Insecticide resistance genes affect Culex quinquefasciatus vector competence for West Nile virus.

Insecticide resistance has been reported to impact the interactions between mosquitoes and the pathogens they transmit. However, the effect on vector competence for arboviruses still remained to be investigated. We examined the influence of two insecticide resistance mechanisms on vector competence of the mosquito Culex quinquefasciatus for two arboviruses, Rift Valley Fever virus (RVFV) and West Nile virus (WNV). Three Cx. quinquefasciatus lines sharing a common genetic background were used: two insecticide-resistant lines, one homozygous for amplification of the Ester2 locus (SA2), the other homozygous for the acetylcholinesterase ace-1 G119S mutation (SR) and the insecticide-susceptible reference line Slab. Statistical analyses revealed no significant effect of insecticide-resistant mechanisms on vector competence for RVFV. However, both insecticide resistance mechanisms significantly influenced the outcome of WNV infections by increasing the dissemination of WNV in the mosquito body, therefore leading to an increase in transmission efficiency by resistant mosquitoes. These results showed that insecticide resistance mechanisms enhanced vector competence for WNV and may have a significant impact on transmission dynamics of arboviruses. Our findings highlight the importance of understanding the impacts of insecticide resistance on the vectorial capacity parameters to assess the overall consequence on transmission.

The evolutionary fate of heterogeneous gene duplications: A precarious overdominant equilibrium between environment, sublethality and complementation.

Gene duplications occur at a high rate. Although most appear detrimental, some homogeneous duplications (identical gene copies) can be selected for beneficial increase in produced proteins. Heterogeneous duplications, which combine divergent alleles of a single locus, are seldom studied due to the paucity of empirical data. We investigated their role in an ongoing adaptive process at the ace-1 locus in Culex pipiens mosquitoes. We assessed the worldwide diversity of the ace-1 alleles (single-copy, susceptible S and insecticide-resistant R, and duplicated D that pair one S and one R copy), analysed their phylogeography and measured their fitness to understand their early dynamics using population genetics models. It provides a coherent and comprehensive evolutionary scenario. We show that D alleles are present in most resistant populations and display a higher diversity than R alleles (27 vs. 4). Most appear to result from independent unequal crossing-overs between local single-copy alleles, suggesting a recurrent process. Most duplicated alleles have a limited geographic distribution, probably resulting from their homozygous sublethality (HS phenotype). In addition, heterozygotes carrying different HS D alleles showed complementation, indicating different recessive lethal mutations. Due to mosaic insecticide control practices, balancing selection (overdominance) plays a key role in the early dynamics of heterogeneous duplicated alleles; it also favours a high local polymorphism of HS D alleles in natural populations (overdominance reinforced by complementation). Overall, our study shows that the evolutionary fate of heterogeneous duplications (and their long-term role) depends on finely balanced selective pressures due to the environment and to their genomic structure.

Stable coexistence of incompatible Wolbachia along a narrow contact zone in mosquito field populations.

In arthropods, the intracellular bacteria Wolbachia often induce cytoplasmic incompatibility (CI) between sperm and egg, which causes conditional embryonic death and promotes the spatial spread of Wolbachia infections into host populations. The ability of Wolbachia to spread in natural populations through CI has attracted attention for using these bacteria in vector-borne disease control. The dynamics of incompatible Wolbachia infections have been deeply investigated theoretically, whereas in natural populations, there are only few examples described, especially among incompatible infected hosts. Here, we have surveyed the distribution of two molecular Wolbachia strains (wPip11 and wPip31) infecting the mosquito Culex pipiens in Tunisia. We delineated a clear spatial structure of both infections, with a sharp contact zone separating their distribution areas. Crossing experiments with isofemale lines from different localities showed three crossing types: wPip11-infected males always sterilize wPip31-infected females; however, while most wPip31-infected males were compatible with wPip11-infected females, a few completely sterilize them. The wPip11 strain was thus expected to spread, but temporal dynamics over 7 years of monitoring shows the stability of the contact zone. We examined which factors may contribute to the observed stability, both theoretically and empirically. Population cage experiments, field samples and modelling did not support significant impacts of local adaptation or assortative mating on the stability of wPip infection structure. By contrast, low dispersal probability and metapopulation dynamics in the host Cx. pipiens probably play major roles. This study highlights the need of understanding CI dynamics in natural populations to design effective and sustainable Wolbachia-based control strategies.

Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species.

BACKGROUND: The maternally inherited bacterium Wolbachia often acts as a subtle parasite that manipulates insect reproduction, resulting potentially in reproductive isolation between host populations. Whilst distinct Wolbachia strains are documented in a group of evolutionarily closely related mosquitoes known as the Culex pipiens complex, their impact on mosquito population genetics remains unclear. To this aim, we developed a PCR-RFLP test that discriminates the five known Wolbachia groups found in this host complex. We further examined the Wolbachia genetic diversity, the variability in the coinherited host mitochondria and their partitioning among members of the Cx. pipiens complex, in order to assess the impact of Wolbachia on host population structure. RESULTS: There was a strong association between Wolbachia and mitochondrial haplotypes indicating a stable co-transmission in mosquito populations. Despite evidence that members of the Cx. pipiens complex are genetically distinct on the basis of nuclear DNA, the association of Wolbachia and mtDNA with members of the Cx. pipiens complex were limited. The Wolbachia wPip-I group, by far the most common, was associated with divergent Cx. pipiens members, including Cx. quinquefasciatus, Cx. pipiens pipiens form pipiens and Cx. pipiens pipiens form molestus. Four other wPip groups were also found in mosquito populations and all were shared between diverse Cx. pipiens members. CONCLUSION: This data overall supports the hypothesis that wPip infections, and their allied mitochondria, are associated with regular transfers between Cx. pipiens members rather than specific host associations. Overall, this is suggestive of a recent and likely ongoing cytoplasmic introgression through hybridization events across the Cx. pipiens complex.

Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito.

The α-proteobacteria Wolbachia are among the most common intracellular bacteria and have recently emerged as important drivers of arthropod biology. Wolbachia commonly act as reproductive parasites in arthropods by inducing cytoplasmic incompatibility (CI), a type of conditional sterility between hosts harboring incompatible infections. In this study, we examined the evolutionary histories of Wolbachia infections, known as wPip, in the common house mosquito Culex pipiens, which exhibits the greatest variation in CI crossing patterns observed in any insect. We first investigated a panel of 20 wPip strains for their genetic diversity through a multilocus scheme combining 13 Wolbachia genes. Because Wolbachia depend primarily on maternal transmission for spreading within arthropod populations, we also studied the variability in the coinherited Cx. pipiens mitochondria. In total, we identified 14 wPip haplotypes, which all share a monophyletic origin and clearly cluster into five distinct wPip groups. The diversity of Cx. pipiens mitochondria was extremely reduced, which is likely a consequence of cytoplasmic hitchhiking driven by a unique and recent Wolbachia invasion. Phylogenetic evidence indicates that wPip infections and mitochondrial DNA have codiverged through stable cotransmission within the cytoplasm and shows that a rapid diversification of wPip has occurred. The observed pattern demonstrates that a considerable degree of Wolbachia diversity can evolve within a single host species over short evolutionary periods. In addition, multiple signatures of recombination were found in most wPip genomic regions, leading us to conclude that the mosaic nature of wPip genomes may play a key role in their evolution.

Rapid evolution of Wolbachia incompatibility types.

In most insects, the endosymbiont Wolbachia induces cytoplasmic incompatibility (CI), an embryonic mortality observed when infected males mate either with uninfected females or with females infected by an incompatible Wolbachia strain. Although the molecular mechanism of CI remains elusive, it is classically viewed as a modification-rescue model, in which a Wolbachia mod function disables the reproductive success of the sperm of infected males, unless eggs are infected and express a compatible resc function. The extent to which the modification-rescue model can predict highly complex CI pattern remains a challenging issue. Here, we show the rapid evolution of the mod-resc system in the Culex pipiens mosquito. We have surveyed four incompatible laboratory isofemale lines over 50 generations and observed in two of them that CI has evolved from complete to partial incompatibility (i.e. the production of a mixture of compatible and incompatible clutches). Emergence of the new CI types depends only on Wolbachia determinants and can be simply explained by the gain of new resc functions. Evolution of CI types in Cx. pipiens thus appears as a gradual process, in which one or several resc functions can coexist in the same individual host in addition to the ones involved in the self-compatibility. Our data identified CI as a very dynamic process. We suggest that ancestral and mutant Wolbachia expressing distinct resc functions can co-infect individual hosts, opening the possibility for the mod functions to evolve subsequently. This gives a first clue towards the understanding of how Wolbachia reached highly complex CI pattern in host populations.

Zika vector competence data reveals risks of outbreaks: the contribution of the European ZIKAlliance project.

First identified in 1947, Zika virus took roughly 70 years to cause a pandemic unusually associated with virus-induced brain damage in newborns. Zika virus is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus, both colonizing a large strip encompassing tropical and temperate regions. As part of the international project ZIKAlliance initiated in 2016, 50 mosquito populations from six species collected in 12 countries were experimentally infected with different Zika viruses. Here, we show that Ae. aegypti is mainly responsible for Zika virus transmission having the highest susceptibility to viral infections. Other species play a secondary role in transmission while Culex mosquitoes are largely non-susceptible. Zika strain is expected to significantly modulate transmission efficiency with African strains being more likely to cause an outbreak. As the distribution of Ae. aegypti will doubtless expand with climate change and without new marketed vaccines, all the ingredients are in place to relive a new pandemic of Zika.

Multiple Wolbachia determinants control the evolution of cytoplasmic incompatibilities in Culex pipiens mosquito populations.

Wolbachia are maternally inherited endosymbionts that can invade arthropod populations through manipulation of their reproduction. In mosquitoes, Wolbachia induce embryonic death, known as cytoplasmic incompatibility (CI), whenever infected males mate with females either uninfected or infected with an incompatible strain. Although genetic determinants of CI are unknown, a functional model involving the so-called mod and resc factors has been proposed. Natural populations of Culex pipiens mosquito display a complex CI relationship pattern associated with the highest Wolbachia (wPip) genetic polymorphism reported so far. We show here that C. pipiens populations from La Réunion, a geographically isolated island in the southwest of the Indian Ocean, are infected with genetically closely related wPip strains. Crossing experiments reveal that these Wolbachia are all mutually compatible. However, crosses with genetically more distant wPip strains indicate that Wolbachia strains from La Réunion belong to at least five distinct incompatibility groups (or crossing types). These incompatibility properties which are strictly independent from the nuclear background, formally establish that in C. pipiens, CI is controlled by several Wolbachia mod/resc factors.

Contrasted transmission efficiency of Zika virus strains by mosquito species Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Reunion Island.

BACKGROUND: Zika virus (ZIKV) is a mosquito-borne flavivirus that recently emerged in the South Pacific islands and Americas where unprecedented outbreaks were reported. Although Aedes aegypti is considered to be the main vector for ZIKV, other mosquito species have been shown to be potential vectors and differences in vector competence with respect to mosquito strain and ZIKV strain have been demonstrated. In this study we compared the vector competence of three mosquito species Aedes albopictus, Ae. aegypti and Culex quinquefasciatus from Reunion Island for three ZIKV strains. METHODS: Five mosquito strains (2 strains of Ae. albopictus, 1 of Ae. aegypti and 2 of Cx. quinquefasciatus) were exposed to three ZIKV strains: one African strain (Dak84) and two Asian strains (PaRi_2015 and MAS66). The vector competence parameters (infection rate, dissemination efficiency and transmission efficiency) and viral loads were examined at 14 and 21 days post-infection. RESULTS: The two Cx. quinquefasciatus strains did not become infected and were therefore unable to either disseminate or transmit any of the three ZIKV strains. Aedes albopictus and Ae. aegypti strains were poorly competent for the two Asian ZIKV strains, while both mosquito species displayed higher infection rates, dissemination and transmission efficiencies for the African ZIKV Dak84 strain. However, this African ZIKV strain was better transmitted by Ae. aegypti as compared to Ae. albopictus. CONCLUSIONS: Our results show that both Ae. albopictus and Ae. aegypti, from Reunion Island, are more likely to be competent for ZIKV in contrast to Cx. quinquefasciatus which appeared refractory to all tested ZIKV strains. This improves our understanding of the role of mosquito species in the risk of the ZIKV emergence on Reunion Island.

High genetic diversity but no geographical structure of Aedes albopictus populations in Réunion Island.

BACKGROUND: In recent years, the Asian tiger mosquito Aedes albopictus has emerged as a species of major medical concern following its global expansion and involvement in many arbovirus outbreaks. On Réunion Island, Ae. albopictus was responsible for a large chikungunya outbreak in 2005-2006 and more recently an epidemic of dengue which began at the end of 2017 and is still ongoing at the time of writing. This dengue epidemic has seen a high number of human cases in south and west coastal regions, while few cases have been reported in the north and east of the island. To better understand the role of mosquito populations in such spatial patterns of dengue virus transmission in Réunion Island, we examined the genetic diversity and population structure of Ae. albopictus sampled across the island. RESULTS: Between November 2016 and March 2017, a total of 564 mosquitoes were collected from 19 locations in three main climatic regions (West, East and Center) of Réunion Island and were genotyped using 16 microsatellite loci. A high genetic diversity was observed with 2-15 alleles per locus and the average number of alleles per population varying between 4.70-5.90. Almost all FIS values were significantly positive and correlated to individual relatedness within populations using a hierarchical clustering approach based on principal components analyses (HCPC). However, the largest part of genetic variance was among individuals within populations (97%) while only 3% of genetic variance was observed among populations within regions. Therefore, no distinguishable population structure or isolation by distance was evidenced, suggesting high rates of gene flow at the island scale. CONCLUSIONS: Our results show high genetic diversity but no genetic structure of Ae. albopictus populations in Réunion Island thus reflecting frequent movements of mosquitoes between populations probably due to human activity. These data should help in the understanding of Ae. albopictus vector capacity and the design of effective mosquito control strategies.

Discovery of Culex pipiens associated tunisia virus: a new ssRNA(+) virus representing a new insect associated virus family.

In the global context of arboviral emergence, deep sequencing unlocks the discovery of new mosquito-borne viruses. Mosquitoes of the species Culex pipiens, C. torrentium, and C. hortensis were sampled from 22 locations worldwide for transcriptomic analyses. A virus discovery pipeline was used to analyze the dataset of 0.7 billion reads comprising 22 individual transcriptomes. Two closely related 6.8 kb viral genomes were identified in C. pipiens and named as Culex pipiens associated tunisia virus (CpATV) strains Ayed and Jedaida. The CpATV genome contained four ORFs. ORF1 possessed helicase and RNA-dependent RNA polymerase (RdRp) domains related to new viral sequences recently found mainly in dipterans. ORF2 and 4 contained a capsid protein domain showing strong homology with Virgaviridae plant viruses. ORF3 displayed similarities with eukaryotic Rhoptry domain and a merozoite surface protein (MSP7) domain only found in mosquito-transmitted Plasmodium, suggesting possible interactions between CpATV and vertebrate cells. Estimation of a strong purifying selection exerted on each ORFs and the presence of a polymorphism maintained in the coding region of ORF3 suggested that both CpATV sequences are genuine functional viruses. CpATV is part of an entirely new and highly diversified group of viruses recently found in insects, and that bears the genomic hallmarks of a new viral family.

Comparison of Irradiation and Wolbachia Based Approaches for Sterile-Male Strategies Targeting Aedes albopictus.

The global expansion of Aedes albopictus together with the absence of vaccines for most of the arboviruses transmitted by this mosquito has stimulated the development of sterile-male strategies aiming at controlling disease transmission through the suppression of natural vector populations. In this context, two environmentally friendly control strategies, namely the Sterile Insect Technique (SIT) and the Wolbachia-based Incompatible Insect Technique (IIT) are currently being developed in several laboratories worldwide. So far however, there is a lack of comparative assessment of these strategies under the same controlled conditions. Here, we compared the mating capacities, i.e. insemination capacity, sterilization capacity and mating competitiveness of irradiated (35 Gy) and incompatible Ae. albopictus males at different ages and ratios under laboratory controlled conditions. Our data show that there was no significant difference in insemination capacity of irradiated and incompatible males, both male types showing lower capacities than untreated males at 1 day but recovering full capacity within 5 days following emergence. Regarding mating competitiveness trials, a global observed trend is that incompatible males tend to induce a lower hatching rate than irradiated males in cage controlled confrontations. More specifically, incompatible males were found more competitive than irradiated males in 5:1 ratio regardless of age, while irradiated males were only found more competitive than incompatible males in the 1:1 ratio at 10 days old. Overall, under the tested conditions, IIT seemed to be slightly more effective than SIT. However, considering that a single strategy will likely not be adapted to all environments, our data stimulates the need for comparative assessments of distinct strategies in up-scaled conditions in order to identify the most suitable and safe sterilizing technology to be implemented in a specific environmental setting and to identify the parameters requiring fine tuning in order to reach optimal release conditions.

Wolbachia-based population control strategy targeting Culex quinquefasciatus mosquitoes proves efficient under semi-field conditions.

In mosquitoes, the maternally inherited bacterial Wolbachia induce a form of embryonic lethality called cytoplasmic incompatibility (CI). This property can be used to reduce the density of mosquito field populations through inundative releases of incompatible males in order to sterilize females (Incompatible Insect Technique, or IIT, strategy). We have previously constructed the LR[wPip(Is)] line representing a good candidate for controlling field populations of the Culex quinquefasciatus mosquito in the islands of the south-western Indian Ocean. The main purpose of the present study was to fill the gap between laboratory experiments and field implementation, i.e. assessing mating competitiveness of these incompatible males under semi-field conditions. In a first set of experiments, we analyzed crossing relationships between LR[wPip(Is)] males and La Réunion field females collected as larvae in 19 distinct localities throughout the island. This investigation revealed total embryonic mortality, confirming the strong sterilizing capacity of LR[wPip(Is)] males. Subsequently, mating competitiveness of LR[wPip(Is)] males was assessed under semi-field conditions in the presence of field males and females from La Réunion. Confrontations were carried out in April and December using different ratios of LR[wPip(Is)] to field males. The results indicated that the LR[wPip(Is)] males successfully compete with field males in mating with field females, displaying even higher competitiveness than field males in April. Our results support the implementation of small-scale field tests in order to assess the feasibility of IIT against Cx. quinquefasciatus in the islands of southwestern Indian Ocean where this mosquito species is a proven competent vector for human pathogens.

Wolbachia divergence and the evolution of cytoplasmic incompatibility in Culex pipiens.

Many insect species harbor Wolbachia bacteria that induce cytoplasmic incompatibility (CI), i.e. embryonic lethality in crosses between infected males and uninfected females, or between males and females carrying incompatible Wolbachia strains. The molecular mechanism of CI remains unknown, but the available data are best interpreted under a modification-rescue model, where a mod function disables the reproductive success of infected males' sperm, unless the eggs are infected and express a compatible resc function. Here we examine the evolution of CI in the mosquito Culex pipiens, harbouring a large number of closely related Wolbachia strains structured in five distinct phylogenetic groups. Specifically, we used a worldwide sample of mosquito lines to assess the hypothesis that genetic divergence should correlate with the divergence of CI properties on a low evolutionary scale. We observed a significant association of Wolbachia genetic divergence with CI patterns. Most Wolbachia strains from the same group were compatible whereas those from different groups were often incompatible. Consistently, we found a strong association between Wolbachia groups and their mod-resc properties. Finally, lines from the same geographical area were rarely incompatible, confirming the conjecture that the spatial distribution of Wolbachia compatibility types should be constrained by selection. This study indicates a clear correlation between Wolbachia genotypes and CI properties, paving the way toward the identification of the molecular basis of CI through comparative genomics.

Cytoplasmic incompatibility as a means of controlling Culex pipiens quinquefasciatus mosquito in the islands of the south-western Indian Ocean.

The use of the bacterium Wolbachia is an attractive alternative method to control vector populations. In mosquitoes, as in members of the Culex pipiens complex, Wolbachia induces a form of embryonic lethality called cytoplasmic incompatibility, a sperm-egg incompatibility occurring when infected males mate either with uninfected females or with females infected with incompatible Wolbachia strain(s). Here we explore the feasibility of the Incompatible Insect Technique (IIT), a species-specific control approach in which field females are sterilized by inundative releases of incompatible males. We show that the Wolbachia wPip(Is) strain, naturally infecting Cx. p. pipiens mosquitoes from Turkey, is a good candidate to control Cx. p. quinquefasciatus populations on four islands of the south-western Indian Ocean (La Réunion, Mauritius, Grande Glorieuse and Mayotte). The wPip(Is) strain was introduced into the nuclear background of Cx. p. quinquefasciatus mosquitoes from La Réunion, leading to the LR[wPip(Is)] line. Total embryonic lethality was observed in crosses between LR[wPip(Is)] males and all tested field females from the four islands. Interestingly, most crosses involving LR[wPip(Is)] females and field males were also incompatible, which is expected to reduce the impact of any accidental release of LR[wPip(Is)] females. Cage experiments demonstrate that LR[wPip(Is)] males are equally competitive with La Réunion males resulting in demographic crash when LR[wPip(Is)] males were introduced into La Réunion laboratory cages. These results, together with the geographic isolation of the four south-western Indian Ocean islands and their limited land area, support the feasibility of an IIT program using LR[wPip(Is)] males and stimulate the implementation of field tests for a Cx. p. quinquefasciatus control strategy on these islands.