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Mosquito-borne viruses-such as Zika, chikungunya, dengue fever, and yellow fever, among others-are of global importance. Although vaccine development for prevention of mosquito-borne arbovirus infections has been a focus, mitigation strategies continue to rely on vector control. However, vector control has failed to prevent recent epidemics and arrest expanding geographic distribution of key arboviruses, such as dengue. As a consequence, there has been increasing necessity to further optimize current strategies within integrated approaches and advance development of alternative, innovative strategies for the control of mosquito-borne arboviruses. This review, intended as a general overview, is one of a series being generated by the Worldwide Insecticide resistance Network (WIN). The alternative strategies discussed reflect those that are currently under evaluation for public health value by the World Health Organization (WHO) and represent strategies of focus by globally recognized public health stakeholders as potential insecticide resistance (IR)-mitigating strategies. Conditions where these alternative strategies could offer greatest public health value in consideration of mitigating IR will be dependent on the anticipated mechanism of action. Arguably, the most pressing need for endorsement of the strategies described here will be the epidemiological evidence of a public health impact. As the burden of mosquito-borne arboviruses, predominately those transmitted by Aedes aegypti and A. albopictus, continues to grow at a global scale, new vector-control tools and integrated strategies will be required to meet public health demands. Decisions regarding implementation of alternative strategies will depend on key ecoepidemiological parameters that each is intended to optimally impact toward driving down arbovirus transmission.

The mosquito microbiome significantly influences vector competence, including in Aedes albopictus , a globally invasive vector. Describing the microbiome and Wolbachia strains of Ae. albopictus from different regions can guide area-specific control strategies. Mosquito samples from Spain and São Tomé were analyzed using 16S rRNA gene sequencing and metagenomic sequencing. Wolbachia infection patterns were observed by sex and population. Female mosquitoes were blood-fed, a factor considered in analyzing their microbiota. Results revealed a dominance of dual Wolbachia infections, strains A and B, in the microbiome of both populations of Ae. albopictus , especially among females. Both populations shared a core microbiome, although 5 and 9 other genera were only present in Spain and São Tomé populations, respectively. Genera like Pelomonas and Nevskia were identified for the first time in Aedes mosquitoes. This study is the first to describe the Ae. albopictus bacteriome in Spain and São Tomé, offering insights for the development of targeted mosquito control strategies. Understanding the specific microbiome composition can help in designing more effective interventions, such as microbiome manipulation and Wolbachia -based approaches, to reduce vector competence and transmission potential of these mosquitoes.

Crimean–Congo hemorrhagic fever virus (CCHFV) causes severe or fatal infections in humans and is geographically widespread. The virus has coevolved with its tick vectors, establishing persistent infections critical to its transmission. This study explored the mechanisms underpinning these persistent infections, using tick cell lines and the Hazara virus (HAZV) as a biosafety level 2 (BSL-2) model for CCHFV. Initially, an RT-qPCR protocol was developed to detect HAZV in tick cells. The study then focused on the production of virus-derived DNA (vDNAs) by tick cells as a defensive response to infection. These vDNAs regulate viral particle production, enabling tick cells to maintain viability and establish persistent infections. The experiments characterized vDNAs production, viral titers, and subcellular localization, and they examined the effect of the reverse transcriptase inhibitor azidothymidine triphosphate (AZT). The results showed that all tested tick cell lines supported HAZV replication, achieving persistent infections without cytopathic effects. vDNAs was detected in both the cytoplasm and nucleus, and its formation was dependent on HAZV infection. Importantly, vDNAs presence was linked to infection persistence; cells treated with AZT exhibited a marked reduction in vDNAs production and an associated increase in viral particle production, which correlated with higher cell death. These findings underscore the critical role of vDNAs in balancing viral replication and promoting long-term cell survival in tick cells, highlighting their importance in the coevolution of tick-borne viruses and their vectors.

Background Tick-borne rickettsioses (TBR) are emerging, neglected, zoonoses, caused by intracellular α-proteobacteria of the genus Rickettsia , that pose a growing public health concern. The aim of the present study was to evaluate rickettsial infections in questing ticks collected from four different ecological areas in mainland Portugal. Methods Over a two-year period, a total of 707 questing ticks were collected. Individual adult ticks and pooled nymphs were submitted to DNA extraction, followed by qPCR assays targeting the gltA rickettsial gene. Positive samples were then submitted to conventional PCR targeting the gltA and the ompA genes for phylogenetic analysis. Results In total, eight tick species were identified: Dermacentor marginatus , Haemaphysalis inermis , Haemaphysalis punctata , Hyalomma lusitanicum , Ixodes frontalis , Ixodes ricinus , Rhipicephalus pusillus , and Rhipicephalus sanguineus sensu lato. Additionally, rickettsial infection was associated with seven of these species, with I. frontalis being the exception. Notably, the prevalence of Rickettsia spp. was 26.35%, with phylogenetic validation confirming infections with R. helvetica , R. massiliae , R. monacensis , Candidatus R. rioja, and R. slovaca . Conclusions The present study highlights the necessity for ongoing surveillance to map and monitor both questing and feeding ticks, along with their vertebrate hosts. Effective control strategies are of utmost importance to mitigate the escalating threat of TBR. Additionally, the present study provides valuable epidemiological insights into TBR in Portugal, including the identification of R. slovaca infecting I. ricinus - an unconventional tick-pathogen relationship - and the first report of Candidatus R. rioja infecting D. marginatus in Portugal. In conclusion, this study contributes with valuable data regarding epidemiological results on ticks and TBR circulating in Portugal, emphasizing the importance of proactive measures to address this emerging public health challenge. Graphical abstract

Since its emergence in 2007 in Micronesia and Polynesia, the arthropod-borne flavivirus Zika virus (ZIKV) has spread in the Americas and the Caribbean, following first detection in Brazil in May 2015. The risk of ZIKV emergence in Europe increases as imported cases are repeatedly reported. Together with chikungunya virus (CHIKV) and dengue virus (DENV), ZIKV is transmitted by Aedes mosquitoes. Any countries where these mosquitoes are present could be potential sites for future ZIKV outbreak. We assessed the vector competence of European Aedes mosquitoes (Aedes aegypti and Aedes albopictus) for the currently circulating Asian genotype of ZIKV. Two populations of Ae. aegypti from the island of Madeira (Funchal and Paul do Mar) and two populations of Ae. albopictus from France (Nice and Bar-sur-Loup) were challenged with an Asian genotype of ZIKV isolated from a patient in April 2014 in New Caledonia. Fully engorged mosquitoes were then maintained in insectary conditions (28°±1°C, 16h:8h light:dark cycle and 80% humidity). 16-24 mosquitoes from each population were examined at 3, 6, 9 and 14 days post-infection to estimate the infection rate, disseminated infection rate and transmission efficiency. Based on these experimental infections, we demonstrated that Ae. albopictus from France were not very susceptible to ZIKV. In combination with the restricted distribution of European Ae. albopictus, our results on vector competence corroborate the low risk for ZIKV to expand into most parts of Europe with the possible exception of the warmest regions bordering the Mediterranean coastline.

Background The Asian tiger mosquito, Aedes albopictus (Skuse, 1894), is a highly invasive species that has successfully colonized many tropical and temperate regions worldwide. Its rapid global spread is strongly associated with human activities and has created favorable conditions for the emergence of human arboviruses in new geographic areas. Methods Mosquito larvae were collected by community health workers from different breeding sites and reared to adults in a field insectary. Adult mosquitoes were morphologically identified to species level. Species identification was confirmed by cytochrome oxidase subunit I DNA barcoding. Results We report the first detection of Aedes albopictus in Angola during an Anopheles stephensi survey conducted in Soyo, Zaire Province. Phylogenetic analysis indicated that the Angolan Ae. albopictus population clusters with sequences from Central African countries, suggesting an introduction from within the continent. Conclusions The presence of Ae. albopictus in Angola highlights the need for enhanced vector surveillance and control measures to prevent the emergence of arboviral diseases. This finding emphasizes the relevance of collaboration between local health authorities, communities, and international organizations in monitoring the spread of invasive mosquito species.

Aedes aegypti is a major mosquito vector of arboviruses, including dengue, chikungunya and Zika. In 2005, Ae. aegypti was identified for the first time in Madeira Island. Despite an initial insecticide-based vector control program, the species expanded throughout the Southern coast of the island, suggesting the presence of insecticide resistance. Here, we characterized the insecticide resistance status and the underlying mechanisms of two populations of Ae. aegypti from Madeira Island, Funchal and Paúl do Mar. WHO susceptibility bioassays indicated resistance to cyfluthrin, permethrin, fenitrothion and bendiocarb. Use of synergists significantly increased mortality rates, and biochemical assays indicated elevated activities of detoxification enzymes, suggesting the importance of metabolic resistance. Microarray-based transcriptome analysis detected significant upregulation in both populations of nine cytochrome P450 oxidase genes (including four known pyrethroid metabolizing enzymes), the organophosphate metabolizer CCEae3a, Glutathione-S-transferases, and multiple putative cuticle proteins. Genotyping of knockdown resistance loci linked to pyrethroid resistance revealed fixation of the 1534C mutation, and presence with moderate frequencies of the V1016I mutation in each population. Significant resistance to three major insecticide classes (pyrethroid, carbamate and organophosphate) is present in Ae. aegypti from Madeira Island, and appears to be mediated by multiple mechanisms. Implementation of appropriate resistance management strategies including rotation of insecticides with alternative modes of action, and methods other than chemical-based vector control are strongly advised to delay or reverse the spread of resistance and achieve efficient control.

Background Malaria is a major public health problem in Angola, with Anopheles gambiae sensu lato (s.l.) and An. funestus s.l. being the primary vectors. This study aimed to clarify the information gaps concerning local Anopheles mosquito populations. Our objectives were to assess their abundance, geographical dispersion, and blood-feeding patterns. We also investigated their insecticide resistance. Molecular methods were used to identify sibling species, determine the origin of blood meals, measure Plasmodium falciparum infection rates, and detect the presence of knockdown resistance (kdr) mutations. Methods Adult mosquitoes were collected indoors using CDC light traps from nine randomly selected households at two sentinel sites with distinct ecological characteristics. The samples were collected from 1 February to 30 June 2022. Anopheles mosquitoes were morphologically identified and subjected to molecular identification. Unfed Anopheles females were tested for the presence of P. falciparum DNA in head and thorax, and engorged females were screened for the source of the blood meals. Additionally, members of An. gambiae complex were genotyped for the presence of the L1014F and L1014S kdr mutations. Results In total, 2226 adult mosquitoes were collected, including 733 Anopheles females. Molecular identification revealed the presence of Anopheles coluzzii , An. gambiae senso stricto (s.s.), An. arabiensis , and An. funestus s.s. Notably, there was the first record of An. coluzzii / An. gambiae s.s. hybrid and An. vaneedeni in Benguela Province. Plasmodium falciparum infection rates for An. coluzzii at the urban sentinel site and An. funestus s.s. at the rural site were 23.1% and 5.7%, respectively. The L1014F kdr mutation was discovered in both resistant and susceptible An. coluzzii mosquitoes, while the L1014S mutation was detected in An. gambiae s.s. for the first time in Benguela Province. No kdr mutations were found in An. arabiensis. Conclusions This study provides valuable insights into the molecular characteristics of malaria vectors from the province of Benguela, emphasising the need for continuous surveillance of local Anopheles populations regarding the establishment of both kdr mutations for tailoring vector control interventions. Graphical abstract

The extensive use of insecticides for vector control has led to the development of insecticide resistance in Aedes aegypti populations on a global scale, which has significantly compromised control actions. Insecticide resistance, and its underlying mechanisms, has been investigated in several countries, mostly in South American and Asian countries. In Africa, however, studies reporting insecticide resistance are rare and data on resistance mechanisms, notably knockdown resistance (kdr) mutations, is scarce. In this study, the recently described V410L kdr mutation is reported for the first time in old world Ae. aegypti populations, namely from Angola and Madeira island. Two additional kdr mutations, V1016I and F1534C, are also reported for the first time in populations from Angola and Cape Verde. Significant associations with the resistance phenotype were found for both V410L and V1016I individually as well as for tri-locus genotypes in the Angolan population. However, no association was found in Madeira island, probably due to the presence of a complex pattern of multiple insecticide resistance mechanisms in the local Ae. aegypti population. These results suggest that populations carrying the same kdr mutations may respond differently to the same insecticide, stressing the need for complementary studies when assessing the impact of kdr resistance mechanisms in the outcome of insecticide-based control strategies.

Background Culex quinquefasciatus plays a crucial role as a vector of West Nile virus (WNV). This mosquito species is widely distributed in Cape Verde, being found in all inhabited islands of the archipelago. However, no data are currently available on the susceptibility of the local mosquito population to WNV. This study aimed to assess the vector competence of Cx. quinquefasciatus mosquitoes from Santiago Island, Cape Verde, for WNV and to explore the potential impact of its native Wolbachia on virus transmission. Methods Wolbachia -infected and uninfected Cx. quinquefasciatus female mosquitoes were exposed to WNV lineage 1 PT6.39 strain using a Hemotek membrane feeding system. Mosquito samples, including the body, legs, wings and saliva, were collected at days 7, 14 and 21 post-infection (dpi) to assess WNV infection through one-step quantitative real-time PCR (RT-qPCR). Results Culex quinquefasciatus from Cape Verde exhibited high susceptibility to the tested strain of WNV. Also, treated females without their native Wolbachia exhibited significantly higher WNV load in their bodies and greater dissemination rate at 7 dpi than their wild-type counterparts carrying Wolbachia . Conclusions The high susceptibility to WNV of Cx. quinquefasciatus from Cape Verde poses a potential risk for virus transmission in the archipelago. However, Wolbachia infection in this mosquito species seems to confer protection against WNV dissemination in the early stages of viral infection. Additional research is required to uncover the mechanisms driving this protection and its potential impact on WNV transmission. Graphical abstract