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Wolbachia in medically important mosquitoes from Cape Verde: prevalence, genetic diversity and role in vector competence
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Wolbachia pipientis é uma bactéria endossimbiótica atualmente utilizada no controlo de doenças transmitidas por vetores, devido à sua capacidade de induzir incompatibilidade citoplasmática e suprimir a replicação de arbovírus em mosquitos vetores. O objetivo deste estudo foi avaliar a prevalência e a diversidade genética de Wolbachia em diferentes espécies de mosquitos (Diptera: Culicidae) em Cabo Verde, bem como avaliar a influência deste endossimbionte na transmissão do vírus do Nilo Ocidental (WNV) pelo vetor Culex quinquefasciatus.
As amostras de mosquitos foram colhidas em seis ilhas de Cabo Verde. A deteção de Wolbachia nos mosquitos foi realizada através da amplificação de um segmento do gene da proteína de superfície (wsp). A identificação da estirpe foi efetuada por Tipagem de Sequência Multilocus (Multilocus Sequence Typing, MLST), utilizando genes constitutivos (gatB, coxA, hcpA, fbpA e ftsZ) e a região hipervariável do wsp (HVR). A classificação dos grupos wPip foi determinada por PCR-RFLP, direcionada para o gene da anquirina (pk1). Para avaliar o impacto da Wolbachia nativa na competência vetorial, fêmeas de Cx. quinquefasciatus, infetadas e não infetadas com Wolbachia, provenientes da ilha de Santiago, foram expostas à estirpe PT6.39 do WNV utilizando um sistema de alimentação por membrana. Posteriormente, a infeção por WNV foi avaliada por One-step RT-qPCR aos 7, 14 e 21 dias pós-infeção (dpi).
Wolbachia foi detetada em Culex tigripes (100% de prevalência), Cx. quinquefasciatus (98,3%), Cx. pipiens s.s. (100%) e em híbridos Cx. pipiens/quinquefasciatus (100%). As análises MLST e filogenética revelaram que a Wolbachia do complexo Cx. pipiens pertence à Sequence Type (ST) 9, ao clado wPip e ao Supergrupo B. A Wolbachia isolada de Cx. tigripes também foi classificada no Supergrupo B, mas formou uma linhagem única, distinta do clado wPip, sem perfil MLST atribuído. Os resultados PCR-RFLP indicaram a presença de wPip-II, wPip-III e wPip-IV em Cabo Verde. As avaliações de competência vetora demonstraram que as fêmeas de Cx. quinquefasciatus desprovidas da sua Wolbachia nativa apresentaram títulos de WNV significativamente mais elevadas nos seus corpos e uma disseminação aumentada do vírus, em comparação com os seus homólogos de tipo selvagem com Wolbachia.
A elevada diversidade dos grupos wPip observada sugere múltiplas introduções de Cx. pipiens s.l. em Cabo Verde e fornece informações cruciais para o desenvolvimento de futuras estratégias de controlo baseadas em Wolbachia no arquipélago. Relata-se a primeira ocorrência de Wolbachia na espécie Culex tigripes, que provavelmente representa uma nova estirpe do endossimbionte, com implicações promissoras para as estratégias de controlo vetorial. A Wolbachia nativa de Cx. quinquefasciatus aparenta restringir a disseminação do vírus do Nilo Ocidental (WNV), sendo necessários estudos adicionais para elucidar o mecanismo subjacente a esta proteção mediada por Wolbachia.
Abstract Wolbachia pipientis is an endosymbiotic bacterium currently employed in the control of vector-borne diseases due to its capacity to induce cytoplasmic incompatibility and suppress arbovirus replication in mosquito vectors. The objective of this study was to evaluate the prevalence and genetic diversity of Wolbachia across different mosquito species (Diptera: Culicidae) in Cape Verde and to assess the potential influence of the endosymbiont on the transmission of West Nile virus (WNV) by the mosquito vector Culex quinquefasciatus. Mosquito samples were collected from six islands in Cape Verde and Wolbachia detection was performed by amplifying a fragment of the surface protein gene (wsp). Strain identification was conducted by multilocus sequence typing (MLST) using housekeeping genes (gatB, coxA, hcpA, fbpA and ftsZ) and the wsp hypervariable region (HVR). Classification of wPip groups was determined by PCR-RFLP, targeting the ankyrin-domain gene pk1. To evaluate the impact of native Wolbachia on vector competence, Wolbachia-infected and uninfected Cx. quinquefasciatus from Santiago Island were exposed to the PT6.39 strain of WNV using a Hemotek membrane feeding system. WNV infection was subsequently evaluated through One-Step RT-qPCR at 7, 14 and 21 days post-infection (dpi). Wolbachia was detected in Culex tigripes (100% prevalence), Cx. quinquefasciatus (98.3%), Cx. pipiens s.s. (100%) and Cx. pipiens/quinquefasciatus hybrids (100%). MLST and phylogenetic analysis revealed that Wolbachia from Cx. pipiens complex belongs to Sequence Type (ST) 9, wPip clade and Supergroup B. Wolbachia isolated from Cx. tigripes was also classified under Supergroup B but formed a unique lineage separate from the wPip clade, with no assigned MLST profile. Results from the PCR-RFLP showed the presence of wPip-II, wPip-III and wPip-IV Wolbachia in Cape Verde. Vector competence assessments demonstrated that Cx. quinquefasciatus females lacking native Wolbachia exhibited significantly higher WNV loads in their bodies and increased virus dissemination compared to their wild-type counterparts harboring Wolbachia. The high diversity of wPip groups observed in this study suggests multiple introductions of Cx. pipiens s.l. in Cape Verde and provides insights for the design of future Wolbachia-based control strategies in the archipelago. The occurrence of Wolbachia in Culex tigripes is reported for the first time, which likely represents a novel strain of the endosymbiont with promising implications for vector control strategies. Native Wolbachia from Cx. quinquefasciatus appears to restrict WNV dissemination and further studies will be needed to elucidate the mechanism underlying this Wolbachia-mediated protection.
Abstract Wolbachia pipientis is an endosymbiotic bacterium currently employed in the control of vector-borne diseases due to its capacity to induce cytoplasmic incompatibility and suppress arbovirus replication in mosquito vectors. The objective of this study was to evaluate the prevalence and genetic diversity of Wolbachia across different mosquito species (Diptera: Culicidae) in Cape Verde and to assess the potential influence of the endosymbiont on the transmission of West Nile virus (WNV) by the mosquito vector Culex quinquefasciatus. Mosquito samples were collected from six islands in Cape Verde and Wolbachia detection was performed by amplifying a fragment of the surface protein gene (wsp). Strain identification was conducted by multilocus sequence typing (MLST) using housekeeping genes (gatB, coxA, hcpA, fbpA and ftsZ) and the wsp hypervariable region (HVR). Classification of wPip groups was determined by PCR-RFLP, targeting the ankyrin-domain gene pk1. To evaluate the impact of native Wolbachia on vector competence, Wolbachia-infected and uninfected Cx. quinquefasciatus from Santiago Island were exposed to the PT6.39 strain of WNV using a Hemotek membrane feeding system. WNV infection was subsequently evaluated through One-Step RT-qPCR at 7, 14 and 21 days post-infection (dpi). Wolbachia was detected in Culex tigripes (100% prevalence), Cx. quinquefasciatus (98.3%), Cx. pipiens s.s. (100%) and Cx. pipiens/quinquefasciatus hybrids (100%). MLST and phylogenetic analysis revealed that Wolbachia from Cx. pipiens complex belongs to Sequence Type (ST) 9, wPip clade and Supergroup B. Wolbachia isolated from Cx. tigripes was also classified under Supergroup B but formed a unique lineage separate from the wPip clade, with no assigned MLST profile. Results from the PCR-RFLP showed the presence of wPip-II, wPip-III and wPip-IV Wolbachia in Cape Verde. Vector competence assessments demonstrated that Cx. quinquefasciatus females lacking native Wolbachia exhibited significantly higher WNV loads in their bodies and increased virus dissemination compared to their wild-type counterparts harboring Wolbachia. The high diversity of wPip groups observed in this study suggests multiple introductions of Cx. pipiens s.l. in Cape Verde and provides insights for the design of future Wolbachia-based control strategies in the archipelago. The occurrence of Wolbachia in Culex tigripes is reported for the first time, which likely represents a novel strain of the endosymbiont with promising implications for vector control strategies. Native Wolbachia from Cx. quinquefasciatus appears to restrict WNV dissemination and further studies will be needed to elucidate the mechanism underlying this Wolbachia-mediated protection.
Descrição
Palavras-chave
Biologia molecular Biologia celular Wolbachia Culex Diversidade genética Competência vetora Vírus do Nilo Ocidental Cabo Verde