A practical limitation to many metabarcoding initiatives is that sampling methods tend to collect many non-target taxa, which become "amplicon noise" that can saturate Next Generation Sequencing results and lead to both financial and resource inefficiencies. An available molecular tool that can significantly decrease these non-target amplicons and decrease the need for pre-DNA-extraction sorting of bycatch is the design of PCR primers tailored to the taxa under investigation. We assessed whether the D2 extension segment of the 28S ribosomal operon can limit this shortcoming within the context of mosquito (Culicidae) monitoring. We designed PCR primers that are fully conserved across mosquitos and exclude from amplification most other taxa likely to be collected with current sampling apparatuses. We show that, given enough sequencing depth, D2 is an effective marker for the detection of mosquito sequences within mock genomic DNA pools. As few as 3,050 quality-filtered Illumina reads were able to recover all 17 species in a bulk pool containing as little as 0.2% of constituent DNA from single taxa. We also mixed these mosquito DNA pools with high concentrations of non-Culicidae bycatch DNA and show that the component mosquito species are generally still recoverable and faithful to their original relative frequencies. Finally, we show that there is little loss of fidelity in abundance parameters when pools from degraded DNA samples were sequenced using the D2 primers.

Anopheles gambiae s.s. is a key vector of Plasmodium parasites. Repellents, which may be a promising alternative to pesticides used to control malaria mosquitoes. Although citronellal is a known mosquito repellent, its repellency characteristics are largely unknown. Determining the specific odorant-binding proteins (OBPs) and odorant receptors (ORs) that detect and transfer the citronellal molecule in A. gambiae s.s. will help to define the mode of action of this compound. In this research, we assessed the repellent activity of citronellal in A. gambiae s.s. using a Y-tube olfactory meter, screened candidate citronellal-binding OBPs and ORs using reverse molecular docking, clarified the binding properties of predicted proteins for citronellal using fluorescence competition binding assay. Results showed that citronellal had a dosage effect on repelling A. gambiae s.s.. The 50% repellent rate was determined to be 4.02 nmol. Results of simulated molecular docking showed that the only proteins that bound tightly with citronellal were AgamOBP4 and AgamORC7. Fluorescence competitive binding assays confirmed the simulations. This research determined that citronellal was captured by AgamOBP4 and transmitted to AgamORC7 in A. gambiae s.s.. Our study will be beneficial in the further understanding the repellent mechanism of citronellal against A. gambiae s.s..

The Asian tiger mosquito Aedes albopictus (Skuse 1894) is assuming an ever-increasing importance as invasive species in Europe and consequently as human health and nuisance concern. In Central Italy, the species has been recently involved in a chikungunya outbreak. A 3 yr Ae. albopictus monitoring was carried out in 21 municipalities of the Lazio region (Central Italy), belonging to three provinces. Samplings were performed on a weekly basis using ovitraps, in order to investigate climatic and spatial variables driving egg abundance and Ae. albopictus period of activity. A temperature of 10.4°C was indicated as lower threshold for the onset of egg-laying activity, together with a photoperiod of 13:11 (L:D) h. The whole oviposition activity lasted 8 mo (May-December), with 95% of eggs laid between early June and mid-November and a peak at the end of August. Egg abundance was positively influenced by accumulated temperature (AT) of the 4 wk preceding sampling and negatively by precipitation during the week before. Egg-laying activity dropped with decreasing AT, increasing rainfall, and with a photoperiod below 10:14 (L:D) h. Our results pinpointed the importance of fine-scaled spatial features on egg abundance. Some of these fine-scaled characteristics have been highlighted, such as the presence of vegetation and human footprint index. Our model estimated an almost doubled maximum number of laid eggs for the maximum value of human footprint. Compelling evidence of the relevance of fine-scaled characteristics was reported, describing cases where human-made breeding sites driven the abundance of Ae. albopictus.

The major drivers of the extensive biodiversity of the Neotropics are proposed to be geological and tectonic events together with Pliocene and Pleistocene environmental and climatic change. Geographical barriers represented by the rivers Amazonas/Solimões, the Andes and the coastal mountain ranges in eastern Brazil have been hypothesized to lead to diversification within the primary malaria vector, Anopheles (Nyssorhynchus) darlingi Root, which primarily inhabits rainforest. To test this biogeographical hypothesis, we analyzed 786 single nucleotide polymorphisms (SNPs) in 12 populations of An. darlingi from across the complex Brazilian landscape. Both model-based (STRUCTURE) and non-model-based (Principal Components and Discriminant Analysis) analysis of population structure detected three major genetic clusters that correspond with newly described Neotropical biogeographical regions: 1) Atlantic Forest province (= southeast population); 2) Parana Forest province (= West Atlantic forest population, with one Chacoan population - SP); and 3) Brazilian dominion population (= Amazonian population with one Chacoan population - TO). Significant levels of pairwise genetic divergences were found among the three clusters, allele sharing among clusters was negligible, and geographical distance did not contribute to differentiation. We infer that the Atlantic forest coastal mountain range limited dispersal between the Atlantic Forest province and the Parana Forest province populations, and that the large, diagonal open vegetation region of the Chacoan dominion dramatically reduced dispersal between the Parana and Brazilian dominion populations. We hypothesize that the three genetic clusters may represent three putative species.

Arthropod-borne diseases represent one of the greatest infection-related threats as a result of climate change and globalization. Repeatedly, arbovirus-infected mosquitoes show behavioral changes whose underlying mechanisms are still largely unknown, but might help to develop control strategies. However, in contrast to well-characterized insects such as fruit flies, little is known about neuroanatomy and neurotransmission in mosquitoes. To overcome this limitation, the study focuses on the immunohistochemical characterization of the nervous system of Culex pipiens biotype molestus in comparison to Drosophila melanogaster using 13 antibodies labeling nervous tissue, neurotransmitters or neurotransmitter-related enzymes. Antibodies directed against γ-aminobutyric acid, serotonin, tyrosine-hydroxylase and glutamine synthetase were suitable for investigations in Culex pipiens and Drosophila melanogaster, albeit species-specific spatial differences were observed. Likewise, similar staining results were achieved for neuronal glycoproteins, axons, dendrites and synaptic zones in both species. Interestingly, anti-phosphosynapsin and anti-gephyrin appear to represent novel markers for synapses and glial cells, respectively. In contrast, antibodies directed against acetylcholine, choline acetyltransferase, elav and repo failed to produce a signal in Culex pipiens comparable to that in Drosophila melanogaster. In summary, present results enable a detailed investigation of the nervous system of mosquitoes, facilitating further studies of behavioral mechanisms associated with arboviruses in the course of vector research.

Aedes albopictus is a highly invasive mosquito species that has become widespread across the globe. In addition, it is an efficient vector of numerous pathogens of medical and veterinary importance, including dengue, chikungunya and Zika viruses. Among others, the vector potential of mosquitoes is influenced by their microbiome. However, this influence is very dynamic and can vary between individuals and life stages. To obtain a rough overview on the microbiome of Ae. albopictus populations in Germany, pooled female and pooled male individuals from seven German locations were investigated by total RNA sequencing. The mosquito specimens had been collected as larvae in the field and processed immediately after adult emergence, i.e. without females having fed on blood. RNA fragments with high degrees of identity to a large number of viruses and microorganisms were identified, including, for example, Wolbachia pipientis and Acinetobacter baumannii, with differences between male and female mosquitoes. Knowledge about the natural occurrence of microorganisms in mosquitoes may be translated into new approaches to vector control, for example W. pipientis can be exploited to manipulate mosquito reproduction and vector competence. The study results show how diverse the microbiome of Ae. albopictus can be, and the more so needs to be adequately analysed and interpreted.

Molecular characterization of the insecticide resistance has become a hot research topic ever since the first disease transmitting arthropod (Anopheles gambiae) genome sequence has unveiled in 2002. A recent publication of the Culex quinquefasciatus genome sequence has opened up new opportunities for molecular and comparative genomic analysis of multiple mosquito genomes to characterize the insecticide resistance. Here, we utilized a whole genome sequence of Cx. quinquefasciatus to identify putatively active members of the detoxification supergene families, namely cytochrome P450s (P450s), glutathione-S-transferases (GSTs), and choline/carboxylesterases (CCEs). The Culex genome analysis revealed 166 P450s, 40 GSTs, and 62 CCEs. Further, the comparative genomic analysis shows that these numbers are considerably higher than the other dipteran mosquitoes. These observed speciesspecific expansions of the detoxification super gene family members endorse the popular understanding of the involvement of these gene families in protecting the organism against multitudinous classes of toxic substances during its complex (aquatic and terrestrial) life cycle. Thus, the generated data set may provide an initial point to start with to characterize the insecticide resistance at a molecular level which could then lead the development of an easy to use molecular marker to monitor the incipient insecticide resistance in field environs.

In the absence of entomological information, tools for predicting Anopheles spp. presence can help evaluate the entomological risk of malaria transmission. Here, we illustrate how species distribution models (SDM) could quantify potential dominant vector species presence in malaria elimination settings. We fitted a 250 m resolution ensemble SDM for Anopheles albimanus Wiedemann. The ensemble SDM included predictions based on seven different algorithms, 110 occurrence records and 70 model projections. SDM covariates included nine environmental variables that were selected based on their importance from an original set of 28 layers that included remotely and spatially interpolated locally measured variables for the land surface of Costa Rica. Goodness of fit for the ensemble SDM was very high, with a minimum AUC of 0.79. We used the resulting ensemble SDM to evaluate differences in habitat suitability (HS) between commercial plantations and surrounding landscapes, finding a higher HS in pineapple and oil palm plantations, suggestive of An. albimanus presence, than in surrounding landscapes. The ensemble SDM suggested a low HS for An. albimanus at the presumed epicenter of malaria transmission during 2018-2019 in Costa Rica, yet this vector was likely present at the two main towns also affected by the epidemic. Our results illustrate how ensemble SDMs in malaria elimination settings can provide information that could help to improve vector surveillance and control.

In the recent time, global attention for the control of vectors has shifted from chemical insecticides to botanicals. In the present investigation, authors attempted to evaluate the efficacy of peel and leaf essential oil (EO) of Citrus aurantifolia against Aedes aegypti.

Nutrients in breeding sites are critical for the survival and development of malaria mosquitoes, having a direct impact on vectorial capacity. Yet, there is a limited understanding about the natural larval diet and its impact on the individual fitness of mosquitoes. Recent studies have shown that gravid Anopheles arabiensis Patton (Diptera: Culicidae) are attracted by and oviposit in grass-associated habitats. The pollen provided by these grasses is a potential source of nutrients for the larvae. Here, we assess the effect of Typha latifolia L. (Poales: Typhaceae), Echinochloa pyramidalis Lamarck, Pennisetum setaceum Forsskål, and Zea mays L. pollen on larval survival and rate of development in An. arabiensis under laboratory conditions. In addition, we characterize the carbon to nitrogen ratio and the size of pollen grains as a measure of diet quality. Carbon-rich pollen with a small grain size (T. latifolia and P. setaceum; 9.7 ± 0.3 × 103 and 5.5 ± 0.2 × 104 µm3, respectively) resulted in enhanced rates of development of An. arabiensis. In contrast, the larva fed on the nitrogen-rich control diet (TetraMin) was slower to develop, but demonstrated the highest larval survival. Larvae fed on carbon-rich and large-grained Z. mays pollen (4.1 ± 0.2 × 105 µm3) survived at similar levels as those fed on the control diet and also took a longer time to develop compared with larvae fed on the other pollens. While males and females did not appear to develop differently on the different pollen diets, males consistently emerged faster than their female counterparts. These results are discussed in relation to integrated vector management.

Dengue represents a serious threat to human health, with billions of people living at risk of the disease. Wolbachia pipientis is a bacterial endosymbiont common to many insect species. Wolbachia transinfections in mosquito disease vectors have great value for disease control given the bacterium's ability to spread into wild mosquito populations, and to interfere with infections of pathogens, such as dengue virus. Aedes fluviatilis is a mosquito with a widespread distribution in Latin America, but its status as a dengue vector has not been clarified. Ae. fluviatilis is also naturally infected by the wFlu Wolbachia strain, which has been demonstrated to enhance infection with the avian malarial parasite Plasmodium gallinaceum. We performed experimental infections of Ae. fluviatilis with DENV-2 and DENV-3 isolates from Brazil via injection or oral feeding to provide insight into its competence for the virus. We also examined the effect of the native Wolbachia infection on the virus using a mosquito line where the wFlu infection had been cleared by antibiotic treatment. Through RT-qPCR, we observed that Ae. fluviatilis could become infected with both viruses via either method of infection, although at a lower rate than Aedes aegypti, the primary dengue vector. We then detected DENV-2 and DENV-3 in the saliva of injected mosquitoes, and observed that injection of DENV-3-infected saliva produced subsequent infections in naïve Ae. aegypti. However, across our data we observed no difference in prevalence of infection and viral load between Wolbachia-infected and -uninfected mosquitoes, suggesting that there is no effect of wFlu on dengue virus. Our results highlight that Ae. fluviatilis could potentially serve as a dengue vector under the right circumstances, although further testing is required to determine if this occurs in the field.

Mosquitoes are insects of medical importance due their role as vectors of different pathogens to humans. There is a lack of information about the evolutionary history and phylogenetic positioning of the majority of mosquito species. Here we characterized the mitogenomes of mosquito species through low-coverage whole genome sequencing and data mining. A total of 37 draft mitogenomes of different species were assembled from which 16 are newly-sequenced species. We datamined additional 49 mosquito mitogenomes, and together with our 37 mitogenomes, we reconstructed the evolutionary history of 86 species including representatives from 15 genera and 7 tribes. Our results showed that most of the species clustered in clades with other members of their own genus with exception of Aedes genus which was paraphyletic. We confirmed the monophyletic status of the Mansoniini tribe including both Coquillettidia and Mansonia genus. The Aedeomyiini and Uranotaeniini were consistently recovered as basal to other tribes in the subfamily Culicinae, although the exact relationships among these tribes differed between analyses. These results demonstrate that low-coverage sequencing is effective to recover mitogenomes, establish phylogenetic knowledge and hence generate basic fundamental information that will help in the understanding of the role of these species as pathogen vectors.

Aedes pulcritarsis is a tree-hole breeding species with its main distribution in the Mediterranean area. Within the scope of two independent monitoring programmes, this mosquito species was detected for the first time in Austria, in the province of Lower Austria (2018, districts Mistelbach and Gaenserndorf; 2020, district Bruck an der Leitha). As the climatic and habitat situation in Central Europe seems to be generally suitable for this species, the most likely explanation for the species not being recorded previously is that it might have been overlooked in the past due to its specialized breeding habitat. However, further research on the distribution of Ae. pulcritarsis in Austria would be needed to support this hypothesis. The results from this study will contribute to the investigation of the northern distribution limit of Ae. pulcritarsis in Europe and possible changes thereof.

Anopheles sinensis (Diptera: Culicidae) is a primary vector of Plasmodium vivax and Brugia malayi in most regions of China. In addition, its phylogenetic relationship with the cryptic species of the Hyrcanus Group is complex and remains unresolved. Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes, of which mitochondrial genome data of An. sinensis is not available.

The tribe Sabethini (Diptera: Culicidae) contains important vectors of the yellow fever virus and presents remarkable morphological and ecological diversity unequalled in other mosquito groups. However, there is limited information about mitochondrial genomes (mitogenomes) from these species. As mitochondrial genetics has been fundamental for posing evolutionary hypotheses and identifying taxonomical markers, in this study we sequenced the first sabethine mitogenomes: Sabethes undosus, Trichoprosopon pallidiventer, Runchomyia reversa, Limatus flavisetosus, and Wyeomyia confusa. In addition, we performed phylogenetic analyses of Sabethini within Culicidae and compared its mitogenomic architecture to that of other insects.

Flaviviruses are important pathogens for humans and animals (Dengue viruses, Yellow fever virus, Zika virus and West Nile virus). Culex flavivirus (CxFV) is an insect-specific virus of the genus Flavivirus, detected in a wide variety of mosquito species.

In Europe, the air-borne accidental introduction of exotic mosquito species (EMS) has been demonstrated using mosquito surveillance schemes at Schiphol International Airport (Amsterdam, The Netherlands). Based upon these findings and given the increasing volume of air transport movements per year, the establishment of EMS after introduction via aircraft is being considered a potential risk. Here we present the airport surveillance results performed by the Centre for Monitoring of Vectors of the Netherlands, by the Monitoring of Exotic Mosquitoes (MEMO) project in Belgium, and by the Public Health England project on invasive mosquito surveillance. The findings of our study demonstrate the aircraft mediated transport of EMS into Europe from a wide range of possible areas in the world. Results show accidental introductions of Aedes aegypti and Ae. albopictus, as well as exotic Anopheles and Mansonia specimens. The findings of Ae. albopictus at Schiphol airport are the first evidence of accidental introduction of the species using this pathway in Europe. Furthermore, our results stress the importance of the use of molecular tools to validate the morphology-based species identifications. We recommend monitoring of EMS at airports with special attention to locations with a high movement of cargo and passengers.

In Sri Lanka, dengue is the most serious arboviral disease. Recent increases in dengue cases suggest a higher infection rate and spread of the disease to new areas. The present study explores gene flow patterns of Ae. aegypti, the main vector of dengue disease, among 10 collection sites including major ports and inland cities using variations at 11 microsatellite loci. Discriminant analysis of principal components (DAPC) and k-means clustering estimated eight genetic clusters. Analysis of Molecular Variance (AMOVA) estimated equal variances among cities and among collections in Colombo, Sri Lanka. Significant evidence, although weak, was detected for isolation by distance. Analysis of gene flow rates and directions using MIGRATE-n indicated that populations throughout the island served as a source of immigrants for Colombo with abundant gene flow among major commercial cities in Sri Lanka, which appear to receive migrant mosquitoes from throughout Sri Lanka. The observed patterns probably arise through human movement of Ae. aegypti during commerce from throughout Sri Lanka into Colombo increasing the risk of spread. The patterns uncovered in this study are significant for global health as Sri Lanka is situated along a key international shipping route.

The mosquito vectors of Plasmodium spp. have largely been overlooked in studies of ecology and evolution of avian malaria and other vertebrates in wildlife.

The Asian mosquito, Aedes albopictus (Skuse), is an invasive mosquito which has become one of the most important vectors of dengue, Zika, and chikungunya viruses worldwide. This species was reported for the first time in Cameroon in early 2000s and became the dominant Aedes species in the urban areas in the southern part of Cameroon but remain poorly characterized. Here, we assessed the susceptibility profile of A. albopictus collected throughout Cameroon and investigated the potential resistance mechanisms involved. Immature stages of A. albopictus were collected between March and July 2017 in 15 locations across Cameroon and reared until G1/G2 generation. Larval, adult bioassays, and synergists [piperonyl butoxide (PBO) and diethyl maleate (DEM)] assays were carried out according to WHO recommendations. F1534C mutation was genotyped in field collected adults (Go) using allele specific PCR. All tested populations were susceptible to both larvicides, temephos and Bacillus thuringiensis israelensis (Bti), after larval bioassays. Adult bioassays revealed a high level of resistance of A. albopictus to 4% DDT with mortality rates ranging from 12.42% in Bafang to 75.04% in Kumba. The resistance was reported also in 0.05% deltamethrin, 0.25% permethrin, and 0.1% propoxur in some locations. A loss of susceptibility to 0.1% bendiocarb was found in one of three populations analysed. A full susceptibility to 1% fenitrothion were observed across the country. A full recovery or partial of susceptibility was observed in A. albopictus when pre-exposed to PBO or DEM and then to DDT and permethrin, respectively. The F1534C kdr mutation was not detected in A. albopictus. This study showed that the susceptibility profile of A. albopictus to insecticide vary according to the sampling location and insecticides used. These findings are useful to planning vector control program against arbovirus vectors in Cameroon and can be used as baseline data for further researches.