Application of computer vision and deep learning models to automatically classify medically important mosquitoes in North Borneo, Malaysia.

Mosquito-borne diseases have emerged in North Borneo in Malaysia due to rapid changes in the forest landscape, and mosquito surveillance is key to understanding disease transmission. However, surveillance programmes involving sampling and taxonomic identification require well-trained personnel, are time-consuming and labour-intensive. In this study, we aim to use a deep leaning model (DL) to develop an application capable of automatically detecting mosquito vectors collected from urban and suburban areas in North Borneo, Malaysia. Specifically, a DL model called MobileNetV2 was developed using a total of 4880 images of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus mosquitoes, which are widely distributed in Malaysia. More importantly, the model was deployed as an application that can be used in the field. The model was fine-tuned with hyperparameters of learning rate 0.0001, 0.0005, 0.001, 0.01 and the performance of the model was tested for accuracy, precision, recall and F1 score. Inference time was also considered during development to assess the feasibility of the model as an app in the real world. The model showed an accuracy of at least 97%, a precision of 96% and a recall of 97% on the test set. When used as an app in the field to detect mosquitoes with the elements of different background environments, the model was able to achieve an accuracy of 76% with an inference time of 47.33 ms. Our result demonstrates the practicality of computer vision and DL in the real world of vector and pest surveillance programmes. In the future, more image data and robust DL architecture can be explored to improve the prediction result.

Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti and Aedes albopictus.

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.

Zika virus in southeastern Senegal: survival of the vectors and the virus during the dry season.

BACKGROUND: Zika virus (ZIKV, genus Flavivirus, family Flaviviridae) is transmitted mainly by Aedes mosquitoes. This virus has become an emerging concern of global public health with recent epidemics associated to neurological complications in the pacific and America. ZIKV is the most frequently amplified arbovirus in southeastern Senegal. However, this virus and its adult vectors are undetectable during the dry season. The aim of this study was to investigate how ZIKV and its vectors are maintained locally during the dry season. METHODS: Soil, sand, and detritus contained in 1339 potential breeding sites (tree holes, rock holes, fruit husks, discarded containers, used tires) were collected in forest, savannah, barren and village land covers and flooded for eggs hatching. The emerging larvae were reared to adult, identified, and blood fed for F1 production. The F0 and F1 adults were identified and tested for ZIKV by Reverse Transcriptase-Real time Polymerase Chain Reaction. RESULTS: A total of 1016 specimens, including 13 Aedes species, emerged in samples collected in the land covers and breeding sites investigated. Ae. aegypti was the dominant species representing 56.6% of this fauna with a high plasticity. Ae. furcifer and Ae. luteocephalus were found in forest tree holes, Ae. taylori in forest and village tree holes, Ae. vittatus in rock holes. ZIKV was detected from 4 out of the 82 mosquito pools tested. Positive pools included Ae. bromeliae (2 pools), Ae. unilineatus (1 pool), and Ae. vittatus (1 pool), indicating that the virus is maintained in these Aedes eggs during the dry season. CONCLUSION: Our investigation identified breeding sites types and land cover classes where several ZIKV vectors are maintained, and their maintenance rates during the dry season in southeastern Senegal. The maintenance of the virus in these vectors in nature could explain its early amplification at the start of the rainy season in this area.

Aedes aegypti spreading in South America: new coldest and southernmost records.

The geographic distribution of Aedes (Stegomyia) aegypti (L.) in South America has been expanding during the last decades. Herein we present two new distribution records that extend its southern limits towards localities with extremer environmental conditions than reported to date. San Antonio Oeste constitutes the southernmost finding for the continent (40º44'S), whereas Tandil is the infested locality with the coldest mean annual temperature in Argentina (14.17ºC). The projection of a previous distribution model for Ae. aegypti predicts these two cities as positive and suggests several other localities with suitable conditions for vector proliferation beyond its assumed distribution limits.

Infestation of an endemic arbovirus area by sympatric populations of Aedes aegypti and Aedes albopictus in Brazil.

BACKGROUND Aedes aegypti and Aedes albopictus are the most important arbovirus vectors in the world. OBJECTIVES This study aimed to investigate and compare the infestation pattern of these species in a neighbourhood of Recife, Brazil, endemic for arboviruses in 2005 (T1) and 2013 (T2). METHODS Infestation, distribution and relative abundance of these sympatric species were recorded by egg collection using a network of 59 sentinel ovitraps (s-ovt) at fixed sampling stations for 12 months in T1 and T2. FINDINGS A permanent occupation pattern was detected which was characterised by the presence of egg-laying females of one or both species with a high ovitrap positivity index (94.3 to 100%) throughout both years analysed. In terms of abundance, the total of eggs collected was lower (p < 0.005) in T2 (146,153) than in T1 (281,103), although ovitraps still displayed a high index of positivity. The spatial distribution showed the presence of both species in 65.1% of the 148 s-ovt assessed, while a smaller number of traps exclusively contained Ae. aegypti (22%) or Ae. albopictus (13.2%) eggs. MAIN CONCLUSIONS Our comparative analysis demonstrated the robustness of the spatial occupation and permanence of Ae. aegypti and Ae. albopictus populations in this endemic urban area.

Nine years of mosquito monitoring in Germany, 2011-2019, with an updated inventory of German culicid species.

Before the background of increasingly frequent outbreaks and cases of mosquito-borne diseases in various European countries, Germany recently realised the necessity of updating decade-old data on the occurrence and spatiotemporal distribution of culicid species. Starting in 2011, a mosquito monitoring programme was therefore launched with adult and immature mosquito stages being collected at numerous sites all over Germany both actively by trapping, netting, aspirating and dipping, and passively by the citizen science project 'Mueckenatlas'. Until the end of 2019, about 516,000 mosquito specimens were analysed, with 52 (probably 53) species belonging to seven genera found, including several species not reported for decades due to being extremely rare (Aedes refiki, Anopheles algeriensis, Culex martinii) or local (Culiseta alaskaensis, Cs. glaphyroptera, Cs. ochroptera). In addition to 43 (probably 44 including Cs. subochrea) out of 46 species previously described for Germany, nine species were collected that had never been documented before. These consisted of five species recently established (Ae. albopictus, Ae. japonicus, Ae. koreicus, An. petragnani, Cs. longiareolata), three species probably introduced on one single occasion only and not established (Ae. aegypti, Ae. berlandi, Ae. pulcritarsis), and a newly described cryptic species of the Anopheles maculipennis complex (An. daciae) that had probably always been present but not been differentiated from its siblings. Two species formerly listed for Germany could not be documented (Ae. cyprius, Ae. nigrinus), while presence is likely for another species (Cs. subochrea), which could not be demonstrated in the monitoring programme as it can neither morphologically nor genetically be reliably distinguished from a closely related species (Cs. annulata) in the female sex. While Cs. annulata males were collected in the present programme, this was not the case with Cs. subochrea. In summary, although some species regarded endemic could not be found during the last 9 years, the number of culicid species that must be considered firmly established in Germany has increased to 51 (assuming Cs. subochrea and Ae. nigrinus are still present) due to several newly emerged ones but also to one species (Ae. cyprius) that must be considered extinct after almost a century without documentation. Most likely, introduction and establishment of the new species are a consequence of globalisation and climate warming, as three of them are native to Asia (Ae. albopictus, Ae. japonicus, Ae. koreicus) and three (Ae. albopictus, An. petragnani, Cs. longiareolata) are relatively thermophilic. Another thermophilic species, Uranotaenia unguiculata, which had been described for southwestern Germany in 1994 and had since been found only at the very site of its first detection, was recently documented at additional localities in the northeastern part of the country. As several mosquito species found in Germany are serious pests or potential vectors of disease agents and should be kept under permanent observation or even be controlled immediately on emergence, the German mosquito monitoring programme has recently been institutionalised and perpetuated.

The invasive Asian tiger mosquito Aedes albopictus in Romania: towards a country-wide colonization?

Currently, five invasive Aedes mosquito species are of concern in Europe according to the European Centre for Disease Prevention and Control: Aedes aegypti, Ae. albopictus, Ae. atropalpus, Ae. japonicus, and Ae. koreicus. Among these, only Ae. albopictus was reported to occur in Romania, in Bucharest. The aim of this study was to update the knowledge on the distribution of this invasive mosquito species in Romania, by investigating new potential locations. Monitoring of Ae. albopictus was carried out between May 2017 and October 2018. Three types of traps (CDC-Gravid Traps, CDC miniature Light Traps, ovitraps) were placed in 53 localities in 13 counties at sites suitable for container-breeding mosquitoes. Collected adult mosquitoes were counted and identified according to morphological criteria. Larvae were found present in domestic containers and rain catch basins. Aedes albopictus adults and eggs were collected in 10 localities in eight counties across Romania. Our study confirms nine new localities and seven counties where Ae. albopictus became established in Romania, highlighting the need for surveillance to further assess the species' distribution and abundance, as well as the pathogen transmission risk related to that vector species.

Aedes aegypti, the dengue fever mosquito in Mexico City. Early invasion and its potential risks.

INTRODUCTION: Mexico City has no endemic presence of Aedes aegypti, and it is therefore free of vector-borne diseases, such as dengue fever, Zika and chikungunya. However, evidence has shown the presence of Aedes aegypti eggs in the city since 2015. OBJECTIVE: To report the constant and increasing presence of Aedes aegypti eggs in Mexico City from 2015 to 2018. METHODS: Surveillance was carried out using ovitraps. Eggs were counted and hatched in order to determine the species. RESULTS: From 2015 to 2018, 378 organisms were identified as Ae. aegypti. In total, 76 Aedes aegypti-positive ovitraps were collected at 50 different places in 11 boroughs of the city. Northeastern Mexico City was the area with the highest number of positive traps. CONCLUSIONS: The results may be indicating a period of early colonization and the probable existence of cryptic colonies of the mosquito; Mexico City could be at risk of experiencing vector-borne epidemics.

INTRODUCCIÓN: La Ciudad de México no tiene presencia endémica de Aedes aegypti, por lo que está libre de enfermedades transmitidas por vector como dengue, Zika y chikunguña. Sin embargo, existe evidencia de la presencia de huevecillos en la urbe desde 2015. OBJETIVO: Reportar la presencia constante y en aumento de huevecillos de Aedes aegypti en la Ciudad de México de 2015 a 2018. MÉTODO: Se realizó vigilancia a través de ovitrampas; se contabilizaron y eclosionaron huevecillos para determinar la especie. RESULTADOS: De 2015 a 2018 fueron identificados 378 organismos como Aedes aegypti. En total fueron colectadas 76 ovitrampas positivas a Aedes aegypti en 50 sitios distintos de 11 alcaldías. El noreste de la Ciudad de México fue el área con mayor positividad. CONCLUSIONES: Los resultados pueden estar indicando un periodo de colonización incipiente y la probable la existencia de colonias crípticas del mosquito, por lo que la Ciudad de México podría estar en riesgo de presentar epidemias de enfermedades transmitidas por vector.

Genome-wide divergence among invasive populations of Aedes aegypti in California.

BACKGROUND: In the summer of 2013, Aedes aegypti Linnaeus was first detected in three cities in central California (Clovis, Madera and Menlo Park). It has now been detected in multiple locations in central and southern CA as far south as San Diego and Imperial Counties. A number of published reports suggest that CA populations have been established from multiple independent introductions. RESULTS: Here we report the first population genomics analyses of Ae. aegypti based on individual, field collected whole genome sequences. We analyzed 46 Ae. aegypti genomes to establish genetic relationships among populations from sites in California, Florida and South Africa. Based on 4.65 million high quality biallelic SNPs, we identified 3 major genetic clusters within California; one that includes all sample sites in the southern part of the state (South of Tehachapi mountain range) plus the town of Exeter in central California and two additional clusters in central California. CONCLUSIONS: A lack of concordance between mitochondrial and nuclear genealogies suggests that the three founding populations were polymorphic for two main mitochondrial haplotypes prior to being introduced to California. One of these has been lost in the Clovis populations, possibly by a founder effect. Genome-wide comparisons indicate extensive differentiation between genetic clusters. Our observations support recent introductions of Ae. aegypti into California from multiple, genetically diverged source populations. Our data reveal signs of hybridization among diverged populations within CA. Genetic markers identified in this study will be of great value in pursuing classical population genetic studies which require larger sample sizes.

Loop-mediated isothermal amplification (LAMP) for the identification of invasive Aedes mosquito species.

Invasive Aedes mosquito species (Diptera: Culicidae) are of public health concern in Europe because they are either recognized or potential vectors of pathogens. Loop-mediated isothermal amplification (LAMP) is a rapid and simple method for amplifying DNA with high specificity and efficiency, with the technique having potential for application in the field, including in high-throughput format. Specific LAMP assays based on rDNA internal transcribed spacers 1 or 2 sequences, considering intraspecies variability at these loci, were developed for Aedes aegypti, Aedes albopictus, Aedes japonicus, Aedes koreicus and the indigenous Aedes geniculatus. No such assays could be developed for Aedes atropalpus and Aedes triseriatus because both loci were too short to serve as target. The assays rely on the clearly visible colour change from violet to sky blue after successful amplification. Sensitivity of egg detection was confirmed with ratios of up to one mosquito egg in 99 other eggs. Simple sample preparation of adults or eggs by mechanical homogenization in water required an additional heat treatment or centrifugation step to avoid non-specific colour changes. Thus, further technical improvements are needed to render these assays truly field-applicable, which would greatly facilitate surveillance of these invasive mosquito species and allow for prompt implementation of control measures.

Aedes koreicus-a new member of the genus Aedes establishing in Germany?

Aedes koreicus, a mosquito species originating from Japan, China, Korea, and parts of Russia, has been sporadically found in Europe since 2008. It is suspected to be a vector of a variety of viruses and nematodes. In Germany, one individual was found in 2015 in the city of Augsburg, situated in the federal state of Bavaria. Based on morphological and molecular species identification, this study reports a new finding of Ae. koreicus, about 370 km northwest from Augsburg. The sampling point is located in the city of Wiesbaden, in the federal state of Hesse, where four individuals were found over a period of 2 months in 2017. The re-finding of the species in the same location in May and July 2018 suggests that (a) the species was able to reproduce and overwinter at this site, and (b) spreading of non-native mosquito species is an ongoing process in Germany, which requires close monitoring.

Monitoring of alien mosquitoes of the genus Aedes (Diptera: Culicidae) in Austria.

Systematic, continuous mosquito surveillance is considered the most reliable tool to predict the spread and establishment of alien mosquito species such as the Asian tiger mosquito (Aedes albopictus), Japanese bush mosquito (Aedes japonicus), and the transmission risk of mosquito-borne arboviruses to humans. Only single individuals of Ae. albopictus have been found in Austria so far. However, it is likely that the species will be able to establish populations in the future due to global trade and traffic as well as increasing temperatures in the course of global climate change. In summer 2017, a project surveilling the oviposition of newly introduced Aedes mosquitoes, using ovitraps, was set up by means of citizen scientists and researchers and was performed in six federal provinces of Austria-Tyrol, Carinthia, Vienna, Lower Austria, Styria, and Burgenland. Eggs of Ae. albopictus were identified in Tyrol during the months August and September, while Ae. japonicus was found in Lower Austria, Styria, and Burgenland. In Vienna and Carinthia, all ovitraps were negative for Aedes eggs; however, Ae. japonicus was found for the first time in Vienna in July 2017 during routine sampling of adult mosquitoes. With this project, we demonstrated the benefits of citizen scientists for ovitrap-based mosquito surveillance. The finding of Ae. albopictus eggs in Northern Tyrol is not yet a proof of the establishment of a self-sustaining population, although it indicates the ongoing introduction of this species along main traffic routes from Italy, where this mosquito is well established. The risk of establishment of the tiger mosquito in the Lower Inn Valley is therefore a given and informing the public about preventive measures to hinder and delay this development is highly recommended.

Rapid evolution and the genomic consequences of selection against interspecific mating.

While few species introduced into a new environment become invasive, those that do provide critical information on ecological mechanisms that determine invasions success and the evolutionary responses that follow invasion. Aedes albopictus (the Asian tiger mosquito) was introduced into the naturalized range of Aedes aegypti (the yellow fever mosquito) in the United States in the mid-1980s, resulting in the displacement of A. aegypti in much of the south-eastern United States. The rapid displacement was likely due to the superior competitive ability of A. albopictus as larvae and asymmetric mating interference competition, in which male A. albopictus mate with and sterilize A. aegypti females, a process called "satyrization." The goal of this study was to examine the genomic responses of a resident species to an invasive species in which the mechanism of character displacement is understood. We used double-digest restriction enzyme DNA sequencing (ddRADseq) to analyse outlier loci between selected and control lines of laboratory-reared A. aegypti females from two populations (Tucson, AZ and Key West, Florida, USA), and individual females classified as either "resisted" or "mated with" A. albopictus males via mating trials of wild-derived females from four populations in Florida. We found significant outlier loci in comparing selected and control lines and between mated and nonmated A. aegypti females in the laboratory and wild-derived populations, respectively. We found overlap in specific outlier loci between different source populations that support consistent genomic signatures of selection within A. aegypti. Our results point to regions of the A. aegypti genome and potential candidate genes that may be involved in mating behaviour, and specifically in avoiding interspecific mating choices.

A distinct group of north European Aedes vexans as determined by mitochondrial and nuclear markers.

The floodwater mosquito Aedes (Aedimorphus) vexans (Meigen, 1830) (Diptera: Culicidae) is common in several areas of Sweden and is predicted to become more abundant in the wake of expected changes in precipitation and temperature caused by climate change. As well as being a nuisance, Ae. vexans can act as a vector of over 30 viruses. In the event of an outbreak of disease caused by a vector-borne virus, knowledge of the distribution, population structure and intermixing of populations from different locations will help direct resources to target locations to prevent spread of the pathogen. The present study analysed individual Ae. vexans from eight locations throughout Sweden. Based on the mitochondrial cytochrome oxidase I (COI) marker, a subset of the analysed mosquitoes cluster apart from the other samples. Similarly, two nuclear loci were sequenced and the same phylogenetic structure observed. These results indicate that this group represents a reproductively isolated population among Ae. vexans. Comparisons with COI sequences held in the Barcode of Life Database (BoLD) for Ae. vexans from around the world show that specimens collected in Belgium and Estonia group together with the Swedish group, suggesting that this genotype is present throughout northern Europe. These results suggest there is a cryptic taxonomic unit related to Ae. vexans in northern Europe.

Transmission potential of Mayaro virus in Florida Aedes aegypti and Aedes albopictus mosquitoes.

Mayaro virus (MAYV) is an emerging mosquito-borne arbovirus present in Central and South America that causes arthralgia and febrile illness. Domestic mosquitoes Aedes aegypti (Diptera: Culicidae) and Aedes albopictus are potential vectors of MAYV that may allow for transmission to humans in urban settings. The present paper assesses susceptibility to infection, disseminated infection and transmission potential in Florida Ae. aegypti and Ae. albopictus for MAYV. Oral infection was significantly higher in Ae. albopictus (85-100%) than in Ae. aegypti (67-82%). Viral dissemination to the haemocoel in Ae. aegypti and Ae. albopictus mosquitoes was rapid and co-occurred with infection of the salivary glands. Rates of disseminated infection were generally higher in Ae. aegypti (45-85%) than in Ae. albopictus (38-76%), although the difference was significant only at 9 days after feeding on MAYV-infected blood. Both mosquito species exhibited low rates of MAYV infection in saliva expectorates. Viral titres in the bodies of mosquitoes increased in line with the number of days post-blood feeding and were higher in Ae. aegypti than in Ae. albopictus. Although Florida mosquito vectors have the potential to transmit MAYV and thus to initiate an urban cycle after having fed on higher titres of MAYV-infected blood, lower viraemia in infected humans is likely to limit transmission potential.

Isolation of an insulin-like peptide from the Asian malaria mosquito, Anopheles stephensi, that acts as a steroidogenic gonadotropin across diverse mosquito taxa.

Many insulin-like peptides (ILPs) have been identified in insects, yet only a few were isolated in their native form for structural and functional studies. Antiserum produced to ILP3 in Aedes aegypti was used in a radioimmunoassay to monitor the purification of an ILP from heads of adult An. stephensi and recognized the ILP in other immunoassays. The structure of the purified peptide matched that predicted for the ILP3 in this species. The native form stimulated ecdysteroid production by ovaries isolated from non-blood fed females. Synthetic forms of An. stephensi ILP3 and ILP4 similarly activated this process in a dose responsive manner. This function was first established for ILP3 and ILP4 homologs in Aedes aegypti, thus suggesting their structural and functional conservation in mosquitoes. We tested the extent of conservation by treating ovaries of An. gambiae, Ae. aegypti, and Culex quinquefasciatus with the An. stephensi ILPs, and both the native and synthetic ILP3 were stimulatory, as was the ILP4. Taken together, these results offer the first evidence for ILP functional conservation across the Anophelinae and Culicinae subfamilies.

Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution.

The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined for the Foshan strain, an established laboratory colony derived from wild mosquitoes from southeastern China, a region within the historical range of the origin of the species. The genome comprises 1,967 Mb, the largest mosquito genome sequenced to date, and its size results principally from an abundance of repetitive DNA classes. In addition, expansions of the numbers of members in gene families involved in insecticide-resistance mechanisms, diapause, sex determination, immunity, and olfaction also contribute to the larger size. Portions of integrated flavivirus-like genomes support a shared evolutionary history of association of these viruses with their vector. The large genome repertory may contribute to the adaptability and success of Ae. albopictus as an invasive species.

Aedes albopictus (Skuse, 1895) (Diptera: Culicidae) in Greece: 13 years of living with the Asian tiger mosquito.

Aedes (Stegomyia) albopictus (Skuse, 1895) (Diptera: Culicidae), commonly known as the Asian tiger mosquito, is an invasive mosquito species of public health significance, well established in many countries worldwide. In Greece, it was first recorded in Corfu and Thesprotia between 2003 and 2004. In the following years, further distribution and establishment of Ae. albopictus in Greece have been confirmed in many Regional Units of the country. In the current study, we report the invasion history of Ae. albopictus in Greece, until 2016. The results from the entomological investigation following imported virus cases in 2014, 2015, and 2016 are also included. Moreover, its presence in Greece is demonstrated in a thematic map based on (a) information provided by pest control companies and/or citizens, (b) the official samples sent to Benaki Phytopathological Institute (BPI) and the National School of Public Health (NSPH), and

Experimental risk assessment for chikungunya virus transmission based on vector competence, distribution and temperature suitability in Europe, 2018.

BackgroundOver the last decade, the abundant distribution of the Asian tiger mosquito Aedes albopictus in southern Europe and the import of chikungunya virus (CHIKV) by infected travellers has resulted in at least five local outbreaks of chikungunya fever in France and Italy. Considering the ongoing spread of Ae. albopictus to central Europe, we performed an analysis of the Europe-wide spatial risk of CHIKV transmission under different temperature conditions. Methods:Ae. albopictus specimens from Germany and Italy were orally infected with CHIKV from an outbreak in France and kept for two weeks at 18 °C, 21 °C or 24 °C. A salivation assay was conducted to detect infectious CHIKV. Results: Analyses of mosquito saliva for infectious virus particles demonstrated transmission rates (TRs) of > 35%. Highest TRs of 50% for the mosquito population from Germany were detected at 18 °C, while the Italian population had highest TRs of 63% at 18 °C and 21 °C, respectively. Temperature data indicated a potential risk of CHIKV transmission for extended durations, i.e. sufficiently long time periods allowing extrinsic incubation of the virus. This was shown for areas already colonised by Ae. albopictus, as well as for large parts of central Europe that are not colonised. Conclusion: The current risk of CHIKV transmission in Europe is not primarily restricted by temperature, which allows extrinsic incubation of the virus, but rather by the vector distribution. Accordingly, all European countries with established populations of Ae. albopictus should implement respective entomological surveillance and monitoring systems, as basis for suitable control measures.

From ground pools to treeholes: convergent evolution of habitat and phenotype in Aedes mosquitoes.

BACKGROUND: Invasive mosquito species are responsible for millions of vector-borne disease cases annually. The global invasive success of Aedes mosquitoes such as Aedes aegypti and Aedes albopictus has relied on the human transport of immature stages in container habitats. However, despite the importance of these mosquitoes and this ecological specialization to their widespread dispersal, evolution of habitat specialization in this group has remained largely unstudied. We use comparative methods to evaluate the evolution of habitat specialization and its potential influence on larval morphology, and evaluate whether container dwelling and invasiveness are monophyletic in Aedes. RESULTS: We show that habitat specialization has evolved repeatedly from ancestral ground pool usage to specialization in container habitats. Furthermore, we find that larval morphological scores are significantly associated with larval habitat when accounting for evolutionary relationships. We find that Ornstein-Uhleinbeck models with unique optima for each larval habitat type are preferred over several other models based predominantly on neutral processes, and that OU models can reliably simulate real morphological data. CONCLUSIONS: Our results demonstrate that multiple lineages of Aedes have convergently evolved a key trait associated with invasive success: the use of container habitats for immature stages. Moreover, our results demonstrate convergence in morphological characteristics as well, and suggest a role of adaptation to habitat specialization in driving phenotypic diversity in this mosquito lineage. Finally, our results highlight that the genus Aedes is not monophyletic.