poblacions locals van mostrar ser competents per a RVFV després d'exposició oral. Per altra banda, ni CxFV ni RVFV van interferir en la seva respectiva infecció, mentre que el flavivirus de mosquit de circulació natural va modular la susceptibilitat d'infecció amb RVFV en Ae. vexans, suggerint el seu potencial ús com agent biològic per a la prevenció de la transmissió de RVFV. Finalment, per a avaluar noves alternatives per a la detecció de virus circulants i vigilància d'arbovirus (Capítol 3), targetes FTA amb esquer de mel van ser exposades a mosquits capturats en el camp durant jornades de vigilància entomològica per a la seva posterior anàlisi mitjançant seqüenciació de nova generació. Virus associats a artròpodes i plantes van ser identificats en les FTAs i es van obtenir genomes virals gairebé complets, el que suggereix una bona preservació d' ARN viral. Per a confirmar la presència de virus associats amb mosquits en els espècimens capturats, els pools de mosquits van ser analitzats utilitzant PCR de transcripció reversa i primers espècie-específics dissenyats a partir de les seqüències obtingudes de les FTAs. Virus relacionats amb Alphamesonivirus, Quaranjavirus i Bunyavirales no classificats van ser detectats en mosquits de Catalunya constituent el primer registre de distribució d'aquests virus en mosquits europeus. La detecció d' ISVs a la saliva de mosquits de forma silvestre demostra la viabilitat d'aquest mètode per a fer monitoreig de la fracció transmissible del viroma de mosquits i a seva utilitat en la vigilància d'arbovirus.En Europa, la amenaza del resurgimiento e introducción de enfermedades transmitidas por mosquitos, como la malaria y la fiebre del Valle de Rift, ha despertado un nuevo interés en el estudio del microbioma asociado con mosquitos autóctonos para un mejor entendimiento de las interacciones mosquito-patógeno con el fin de desarrollar herramientas de vigilancia vectorial y control ecológicamente más adecuadas. Consecuentemente, la presente tesis se enfocó en: i) explorar la microbiota de Anopheles atroparvus, ii) evaluar la influencia de flavivirus insecto-específico en la competencia vectorial de Culex pipiens y Aedes vexans de Europa para la transmisión de RVFV, y iii) Aplicar metagenómica en tarjetas FTA como un nuevo método para la detección de virus y vigilancia de arbovirus. En el primer capítulo, se estableció una colonia de laboratorio de An. atroparvus del Delta del Ebro y se actualizó su protocolo de cría. El secuenciamiento del gen bacteriano 16S rRNA mostró que el ambiente de cría, fisiología y hábitos de forrajeo influyeron en la microbiota de mosquitos tanto de campo como de laboratorio. Los análisis de diversidad mostraron variación entre los diferentes estadios de desarrollo silvestres y un declive de diversidad en hembras de la décima generación de laboratorio, sin embargo, una fracción significativa de la microbiota de hembras silvestres fue conservada. Finalmente, proteobacterias Gram-negativas predominaron en la microbiota de An. atroparvus, entre las cuales Pseudomonas, Asaia y Serratia fueron identificadas como candidatas potenciales para control vectorial local. Para evaluar la influencia de ISFVs en la competencia vectorial frente a la transmisión de RVFV (Capítulo 2), primero se estudió la infección de Culex flavivirus (CxFV) en Cx. pipiens a través de exposición oral e inoculación intratorácica. CxFV infectó a Cx. pipiens después de inoculaciones intratorácicas pero no a través de la exposición oral. Los ensayos de competencia vectorial frente a RVFV se realizaron en co-infección con CxFV y un flavivirus de mosquito de circulación natural, respectivamente, en Cx. pipiens y Ae. vexans. Ambas poblaciones mostraron ser competentes para RVFV después de exposición oral. Por otra parte, CxFV ni RVFV interfirieron con su respectiva infección, mientras que el flavivirus de mosquito moduló la susceptibilidad de infección con RVFV en Ae. vexans, sugiriendo su potencial uso como agente biológico para la prevención de la transmisión de RVFV. Finalmente, para evaluar nuevas alternativas para la detección de virus circulantes y vigilancia de arbovirus (Capítulo 3), tarjetas FTA con cebo de miel fueron expuestas a mosquitos capturados en el campo durante jornadas de vigilancia entomológica para su posterior análisis mediante NGS. Virus asociados a artrópodos y plantas fueron identificados en las FTAs y genomas virales casi completos fueron obtenidos, lo que sugiere una buena preservación de ARN viral. Para confirmar la presencia de virus asociados con mosquitos en los especímenes capturados, los pools de mosquitos fueron analizados usando RT-PCR y primers especie-específicos diseñados a partir de las secuencias obtenidas de las FTAs. Virus relacionados con Alphamesonivirus, Quaranjavirus y Bunyavirales no clasificados fueron detectados en mosquitos de Cataluña constituyendo el primer registro de distribución de estos virus en mosquitos europeos. La detección de ISVs en la saliva de mosquitos de forma silvestre demuestra la viabilidad de este método para monitorear la fracción transmisible del virroma de mosquitos y su utilidad en la vigilancia de arbovitus. El presente trabajo contribuye con información para un mejor entendimiento de los factores detrás de la estructura del microbioma de mosquitos locales y su potencial influencia en la competencia vectorial frente a arbovirus. Adicionalmente, provee un nuevo método para complementar la vigilancia de arbovirus en zonas susceptibles, así como, para la detección de nuevos virus circulantes y potencialmente patogénicosThe threat of the resurgence or introduction of mosquito-borne diseases, such as malaria and Rift Valley fever, into the European continent has awakened new interests in studying the microbiome associated to autochthonous mosquitoes for better understanding mosquito-pathogen interaction and developing ecologically adequate vector surveillance and control tools. Consequently, this thesis aimed to i) explore the microbiota of Anopheles atroparvus, a vector involved in malaria transmission in Europe, ii) assess the influence of insect-specific flaviviruses on the vector competence of European Culex pipiens and Aedes vexans for the transmission of RVFV, and iii) apply metagenomics on FTA cards as a new approach for virus detection and arbovirus surveillance. In the first chapter, a laboratory colony of An. atroparvus from the Ebro Delta was established, and its rearing protocol updated. Sequencing of the bacterial 16S rRNA gene showed that the breeding environment, physiology and foraging habits influenced the microbiota of field-caught and laboratory-colonized mosquitoes. Diversity analyses showed inter-sample variation among sylvan developmental stages and a diversity decline in adult females after ten-laboratory generations. Nonetheless, a significant fraction of the microbiota was conserved from wild-caught specimens until the tenth laboratory-generation. Environmentally acquired Gram-negative proteobacteria dominated the microbiota of this anopheles population, among which, Pseudomonas, Asaia and Serratia were identified as potential candidates to be studied for local vector control. To assess the influence of ISFVs on the vector competence for the transmission of RVFV (Chapter 2), the infection of Culex flavivirus (CxFV) was first studied in Cx. pipiens by oral exposure and intrathoracic inoculations. CxFV infected Cx. pipiens after intrathoracic inoculations but not after oral exposure. Then, RVFV vector competence assays in co-infection with CxFV and a mosquito-flavivirus of natural circulation were conducted in Cx. pipiens and Ae. vexans respectively. Both Catalonian species showed to be competent vectors for RVFV after oral exposure. CxFV nor RVFV interfered with each other's infection, while, naturally infecting mosquito-flavivirus modulated RVFV infection susceptibility in Ae. vexans, suggesting its potential use as bio-agent for preventing RVFV transmission. Finally, to assess new alternatives for circulating viruses' detection and arboviral surveillance (Chapter 3), next generation sequencing was applied on honey-baited FTA cards that were exposed to field-captured mosquitoes during entomological surveys. Arthropod- and plant-infecting viruses were identified on FTAs and near-complete viral genomes were obtained suggesting good quality preservation of viral RNAs. To confirm the presence of mosquito-associated viruses in the captured specimens, mosquito pools were screened using reverse-transcription PCRs and species-specific primers designed from the sequences obtained from the FTAs. Viruses related to Alphamesonivirus, Quaranjavirus and unclassified Bunyavirales were detected in Catalonian mosquitoes. These findings constitute the first distribution record of these insect-specific viruses in European mosquitoes. Detecting ISVs in mosquitoes' saliva in field conditions demonstrate the feasibility of this approach to monitor the transmissible fraction of the mosquitoes' virome and its suitability for arbovirus surveillance. Overall, the present work contributes with valuable information for better understanding the factors behind the structure of the microbiome of local vector mosquitoes, its potential influence in vector competence, and provides a new approach to complement arbovirus surveillance in susceptible areas and to detect circulating and new potentially pathogenic viruses

The microbiome and vector mosquitoes: New Insights for Malaria and Arbovirus Control and Surveillance

A Europa, l’amenaça del ressorgiment i introducció de malalties transmeses per mosquits, com la malària i la febre de la vall del Rift, ha despertat un nou interès en l’estudi del microbioma associat amb mosquits autòctons per a una millor comprensió de les interaccions mosquit–patogen amb la finalitat de desenvolupar eines de vigilància vectorial i control ecològicament més adequades - En conseqüència, la tesi present es va enfocar en i) explorar la microbiota de l’Anopheles atroparvus, ii) avaluar la influencia de flavivirus insecto-específic en la competència vectorial del Culex pipiens i de l’Aedes vexans d’ Europa per a la transmissió de RVFV, i iii) Aplicar metagenòmica a targetes FTA com a nou mètode per a la detecció de virus i vigilància d’arbovirus. Al primer capítol, es va establir una colònia de laboratori d’ An. atroparvus del Delta de l’ Ebre i es va actualitzar el seu protocol de cria. El seqüènciament del gen bacterià 1S rRNA va mostrar que l’ambient de cria, fisiologia i hàbits de farratge va influir en la microbiota tant de mosquits de camp com de laboratori. Addicionalment, els anàlisis de diversitat van mostrar variació entre els diferents estadis de desenvolupament silvestres i un declivi de la diversitat en el grup de femelles de la desena generació de laboratori F10, tot i això, van conservar una fracció significativa de la microbiota de les femelles silvestres. Finalment, proteobactèries Gram-negatives van predominar en la microbiota de l’ An. atroparvus, entre les quals Pseudomonas, Asaia i Serratia van ser identificades com a candidates potencials per al control vectorial local. Per avaluar la influencia de ISFVs en la competència vectorial enfront de la transmissió de RVFV (Capítol 2), primer es va estudiar la infecció de Culex flavivirus (CxFV) a Cx. Pipiens a través d’exposició oral i inoculació intratoràcica. CxFV va infectar a Cx. Pipiens després d’inoculacions intratoràciques però no a través de l’exposició oral. Posteriorment, els assajos de competència vectorial enfront de RVFV es van realitzar en co-infecció amb CxFV i un flavivirus de mosquit de circulació natural, respectivament, en Cx. pipiens i Ae. vexans. Ambdues poblacions locals van mostrar ser competents per a RVFV després d’exposició oral. Per altra banda, ni CxFV ni RVFV van interferir en la seva respectiva infecció, mentre que el flavivirus de mosquit de circulació natural va modular la susceptibilitat d’infecció amb RVFV en Ae. vexans, suggerint el seu potencial ús com agent biològic per a la prevenció de la transmissió de RVFV. Finalment, per a avaluar noves alternatives per a la detecció de virus circulants i vigilància d’arbovirus (Capítol 3), targetes FTA amb esquer de mel van ser exposades a mosquits capturats en el camp durant jornades de vigilància entomològica per a la seva posterior anàlisi mitjançant seqüenciació de nova generació. Virus associats a artròpodes i plantes van ser identificats en les FTAs i es van obtenir genomes virals gairebé complets, el que suggereix una bona preservació d’ ARN viral. Per a confirmar la presència de virus associats amb mosquits en els espècimens capturats, els pools de mosquits van ser analitzats utilitzant PCR de transcripció reversa i primers espècie-específics dissenyats a partir de les seqüències obtingudes de les FTAs. Virus relacionats amb Alphamesonivirus, Quaranjavirus i Bunyavirales no classificats van ser detectats en mosquits de Catalunya constituent el primer registre de distribució d’aquests virus en mosquits europeus. La detecció d’ ISVs a la saliva de mosquits de forma silvestre demostra la viabilitat d’aquest mètode per a fer monitoreig de la fracció transmissible del viroma de mosquits i a seva utilitat en la vigilància d’arbovirus.En Europa, la amenaza del resurgimiento e introducción de enfermedades transmitidas por mosquitos, como la malaria y la fiebre del Valle de Rift, ha despertado un nuevo interés en el estudio del microbioma asociado con mosquitos autóctonos para un mejor entendimiento de las interacciones mosquito-patógeno con el fin de desarrollar herramientas de vigilancia vectorial y control ecológicamente más adecuadas. Consecuentemente, la presente tesis se enfocó en: i) explorar la microbiota de Anopheles atroparvus, ii) evaluar la influencia de flavivirus insecto-específico en la competencia vectorial de Culex pipiens y Aedes vexans de Europa para la transmisión de RVFV, y iii) Aplicar metagenómica en tarjetas FTA como un nuevo método para la detección de virus y vigilancia de arbovirus. En el primer capítulo, se estableció una colonia de laboratorio de An. atroparvus del Delta del Ebro y se actualizó su protocolo de cría. El secuenciamiento del gen bacteriano 16S rRNA mostró que el ambiente de cría, fisiología y hábitos de forrajeo influyeron en la microbiota de mosquitos tanto de campo como de laboratorio. Los análisis de diversidad mostraron variación entre los diferentes estadios de desarrollo silvestres y un declive de diversidad en hembras de la décima generación de laboratorio, sin embargo, una fracción significativa de la microbiota de hembras silvestres fue conservada. Finalmente, proteobacterias Gram-negativas predominaron en la microbiota de An. atroparvus, entre las cuales Pseudomonas, Asaia y Serratia fueron identificadas como candidatas potenciales para control vectorial local. Para evaluar la influencia de ISFVs en la competencia vectorial frente a la transmisión de RVFV (Capítulo 2), primero se estudió la infección de Culex flavivirus (CxFV) en Cx. pipiens a través de exposición oral e inoculación intratorácica. CxFV infectó a Cx. pipiens después de inoculaciones intratorácicas pero no a través de la exposición oral. Los ensayos de competencia vectorial frente a RVFV se realizaron en co-infección con CxFV y un flavivirus de mosquito de circulación natural, respectivamente, en Cx. pipiens y Ae. vexans. Ambas poblaciones mostraron ser competentes para RVFV después de exposición oral. Por otra parte, CxFV ni RVFV interfirieron con su respectiva infección, mientras que el flavivirus de mosquito moduló la susceptibilidad de infección con RVFV en Ae. vexans, sugiriendo su potencial uso como agente biológico para la prevención de la transmisión de RVFV. Finalmente, para evaluar nuevas alternativas para la detección de virus circulantes y vigilancia de arbovirus (Capítulo 3), tarjetas FTA con cebo de miel fueron expuestas a mosquitos capturados en el campo durante jornadas de vigilancia entomológica para su posterior análisis mediante NGS. Virus asociados a artrópodos y plantas fueron identificados en las FTAs y genomas virales casi completos fueron obtenidos, lo que sugiere una buena preservación de ARN viral. Para confirmar la presencia de virus asociados con mosquitos en los especímenes capturados, los pools de mosquitos fueron analizados usando RT-PCR y primers especie-específicos diseñados a partir de las secuencias obtenidas de las FTAs. Virus relacionados con Alphamesonivirus, Quaranjavirus y Bunyavirales no clasificados fueron detectados en mosquitos de Cataluña constituyendo el primer registro de distribución de estos virus en mosquitos europeos. La detección de ISVs en la saliva de mosquitos de forma silvestre demuestra la viabilidad de este método para monitorear la fracción transmisible del virroma de mosquitos y su utilidad en la vigilancia de arbovitus. El presente trabajo contribuye con información para un mejor entendimiento de los factores detrás de la estructura del microbioma de mosquitos locales y su potencial influencia en la competencia vectorial frente a arbovirus. Adicionalmente, provee un nuevo método para complementar la vigilancia de arbovirus en zonas susceptibles, así como, para la detección de nuevos virus circulantes y potencialmente patogénicosThe threat of the resurgence or introduction of mosquito-borne diseases, such as malaria and Rift Valley fever, into the European continent has awakened new interests in studying the microbiome associated to autochthonous mosquitoes for better understanding mosquito-pathogen interaction and developing ecologically adequate vector surveillance and control tools. Consequently, this thesis aimed to i) explore the microbiota of Anopheles atroparvus, a vector involved in malaria transmission in Europe, ii) assess the influence of insect-specific flaviviruses on the vector competence of European Culex pipiens and Aedes vexans for the transmission of RVFV, and iii) apply metagenomics on FTA cards as a new approach for virus detection and arbovirus surveillance. In the first chapter, a laboratory colony of An. atroparvus from the Ebro Delta was established, and its rearing protocol updated. Sequencing of the bacterial 16S rRNA gene showed that the breeding environment, physiology and foraging habits influenced the microbiota of field-caught and laboratory-colonized mosquitoes. Diversity analyses showed inter-sample variation among sylvan developmental stages and a diversity decline in adult females after ten-laboratory generations. Nonetheless, a significant fraction of the microbiota was conserved from wild-caught specimens until the tenth laboratory-generation. Environmentally acquired Gram-negative proteobacteria dominated the microbiota of this anopheles population, among which, Pseudomonas, Asaia and Serratia were identified as potential candidates to be studied for local vector control. To assess the influence of ISFVs on the vector competence for the transmission of RVFV (Chapter 2), the infection of Culex flavivirus (CxFV) was first studied in Cx. pipiens by oral exposure and intrathoracic inoculations. CxFV infected Cx. pipiens after intrathoracic inoculations but not after oral exposure. Then, RVFV vector competence assays in co-infection with CxFV and a mosquito-flavivirus of natural circulation were conducted in Cx. pipiens and Ae. vexans respectively. Both Catalonian species showed to be competent vectors for RVFV after oral exposure. CxFV nor RVFV interfered with each other’s infection, while, naturally infecting mosquito-flavivirus modulated RVFV infection susceptibility in Ae. vexans, suggesting its potential use as bio-agent for preventing RVFV transmission. Finally, to assess new alternatives for circulating viruses’ detection and arboviral surveillance (Chapter 3), next generation sequencing was applied on honey-baited FTA cards that were exposed to field-captured mosquitoes during entomological surveys. Arthropod- and plant-infecting viruses were identified on FTAs and near-complete viral genomes were obtained suggesting good quality preservation of viral RNAs. To confirm the presence of mosquito-associated viruses in the captured specimens, mosquito pools were screened using reverse-transcription PCRs and species-specific primers designed from the sequences obtained from the FTAs. Viruses related to Alphamesonivirus, Quaranjavirus and unclassified Bunyavirales were detected in Catalonian mosquitoes. These findings constitute the first distribution record of these insect-specific viruses in European mosquitoes. Detecting ISVs in mosquitoes’ saliva in field conditions demonstrate the feasibility of this approach to monitor the transmissible fraction of the mosquitoes’ virome and its suitability for arbovirus surveillance. Overall, the present work contributes with valuable information for better understanding the factors behind the structure of the microbiome of local vector mosquitoes, its potential influence in vector competence, and provides a new approach to complement arbovirus surveillance in susceptible areas and to detect circulating and new potentially pathogenic viruses.Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina i Sanitat Animal