Estimating the potential risk of transmission of arboviruses in the Americas and Europe: a modelling study.

BACKGROUND: Estimates of the spatiotemporal distribution of different mosquito vector species and the associated risk of transmission of arboviruses are key to design adequate policies for preventing local outbreaks and reducing the number of human infections in endemic areas. In this study, we quantified the abundance of Aedes albopictus and Aedes aegypti and the local transmission potential for three arboviral infections at an unprecedented spatiotemporal resolution in areas where no entomological surveillance is available. METHODS: We developed a computational model to quantify the daily abundance of Aedes mosquitoes, leveraging temperature and precipitation records. The model was calibrated on mosquito surveillance data collected in 115 locations in Europe and the Americas between 2007 and 2018. Model estimates were used to quantify the reproduction number of dengue virus, Zika virus, and chikungunya in Europe and the Americas, at a high spatial resolution. FINDINGS: In areas colonised by both Aedes species, A aegypti was estimated to be the main vector for the transmission of dengue virus, Zika virus, and chikungunya, being associated with a higher estimate of R0 when compared with A albopictus. Our estimates highlighted that these arboviruses were endemic in tropical and subtropical countries, with the highest risks of transmission found in central America, Venezuela, Colombia, and central-east Brazil. A non-negligible potential risk of transmission was also estimated for Florida, Texas, and Arizona (USA). The broader ecological niche of A albopictus could contribute to the emergence of chikungunya outbreaks and clusters of dengue autochthonous cases in temperate areas of the Americas, as well as in mediterranean Europe (in particular, in Italy, southern France, and Spain). INTERPRETATION: Our results provide a comprehensive overview of the transmission potential of arboviral diseases in Europe and the Americas, highlighting areas where surveillance and mosquito control capacities should be prioritised. FUNDING: EU and Ministero dell'Università e della Ricerca, Italy (Piano Nazionale di Ripresa e Resilienza Extended Partnership initiative on Emerging Infectious Diseases); EU (Horizon 2020); Ministero dell'Università e della Ricerca, Italy (Progetti di ricerca di Rilevante Interesse Nazionale programme); Brazilian National Council of Science, Technology and Innovation; Ministry of Health, Brazil; and Foundation of Research for Minas Gerais, Brazil.

Shallow Whole-Genome Sequencing of Aedes japonicus and Aedes koreicus from Italy and an Updated Picture of Their Evolution Based on Mitogenomics and Barcoding.

Aedes japonicus and Aedes koreicus are two invasive mosquitoes native to East Asia that are quickly establishing in temperate regions of Europe. Both species are vectors of arboviruses, but we currently lack a clear understanding of their evolution. Here, we present new short-read, shallow genome sequencing of A. japonicus and A. koreicus individuals from northern Italy, which we used for downstream phylogenetic and barcode analyses. We explored associated microbial DNA and found high occurrences of Delftia bacteria in both samples, but neither Asaia nor Wolbachia. We then assembled complete mitogenomes and used these data to infer divergence times estimating the split of A. japonicus from A. koreicus in the Oligocene, which was more recent than that previously reported using mitochondrial markers. We recover a younger age for most other nodes within Aedini and other Culicidae. COI barcoding and phylogenetic analyses indicate that A. japonicus yaeyamensis, A. japonicus amamiensis, and the two A. koreicus sampled from Europe should be considered as separate species within a monophyletic species complex. Our studies further clarify the evolution of A. japonicus and A. koreicus, and indicate the need to obtain whole-genome data from putative species in order to disentangle their complex patterns of evolution.

dynamAedes: a unified modelling framework for invasive Aedes mosquitoes.

Mosquito species belonging to the genus Aedes have attracted the interest of scientists and public health officers because of their capacity to transmit viruses that affect humans. Some of these species were brought outside their native range by means of trade and tourism and then colonised new regions thanks to a unique combination of eco-physiological traits. Considering mosquito physiological and behavioural traits to understand and predict their population dynamics is thus a crucial step in developing strategies to mitigate the local densities of invasive Aedes populations. Here, we synthesised the life cycle of four invasive Aedes species (Ae. aegypti, Ae. albopictus, Ae. japonicus and Ae. koreicus) in a single multi-scale stochastic modelling framework which we coded in the R package dynamAedes. We designed a stage-based and time-discrete stochastic model driven by temperature, photo-period and inter-specific larval competition that can be applied to three different spatial scales: punctual, local and regional. These spatial scales consider different degrees of spatial complexity and data availability by accounting for both active and passive dispersal of mosquito species as well as for the heterogeneity of the input temperature data. Our overarching aim was to provide a flexible, open-source and user-friendly tool rooted in the most updated knowledge on the species' biology which could be applied to the management of invasive Aedes populations as well as to more theoretical ecological inquiries.

Diapause characterization in the invasive alien mosquito species Aedes koreicus: a laboratory experiment.

Aedes koreicus is an invasive alien mosquito species native to Asia now introduced in several European countries, including northern Italy. In this temperate region, mosquito populations survive cold winter temperatures thanks to diapausing eggs or adults, depending on the species. In its native area, Ae. koreicus was reported to overwinter in the egg stage, but to the best of our knowledge, it is not confirmed whether overwintering eggs are actually diapausing or only in a quiescence stage, i.e., they might hatch as soon as external conditions are favorable. Based on previous laboratory studies, we established a diapausing Ae. koreicus colony, maintained at 21 °C with a photoperiod of 12L:12D. Females were allowed to lay eggs, which were consequently placed in water at different time intervals after oviposition, from 30 days to 5 months. We found that diapausing eggs younger than 3 months have a poor hatching rate, while after about 100 days we observed that almost all eggs hatched. Our findings highlight that water immersion alone did not lead to the hatching of eggs, as age was found to be a significantly important factor. We thus confirm effective diapause, occurring at the egg stage, for Ae. koreicus in a recently invaded area. Moreover, our quantification of diapause duration and hatching success might help in better designing future experiments and improving modeling efforts.

First report of the blood-feeding pattern in Aedes koreicus, a new invasive species in Europe.

Aedes koreicus is an invasive mosquito species which has been introduced into several European countries. Compared to other invasive Aedes mosquitoes, little is known of its biology and ecology. To determine Ae. koreicus' vectorial capacity, it is essential to establish its feeding patterns and level of anthropophagy. We report on the blood-feeding patterns of Ae. koreicus, examining the blood meal origin of engorged females and evaluating the influence of different biotic and abiotic factors on feeding behavior. Mosquitoes were collected in 23 sites in northern Italy by manual aspiration and BG-sentinel traps; host availability was estimated by survey. The source of blood meals was identified using a nested PCR and by targeting and sequencing the cytochrome c oxidase subunit I gene. In total, 352 Ae. koreicus engorged females were collected between 2013 and 2020 and host blood meals were determined from 299 blood-fed mosquitoes (84.9%). Eleven host species were identified, with the highest prevalences being observed among roe deer (Capreolus capreolus) (N = 189, 63.2%) and humans (N = 46, 15.4%). Blood meals were mostly taken from roe deer in forested sites and from humans in urban areas, suggesting that this species can feed on different hosts according to local abundance. Two blood meals were identified from avian hosts and one from lizard. Ae. koreicus' mammalophilic feeding pattern suggests that it may be a potential vector of pathogens establishing transmission cycles among mammals, whereas its role as a bridge vector between mammals and birds could be negligible.

Aedes koreicus, a vector on the rise: Pan-European genetic patterns, mitochondrial and draft genome sequencing.

BACKGROUND: The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species' biology, behavior, and dispersal patterns were poorly investigated to date. METHODOLOGY/PRINCIPAL FINDINGS: To understand the species' population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. CONCLUSIONS/SIGNIFICANCE: Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of 'Single Nucleotide Polymorphism' markers. Considering its public health importance, it is crucial to further investigate the species' population genetic dynamic, including a larger sampling and additional genomic markers.

First evidence of pyrethroid resistance in Italian populations of West Nile virus vector Culex pipiens.

Culex pipiens (Linnaeus), one of the most abundant mosquito species in Europe, plays a crucial role in the endemic transmission of West Nile virus and caused the large outbreak with >1600 human cases in 2018. Although evidence of resistance to pyrethroids has been reported for Cx. pipiens populations from Spain and Greece, resistance monitoring has been largely neglected in Italy. Herein, we investigate susceptibility of Italian Cx. pipiens populations to the pyrethroids permethrin and deltamethrin. Results from WHO-tube-bioassays revealed mortalities ranging from 14-54%, indicating high levels of resistance, in four out of 10 populations exposed to permethrin (0.75%) and of 63% in one of three populations exposed to deltamethrin (0.05%). Reduced susceptibility (mortality<98%) was detected in almost all other populations. A clear association is shown between the resistant phenotype and the presence of kdr-alleles in position 1014 of the VSSC, strongly suggesting its role in reducing susceptibility. The study provides the first evidence of pyrethroid-resistance in Italian Cx. pipiens populations and reports levels of resistance paralleled in the European region only in Turkey. This highlights the urgent need to implement insecticide-resistance management plans to restore the efficacy of the nowadays only chemical weapon available to control arbovirus transmission in Europe.

AIMSurv: First pan-European harmonized surveillance of Aedes invasive mosquito species of relevance for human vector-borne diseases.

Human and animal vector-borne diseases, particularly mosquito-borne diseases, are emerging or re-emerging worldwide. Six Aedes invasive mosquito (AIM) species were introduced to Europe since the 1970s: Aedes aegypti, Ae. albopictus, Ae. japonicus, Ae. koreicus, Ae. atropalpus and Ae. triseriatus. Here, we report the results of AIMSurv2020, the first pan-European surveillance effort for AIMs. Implemented by 42 volunteer teams from 24 countries. And presented in the form of a dataset named "AIMSurv Aedes Invasive Mosquito species harmonized surveillance in Europe. AIM-COST Action. Project ID: CA17108". AIMSurv2020 harmonizes field surveillance methodologies for sampling different AIMs life stages, frequency and minimum length of sampling period, and data reporting. Data include minimum requirements for sample types and recommended requirements for those teams with more resources. Data are published as a Darwin Core archive in the Global Biodiversity Information Facility- Spain, comprising a core file with 19,130 records (EventID) and an occurrences file with 19,743 records (OccurrenceID). AIM species recorded in AIMSurv2020 were Ae. albopictus, Ae. japonicus and Ae. koreicus, as well as native mosquito species.

A Novel Allele Specific Polymerase Chain Reaction (AS-PCR) Assay to Detect the V1016G Knockdown Resistance Mutation Confirms Its Widespread Presence in Aedes albopictus Populations from Italy.

Polymerase chain reaction (PCR)-based genotyping of mutations in the voltage-sensitive sodium channel (vssc) associated with resistance to pyrethroid insecticides is widely used and represents a potential early warning and monitoring system for insecticide resistance arising in mosquito populations, which are vectors of different human pathogens. In the secondary vector Aedes albopictus-an Asian species that has invaded and colonized the whole world, including temperate regions-sequencing of domain II of the vssc gene is still needed to detect the V1016G mutation associated with pyrethroid resistance. In this study we developed and tested a novel allele-specific PCR (AS-PCR) assay to genotype the V1016G mutation in this species and applied it to the analysis of wild populations from Italy. The results confirm the high accuracy of the novel AS-PCR and highlight frequencies of the V1016G allele as >5% in most sampling sites, with peaks of 20-45% in coastal touristic sites where pyrethroid treatments are extensively implemented, mostly for mosquito nuisance reduction. The high frequency of this mutation observed in Italian Ae. albopictus populations should serve as a warning bell, advocating for increased monitoring and management of a phenomenon which risks neutralizing the only weapon today available to counteract (risks of) arbovirus outbreaks.

Influence of Temperature on the Life-Cycle Dynamics of Aedes albopictus Population Established at Temperate Latitudes: A Laboratory Experiment.

The mosquito species Aedes albopictus has successfully colonized many areas at temperate latitudes, representing a major public health concern. As mosquito bionomics is critically affected by temperature, we experimentally investigated the influence of different constant rearing temperatures (10, 15, 25, and 30 °C) on the survival rates, fecundity, and developmental times of different life stages of Ae. albopictus using a laboratory colony established from specimens collected in northern Italy. We compared our results with previously published data obtained with subtropical populations. We found that temperate Ae. albopictus immature stages are better adapted to colder temperatures: temperate larvae were able to develop even at 10 °C and at 15 °C, larval survivorship was comparable to the one observed at warmer conditions. Nonetheless, at these lower temperatures, we did not observe any blood-feeding activity. Adult longevity and fecundity were substantially greater at 25 °C with respect to the other tested temperatures. Our findings highlight the ability of Ae. albopictus to quickly adapt to colder environments and provide new important insights on the bionomics of this species at temperate latitudes.

Geographical Distribution of Ljungan Virus in Small Mammals in Europe.

Ljungan virus (LV), which belongs to the Parechovirus genus in the Picornaviridae family, was first isolated from bank voles (Myodes glareolus) in Sweden in 1998 and proposed as a zoonotic agent. To improve knowledge of the host association and geographical distribution of LV, tissues from 1685 animals belonging to multiple rodent and insectivore species from 12 European countries were screened for LV-RNA using reverse transcriptase (RT)-PCR. In addition, we investigated how the prevalence of LV-RNA in bank voles is associated with various intrinsic and extrinsic factors. We show that LV is widespread geographically, having been detected in at least one host species in nine European countries. Twelve out of 21 species screened were LV-RNA PCR positive, including, for the first time, the red vole (Myodes rutilus) and the root or tundra vole (Alexandromys formerly Microtus oeconomus), as well as in insectivores, including the bicolored white-toothed shrew (Crocidura leucodon) and the Valais shrew (Sorex antinorii). Results indicated that bank voles are the main rodent host for this virus (overall RT-PCR prevalence: 15.2%). Linear modeling of intrinsic and extrinsic factors that could impact LV prevalence showed a concave-down relationship between body mass and LV occurrence, so that subadults had the highest LV positivity, but LV in older animals was less prevalent. Also, LV prevalence was higher in autumn and lower in spring, and the amount of precipitation recorded during the 6 months preceding the trapping date was negatively correlated with the presence of the virus. Phylogenetic analysis on the 185 base pair species-specific sequence of the 5' untranslated region identified high genetic diversity (46.5%) between 80 haplotypes, although no geographical or host-specific patterns of diversity were detected.

Tick-borne encephalitis foci in northeast Italy revealed by combined virus detection in ticks, serosurvey on goats and human cases.

Tick-borne encephalitis (TBE) is a severe zoonotic neurological disease endemic in northeast Italy since 1992. In the Province of Trento, a sharp increase in TBE incidence has been recorded since 2012, despite the vaccination efforts. To assess current TBE infection hazard in this area, we applied an integrated approach combining the distribution of human cases, the seroprevalence of tick-borne encephalitis virus (TBEV) in sentinel hosts and the screening of questing ticks for TBEV. A total of 706 goat sera from 69 farms were screened for TBEV-specific antibodies resulting in 5 positive farms, while the location of human cases was provided by the local Public Health Agency. Tick sampling was concentrated in areas where TBEV circulation was suggested by either seroprevalence in goats or human cases, resulting in 2,410 Ixodes ricinus collected and analyzed by real-time RT-PCR. Four tick samples from 2 areas with record of human cases were positive to TBEV corresponding to a 0.17% prevalence in the region, while risk areas suggested by serology on goats were not confirmed by tick screening. Our results revealed an increase in TBEV prevalence in ticks and the emergence of new active TBE foci, compared to previous surveys, and demonstrated the importance of an integrated approach for TBE risk assessment. A phylogenetic analysis of the partial E gene confirmed that the European TBEV subtype is circulating in northeast Italy and suggested that the different Italian TBEV strains originated independently as a result of different introductions from neighbouring countries, presumably through migratory birds.

First report of the influence of temperature on the bionomics and population dynamics of Aedes koreicus, a new invasive alien species in Europe.

BACKGROUND: Aedes koreicus was detected in northern Italy for the first time in 2011, and it is now well established in several areas as a new invasive mosquito species. Data regarding the influence of temperature on mosquito survival and development are not available yet for this species. METHODS: We experimentally investigated the influence of different constant rearing temperatures (between 4 and 33 °C) on the survival rates and developmental times of different life stages of Ae. koreicus under laboratory conditions. The resulting data were subsequently used to inform a mathematical model reproducing the Ae. koreicus life-cycle calibrated to counts of adult females captured in the field in the autonomous province of Trento (northern Italy) between 2016 and 2018. RESULTS: We found that temperatures above 28 °C are not optimal for the survival of pupae and adults, whereas temperate conditions of 23-28 °C seem to be very favorable, explaining the recent success of Ae. koreicus at establishing into new specific areas. Our results indicate that Ae. koreicus is less adapted to local climatic conditions compared to Ae. albopictus, another invasive species which has been invading the area for the last three decades. Warmer seasons, which are more likely to occur in the future because of climate change, might extend the breeding time and therefore increase the abundance of Ae. koreicus in the study region. CONCLUSIONS: Our findings provide, to our knowledge, the first evidence on how temperature influences the bionomics and dynamics of Ae. koreicus and highlight the need for further studies on the phenology of this species in temperate areas of Europe.

Variation in Wolbachia cidB gene, but not cidA, is associated with cytoplasmic incompatibility mod phenotype diversity in Culex pipiens.

Endosymbiotic Wolbachia bacteria are, to date, considered the most widespread symbionts in arthropods and are the cornerstone of major biological control strategies. Such a high prevalence is based on the ability of Wolbachia to manipulate their hosts' reproduction. One manipulation called cytoplasmic incompatibility (CI) is based on the death of the embryos generated by crosses between infected males and uninfected females or between individuals infected with incompatible Wolbachia strains. CI can be seen as a modification-rescue system (or mod-resc) in which paternal Wolbachia produce mod factors, inducing embryonic defects, unless the maternal Wolbachia produce compatible resc factors. Transgenic experiments in Drosophila melanogaster and Saccharomyces cerevisiae converged towards a model where the cidB Wolbachia gene is involved in the mod function while cidA is involved in the resc function. However, as cidA expression in Drosophila males was required to observe CI, it has been proposed that cidA could be involved in both resc and mod functions. A recent correlative study in natural Culex pipiens mosquito populations has revealed an association between specific cidA and cidB variations and changes in mod phenotype, also suggesting a role for both these genes in mod diversity. Here, by studying cidA and cidB genomic repertoires of individuals from newly sampled natural C. pipiens populations harbouring wPipIV strains from North Italy, we reinforce the link between cidB variation and mod phenotype variation fostering the involvement of cidB in the mod phenotype diversity. However, no association between any cidA variants or combination of cidA variants and mod phenotype variation was observed. Taken together our results in natural C. pipiens populations do not support the involvement of cidA in mod phenotype variation.

Parasites and wildlife in a changing world: The vector-host- pathogen interaction as a learning case.

In the Anthropocene context, changes in climate, land use and biodiversity are considered among the most important anthropogenic factors affecting parasites-host interaction and wildlife zoonotic diseases emergence. Transmission of vector borne pathogens are particularly sensitive to these changes due to the complexity of their cycle, where the transmission of a microparasite depends on the interaction between its vector, usually a macroparasite, and its reservoir host, in many cases represented by a wildlife vertebrate. The scope of this paper focuses on the effect of some major, fast-occurring anthropogenic changes on the vectorial capacity for tick and mosquito borne pathogens. Specifically, we review and present the latest advances regarding two emerging vector-borne viruses in Europe: Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV). In both cases, variation in vector to host ratio is critical in determining the intensity of pathogen transmission and consequently infection hazard for humans. Forecasting vector-borne disease hazard under the global change scenarios is particularly challenging, requiring long term studies based on a multidisciplinary approach in a One-Health framework.

Changes in host densities and co-feeding pattern efficiently predict tick-borne encephalitis hazard in an endemic focus in northern Italy.

Tick-borne encephalitis is an important zoonosis in many parts of north-western, central and eastern Europe, Russia and the Far East, with considerable altitudinal and latitudinal shifts described during recent decades. The reported routes of transmission for TBE virus include the saliva-activated non-viraemic transmission between co-feeding ticks taking place on rodent hosts. During the period 2001-2014, a population of the yellow-necked mouse (Apodemus flavicollis), which is considered among the most efficient TBE competent host, especially in central and western Europe, was intensively live-trapped in a known TBE focus in the Province of Trento, Italy. Individual live-trapped mice were checked for the number and position of feeding ticks and serologically screened for TBEv antibodies. A combined effect of climatic conditions and density of both roe deer and mice on the number of co-feeding tick groups was observed. Specifically, the occurrence of co-feeding ticks on mice during the questing season was affected by autumnal cooling in the previous season. On the other hand, co-feeding occurrence was also positively associated with roe deer abundance, while mouse density showed a hump-shaped pattern. Individual features of A. flavicollis such as weight and sex also affected co-feeding occurrence with the heaviest (breeding adult) males carrying more co-feeding ticks. We also found that the overall number of co-feeding ticks on mice positively affected TBEv antibody detection in this species the following year. In conclusion, a specific combination of climatic conditions in conjunction with certain rodent and roe deer densities are the principal determinants of the number of co-feeding ticks on A. flavicollis and, consequently, TBEv circulation. These variables can be used to provide an early warning signal for a TBE hazard, thus representing a useful tool for Public Health authorities to prepare action for prevention and control within TBEv circulation areas.

Investigation on potential malaria vectors (Anopheles spp.) in the Province of Trento, Italy.

BACKGROUND: Europe and Italy were declared malaria free since the 1970s although the presence of competent vectors and the high number of yearly imported malaria cases make this disease a potential rising health issue. In September 2017, a cryptic fatal case of Plasmodium falciparum malaria in the Province of Trento, Italy, raised the concern of health authorities on the possible resurgence of this disease in the Mediterranean Basin. METHODS: An entomological surveillance by means of BG traps, CDC light traps and larval search was performed. Sites were chosen among urban and suburban environments (e.g. private houses, public parks, schools, cemeteries, ecotone urban/forest, farms), ranging from an altitude of 91 to 1332 m above sea level. All the mosquitoes collected were morphologically identified and about half of them (103; 49%) were confirmed with the sequencing analysis of the rRNA internal transcribed spacer 2 (ITS-2). RESULTS: In the present study 287 sites were screened for the presence of Anopheles spp. and 211 specimens were collected and identified. Hundred-eighteen individuals (56%) belonged to Anopheles plumbeus, 56 (26.5%) to Anopheles maculipennis complex, 10 (4.7%) to Anopheles claviger and 27 were identified only at genus level. This is the first record for the presence of An. plumbeus in the study area. CONCLUSIONS: The presence of Anopheles spp. mosquitoes in the Province of Trento, Italy, has been updated with the occurrence of An. plumbeus. The risk of malaria endemicity in the area is to be considered very low, but urban and peri-urban habitat may act as potential breeding sites for the presence of mosquito vectors and should be constantly monitored.

Changes in Microbiota Across Developmental Stages of Aedes koreicus, an Invasive Mosquito Vector in Europe: Indications for Microbiota-Based Control Strategies.

Since it has been understood that gut microbiota of vector mosquitoes can influence their vector competence, efforts have been undertaken to develop new control strategies based on host microbiota manipulation, and aimed at suppressing the vector population or replacing it with a less competent one. For the proper design of such control strategies it is necessary to know the microbiota composition of the target vector species, how it is acquired, and how it changes throughout the host's life cycle. In this study, 16S rRNA amplicon high-throughput sequencing was used to characterize the changes in microbiota from the aquatic environment (larval breeding sites) to the different developmental stages of field-collected Aedes koreicus in Italy, an emerging invasive mosquito species in Europe and a potential vector of several pathogens. The bacterial communities of the aquatic breeding sites, larvae, pupae and adults showed distinctive structures to one another. Indeed, 84% of community members were unique to a given sample type. Nevertheless, almost 40% of the sequences generated were assigned to bacteria detected in all sample types, suggesting the importance of bacteria transstadially transmitted from water to the adult stage in constituting mosquito microbiota. Among these, genus C39 largely constituted water microbiota, family Burkholderiaceae was the most abundant in larvae and pupae, and genus Asaia dominated adult communities. In addition, Asaia constituted a core microbiota across all sample types. Our results suggest that the microbiota of Ae. koreicus mosquitoes is composed by a community which derives from the aquatic bacteria of the larval breeding sites, is then filtered by the larval gut, where only certain members are able to persist, rearranged by metamorphosis and finally modified by the change in diet at the adult stage. Understanding how the microbiota of Ae. koreicus changes through the mosquito life cycle represents a first step in selecting bacterial candidates for use in microbiota-based intervention measures for this species. The properties which Asaia exhibits in this species, such as dominance, high prevalence and transstadial transmission, prevent the use of Wolbachia but make Asaia an ideal candidate for paratransgenesis.

Reduced diversity of gut microbiota in two Aedes mosquitoes species in areas of recent invasion.

Aedes mosquitoes are considered highly successful global invasive species and vectors of several pathogens of relevance for public health. Their midgut's microbiota can play an important role in affecting not only their vectorial competence but also their fitness, physiology, food digestion, metabolism, immunity and adaptation to new environmental conditions. Using high-throughput sequencing we compared the microbiota of Aedes albopictus collected in Italy with those reported in populations from France and Vietnam. We also analysed Aedes koreicus gut microbiota for the first time. We found remarkable individual difference along with common bacterial taxa in both species. Ae. albopictus collected in Italy had a lower richness and a different composition of microbiota in respect to specimens collected in France and Vietnam. It also showed a core microbiota formed mainly of bacteria of the genus Pseudomonas. Overall, the two Aedes species (Ae. albopictus and Ae. koreicus) collected in Italy, showed a large core microbiota with 75.98% of the identified Operational Taxonomic Units. Furthermore, Ae. albopictus had 2.5% prevalence of Wolbachia and 0.07% of Asaia spp, while Ae. koreicus had 14.42% of Asaia spp. and no Wolbachia. This study provides new informations on the spatial variation of the midgut bacterial communities in mosquitoes of medical relevance within areas of recent invasion and provide the basis for further studies aimed at assessing the effects of such variation on vectorial capacity for a range of pathogens.

Effect of Climate and Land Use on the Spatio-Temporal Variability of Tick-Borne Bacteria in Europe.

The incidence of tick-borne diseases caused by Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. has been rising in Europe in recent decades. Early pre-assessment of acarological hazard still represents a complex challenge. The aim of this study was to model Ixodes ricinus questing nymph density and its infection rate with B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in five European countries (Italy, Germany, Czech Republic, Slovakia, Hungary) in various land cover types differing in use and anthropisation (agricultural, urban and natural) with climatic and environmental factors (Normalized Difference Vegetation Index (NDVI), Normalized Difference Water Index (NDWI), Land Surface Temperature (LST) and precipitation). We show that the relative abundance of questing nymphs was significantly associated with climatic conditions, such as higher values of NDVI recorded in the sampling period, while no differences were observed among land use categories. However, the density of infected nymphs (DIN) also depended on the pathogen considered and land use. These results contribute to a better understanding of the variation in acarological hazard for Ixodes ricinus transmitted pathogens in Central Europe and provide the basis for more focused ecological studies aimed at assessing the effect of land use in different sites on tick-host pathogens interaction.