Mosquito vector competence is usually represented as a process in which, once virus is detected in saliva, mosquitoes are assumed to remain infectious for life, implying an irreversible transition to the transmitting state. However, some experiments report declines in the proportion of transmitting mosquitoes at late times post-exposure, suggesting transmission capacity may not be permanent. To investigate this hypothesis, we extended a previously developed stochastic intra-vector viral dynamics model by introducing transmission states allowing either permanent cessation or temporary interruption of transmission. We fitted three competing models to longitudinal data from 52 vector competence conditions covering chikungunya, dengue, Zika, West Nile, and Rift Valley fever viruses, using Approximate Bayesian Computation with Sequential Monte Carlo inference. Among the 10 conditions showing declines in transmission, models allowing exit from the transmitting state provided a better fit in 7 cases, with clear improvement in 5. In these conditions, allowing interruption of transmission increased posterior estimates of the proportion of mosquitoes that crossed all intra-mosquito barriers, whereas estimates of infected and disseminated state durations were largely unchanged. In cases where intermittent transmission was selected, its performance was similar to that of permanent cessation, with non-transmitting periods lasting several days. These results indicate that the assumption of lifelong mosquito infectiousness does not always provide the best explanation for vector competence data and may lead to underestimation of the proportion of mosquitoes that become capable of transmission. Incorporating time-varying transmission competence into intra-vector models could improve interpretation of vector competence experiments and refine epidemiological representations of arbovirus transmission.

Ecological niches present unique environmental and biological trademarks such as abiotic conditions, nutrient availability, and trophic interactions that may impact the ecology of living organisms. Female mosquitoes deposit their eggs in aquatic niches with fluctuating diet sources and microbial communities. However, how niche’s diet and microbial composition impact mosquito performance (i.e., traits that maximize mosquito fitness) are not well understood. In this study, we focused on the Asian tiger mosquito, Aedes albopictus, one of the most invasive species in the world and a competent vector for human pathogens. To remove any external microbes, Ae. albopictus eggs were surface-sterilized then hatching larvae were exposed to a gradient of bacterial inoculum (i.e., initial microbial load) and diet concentrations while their impact on mosquito performance traits during juvenile development was measured. Our results showed that Ae. albopictus larvae develop faster and give larger adults when exposed to microbiota in rearing water. However, mosquito performance, up to the adult stage, depends on both bacterial inoculum size and diet concentration in the aquatic habitat. Upon low inoculum size, larvae survived better if the diet was in sufficient amounts whereas a higher inoculum size was associated with optimal larvae survival only in the presence of the lower amount of diet. Inoculum size, and to a lesser extent diet concentration, shaped bacterial community structure and composition of larval-rearing water allowing the identification of bacterial taxa for which their abundance in larvae-rearing water correlated with niche parameters and/or larval traits. Our work demonstrates that both diet concentration and bacterial inoculum size impact mosquito performance possibly by shaping bacterial community structure in the larval habitat, which accounts for a large part of the juvenile’s microbiota. Host-microbe interactions influence several mosquito life-history traits, and our work reveals that niche parameters such as inoculum size and diet concentration could have numerous implications on the microbiota assembly and host evolutionary trajectory. This underlies that host-microbe-environment interactions are an important yet overlooked factor of mosquito adaptation to its local environment, with potential future implications for vector control and vector ecology.

Arboviruses represent a significant threat to the health of humans, animals, and plants worldwide. Mechanistic modeling has proven useful for elucidating the transmission, anticipating the spread, and predicting the response of arboviruses to control measures. However, most models approximate the intra-vector infection dynamic (IVD), which occurs during the extrinsic incubation period (EIP), by a single stage with an average duration. At the end of this stage, all exposed vectors are expected to become infectious. Strong assumption is hidden behind this: that the EIP is exponentially distributed in the vector population. To assess the validity of this assumption, we developed a stochastic compartmental model that represents successive IVD stages, associated with the crossing or not of the three within-mosquito barriers (infection, dissemination, and transmission). We calibrated the model using an ABC-SMC (Approximate Bayesian Computation - Sequential Monte Carlo) method, which includes model selection. We searched for literature data on experimental infections of Aedes mosquitoes infected by dengue, chikungunya, or Zika viruses. We demonstrated the large discrepancy between the exponential hypothesis and observed EIP distributions for dengue and Zika viruses, and identified more relevant EIP distributions. This work provides a generic modeling framework that can be applied to other arboviruses for which similar data are available. Our model also can be linked to population-scale models to aid future arbovirus control efforts.

The Asian tiger mosquito, Aedes albopictus, is an invasive species that spreads diseases like chikungunya and has caused outbreaks worldwide. Studies show that mosquito-associated microbes can affect disease transmission. One of those microbes, the parasite Ascogregarina taiwanensis, is common in native and settled mosquito populations (>3 years after introduction) but rare in recently introduced ones. We found that this parasite slows down the spread of the chikungunya virus within the mosquito and decreases its transmission rate by half. Unparasitized mosquitoes spread the virus more easily, suggesting that changes in mosquito-associated microbes could impact disease outbreaks and public health.

While locally-acquired dengue virus (DENV) human infections occur in mainland France since 2010, data to identify the mosquito species involved and to trace the virus are frequently lacking. Supported by a local network gathering public health agencies and research laboratories, we analysed, in late summer 2023, mosquitoes from privately-owned traps within a French urban neighbourhood affected by a dengue cluster. The cluster, in Auvergne-Rhône-Alpes, comprised three cases, including two autochthonous ones. Upon return from a recent visit to the French Caribbean Islands, the third case had consulted healthcare because of dengue-compatible symptoms, but dengue had not been recognised. For the two autochthonous cases, DENV-specific antibodies in serum or a positive quantitative PCR for DENV confirmed DENV infection. The third case had anti-flavivirus IgMs. No DENV genetic sequences were obtained from affected individuals but Aedes albopictus mosquitoes trapped less than 200 m from the autochthonous cases' residence contained DENV. Genetic data from the mosquito-derived DENV linked the cluster to the 2023-2024 dengue outbreak in the French Caribbean Islands. This study highlights the importance of raising mosquito-borne disease awareness among healthcare professionals. It demonstrates Ae. albopictus as a DENV vector in mainland France and the value of private mosquito traps for entomo-virological surveillance.

Vector competence assays allow to measure, in the laboratory, the ability of a mosquito to get infected and then retransmit an arbovirus while mimicking natural vector infection route. Aedes aegypti is a major vector of arboviruses worldwide and thus a reference species used in vector competence assays. Rift Valley fever virus (RVFV) is a major public health threat, mostly in Africa, that infects humans and animals through the bite of mosquito vectors. Here, we describe vector competence assay of Aedes aegypti mosquitoes for RVFV, from mosquito exposure to the virus through an infectious artificial blood meal to the measurement of virus prevalence in the mosquito's body, head, and saliva.

Arbovirus emergence and epidemic potential, as approximated by the vectorial capacity formula, depends on host and vector parameters, including the vector’s intrinsic ability to replicate then transmit the pathogen known as vector competence. Vector competence is a complex, time-dependent, quantitative phenotype influenced by biotic and abiotic factors. A combination of experimental and modelling approaches is required to assess arbovirus intra-vector dynamics and estimate epidemic potential. In this study, we measured infection, dissemination, and transmission dynamics of chikungunya virus (CHIKV) in a field-derived Aedes albopictus population (Lyon, France) after oral exposure to a range of virus doses spanning human viraemia. Statistical modelling indicates rapid and efficient CHIKV progression in the vector mainly due to an absence of a dissemination barrier, with 100% of the infected mosquitoes ultimately exhibiting a disseminated infection, regardless of the virus dose. Transmission rate data revealed a time-dependent, but overall weak, transmission barrier, with individuals transmitting as soon as 2 days post-exposure (dpe) and >50% infectious mosquitoes at 6 dpe for the highest dose. Based on these experimental intra-vector dynamics data, epidemiological simulations conducted with an agent-based model showed that even at low mosquito biting rates, CHIKV could trigger outbreaks locally. Together, this reveals the epidemic potential of CHIKV upon transmission by Aedes albopictus in mainland France.

Along their life cycle, mosquitoes colonize different ecological niches with various microorganisms and diet sources that likely modulate their performance (i.e. a set of mosquito fitness-related traits). However, which ecological parameters and how their variations modulate mosquito performance is not completely understood. In this study, we used Ae. albopictus surface-sterilized eggs re-associated or not to conventional bacterial microbiota upon a range of diet concentrations and addressed the impact of microbial inoculum and diet concentration variation on several mosquito performance traits. Results showed that mosquito juvenile survival depends on the interaction between bacterial inoculum load and diet concentration in the breeding water. Exposure to bacteria in rearing water shorten larval development time although it impacted larvae survival in an inoculum and diet concentration-dependent manner. Bacterial composition of larval rearing water was mainly structured by the bacterial inoculum concentration, with some Operational Taxonomic Units abundances correlating with larval traits. Ae. albopictus survival, development and bacterial community patterns upon gradients of diet and bacterial inoculum illustrated the complex impact of diet-microbiota interaction on mosquito performance. These findings argue the importance of deciphering host-microbe-environment interactions and open promising perspectives to improve Ae. albopictus control measures in the field.IMPORTANCE Microbiota is increasingly recognized as a driving force of metazoan biology, impacting diverse traits including nutrition, behaviour or reproduction. The microbial impact on host nutrition is among the most studied host-microbe interactions although it remains poorly understood in arthropod vectors like mosquitoes. Here, we manipulated mosquito microbiota using gnotobiology to decipher the impact of bacteria and diet on the Asian tiger mosquito, Ae. albopictus. These results are key to understand the link between diet and bacteria concentration on juvenile mosquitoes as well as carry-over effects in adults. They unveil some specific aspects of mosquito-bacteria interactions while opening interesting avenues for vector management of this vector of arboviruses.

Arbovirus emergence and epidemic potential, as approximated by the vectorial capacity formula, depends on several host and vector parameters including vector intrinsic ability to transmit the pathogen. Such ability, called vector competence is influenced by biotic (e.g. virus and vector genotype) and abiotic (e.g. temperature). Vector competence is often evaluated as a qualitative phenotype although it is a multistep, time-dependent, quantitative phenotype. Combination of experimental and modelling approaches can i) capture intra-vector dynamics of arboviral infection and ii) use data to estimate arbovirus epidemic potential. Here, we measured individual Aedes albopictus (Lyon, France) mosquitoes infection, dissemination, and transmission rate upon oral exposure to chikungunya virus (CHIKV, La Reunion Island isolate) at eleven time-points from day 2 to day 20 post-exposure (dpe) for a range of CHIKV infectious doses spanning human viremia. Statistical modelisation indicates an explosive CHIKV intra-vector dynamics mainly due to an absence of dissemination barrier with 100% of the infected mosquitoes ultimately exhibiting a disseminated infection regardless of the viral dose. Transmission rate data revealed a time and dose-dependent but overall weak transmission barrier with individuals transmitting as soon as 2 dpe and >50% infectious mosquitoes at 6 dpe for the highest dose. Epidemiological simulations conducted with an Agent-Based Model based on experimental intra-vector dynamics data showed that even at low mosquito biting rates, CHIKV triggers explosive outbreaks. Together, this reveals the high epidemic potential of this CHIKV isolate with this French metropolitan population of Aedes albopictus.

Mosquitoes are considered one of the most important threats worldwide due to their ability to vector pathogens. They are responsible for the transmission of major pathogens such as malaria, dengue, zika, or chikungunya. Due to the lack of treatments or prophylaxis against many of the transmitted pathogens and an increasing prevalence of mosquito resistance to insecticides and drugs available, alternative strategies are now being explored. Some of these involve the use of microorganisms as promising agent to limit the fitness of mosquitoes, attract or repel them, and decrease the replication and transmission of pathogenic agents. In recent years, the importance of microorganisms colonizing the habitat of mosquitoes has particularly been investigated since they appeared to play major roles in their development and diseases transmission. In this issue, we will synthesize researches investigating how microorganisms present within water habitats may influence breeding site selection and oviposition strategies of gravid mosquito females. We will also highlight the impact of such microbes on the fate of females' progeny during their immature stages with a specific focus on egg hatching, development rate, and larvae or pupae survival.

Diseases caused by mosquito-borne viruses have been on the rise for the last decades, and novel methods aiming to use laboratory-engineered mosquitoes that are incapable of carrying viruses have been developed to reduce pathogen transmission. This has stimulated efforts to identify optimal target genes that are naturally involved in mosquito antiviral defenses or required for viral replication. Here, we investigated the role of a member of the Tudor protein family, Tudor-SN, upon dengue virus infection in the mosquito Aedes aegypti. Tudor-SN knockdown reduced dengue virus replication in the midgut of Ae. aegypti females. In immunofluorescence assays, Tudor-SN localized to the nucleolus in both Ae. aegypti and Aedes albopictus cells. A reporter assay and small RNA profiling demonstrated that Tudor-SN was not required for RNA interference function in vivo. Collectively, these results defined a novel proviral role for Tudor-SN upon early dengue virus infection of the Ae. aegypti midgut.

The fruit fly Drosophila melanogaster is a model organism to study several aspects of metazoan biology. Most of the work has been conducted in adult fruit flies, including laboratory and field-derived specimens, but Drosophila melanogaster larvae recently became a valuable model to better understand animal physiology, development, or host-microbe interactions. While adult flies can be easily assigned to a given Drosophila species based on morphological characteristics, such visual identification is more intricate at the larval stage. This could explain the limited number of studies focusing on larvae, especially field-derived samples. Here, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay that discriminates D. melanogaster from other ecologically relevant Drosophila species at the larval stage. The method, which targets the cytochrome oxidase I (COI) gene, was validated using laboratory-derived larvae from seven D. melanogaster populations originating from different geographic areas as well as six Drosophila species. We further validated this PCR-RFLP assay in a natural context, by identifying wild larvae collected in two locations in France. Notably, among all PCR-RFLP profiles that matched the D. melanogaster species, 100% were correctly identified, as confirmed by COI sequencing. In summary, our work provides a rapid, simple, and accurate molecular tool to identify D. melanogaster from field-collected larvae.

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution.

Dengue virus (DENV) causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP), which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation.

Wolbachia, a widespread endosymbiont of terrestrial arthropods, can protect its host against viral and parasitic infections, a phenotype called "pathogen blocking". However, in some cases Wolbachia may have no effect or even enhance pathogen infection, depending on the host-Wolbachia-pathogen combination. The tiger mosquito Aedes albopictus is naturally infected by two strains of Wolbachia, wAlbA and wAlbB, and is a competent vector for different arboviruses such as dengue virus (DENV) and chikungunya virus (CHIKV). Interestingly, it was shown in some cases that Ae. albopictus native Wolbachia strains are able to inhibit DENV transmission by limiting viral replication in salivary glands, but no such impact was measured on CHIKV replication in vivo. To better understand the Wolbachia/CHIKV/Ae. albopictus interaction, we generated a cellular model using Ae. albopictus derived C6/36 cells that we infected with the wAlbB strain. Our results indicate that CHIKV infection is negatively impacted at both RNA replication and virus assembly/secretion steps in presence of wAlbB. Using FISH, we observed CHIKV and wAlbB in the same mosquito cells, indicating that the virus is still able to enter the cell in the presence of the bacterium. Further work is needed to decipher molecular pathways involved in Wolbachia-CHIKV interaction at the cellular level, but this cellular model can be a useful tool to study the mechanism behind virus blocking phenotype induced by Wolbachia. More broadly, this put into question the ecological role of Wolbachia symbiont in Ae. albopictus, but also the ability of the CHIKV to counteract Wolbachia's antiviral potential in vivo.

BACKGROUND:The chikungunya (CHIK) outbreak that struck La Reunion Island in 2005 was preceded by few human cases of Dengue (DEN), but which surprisingly did not lead to an epidemic as might have been expected in a non-immune population. Both arboviral diseases are transmitted to humans by two main mosquito species, Aedes aegypti and Aedes albopictus. In the absence of the former, Ae. albopictus was the only species responsible for viral transmission on La Reunion Island. This mosquito is naturally super-infected with two Wolbachia strains, wAlbA and wAlbB. While Wolbachia does not affect replication of CHIK virus (CHIKV) in Ae. albopictus, a similar effect was not observed with DEN virus (DENV).METHODS/PRINCIPAL FINDINGS:To understand the weak vectorial status of Ae. albopictus towards DENV, we used experimental oral infections of mosquitoes from La Reunion Island to characterize the impact of Wolbachia on DENV infection. Viral loads and Wolbachia densities were measured by quantitative PCR in different organs of Ae. albopictus where DENV replication takes place after ingestion. We found that: (i) Wolbachia does not affect viral replication, (ii) Wolbachia restricts viral density in salivary glands, and (iii) Wolbachia limits transmission of DENV, as infectious viral particles were only detected in the saliva of Wolbachia-uninfected Ae. albopictus, 14 days after the infectious blood-meal.CONCLUSIONS:We show that Wolbachia does not affect the replication of DENV in Ae. albopictus. However, Wolbachia is able to reduce viral infection of salivary glands and limit transmission, suggesting a role of Wolbachia in naturally restricting the transmission of DENV in Ae. albopictus from La Reunion Island. The extension of this conclusion to other Ae. albopictus populations should be investigated.