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BackgroundIn 2007, the first outbreak of Chikungunya in Italy generated great alarm, highlighting the health risks caused by exotic species recently introduced in Europe and the need to strengthen control actions against the vectors. Besides health risks, mosquitoes cause nuisance, and citizens are required to adopt control measures. While the economic aspects of mosquito control by public agencies have been investigated, the scientific literature on the costs of mosquito protection incurred by families is scarce. This study assessed the households' expenditure on protective measures against mosquitoes in Emilia-Romagna, a region in Northern Italy.Methodology/principal findingsA phone questionnaire survey was conducted to collect data on the annual expenditure incurred by households for self-protection against mosquitos in relation to the perceived level of nuisance and the household and dwelling characteristics. Univariate and multivariate analyses were conducted to identify the main determinants influencing such expenditure, which resulted affected by dwelling characteristics, presence of children under 6 years of age, and health concerns of family members. The average annual household expenditure was estimated at 84.63 euros, about 30 times higher than the expenditure per household supported by regional and local administrations for interventions against mosquitoes in public areas, as calculated in a previous study.Conclusion/significanceHousehold expenditure is mainly aimed at providing a direct defense against mosquito bites (mosquito nets, adulticides, skin-on repellents, etc.) while spending for more effective measures addressed to reduce mosquito density results marginal: e.g., only 3.5% of the total expenditure was dedicated to larval control. Control activities that lower the mosquito density in both private and public areas could reduce the use of household insecticides in urban environments and the related costs, and the risk of spread of imported arboviruses as well.

The "Sterile Insect Technique" (SIT), a promising method to control Aedes albopictus, the Asian tiger mosquito, is gaining increasing interest. Recently, the role of microbiota in mosquito fitness received attention, but the link between microbiota and larval diet in mass rearing programs for SIT remains largely unexplored. We characterized the microbiota of four larval instars, pupae and eggs of non-wild (NW) lab-reared Ae. albopictus fed with a diet based on Black soldier fly (Hermetia illucens) larvae powder and fish food KOI pellets. We compared it with wild (W) field-collected individuals and the bacterial community occurring in rearing water-diet (DIET). A total of 18 bacterial classes with > 0.10% abundance were found overall in the samples, with seven classes being especially abundant. Overall, the microbiota profile significantly differed among NW, W and DIET. Verrucomicrobiae were significantly more abundant in W and DIET, Bacteroidia were more abundant in NW and DIET, and Gammaproteobacteria were only more abundant in W than in DIET. W-eggs microbiota differed from all the other groups. Large differences also appeared at the bacterial genus-level, with the abundance of 14 genera differing among groups. Three ASVs of Acinetobacter, known to have positive effects on tiger mosquitoes, were more abundant in NW than in W, while Serratia, known to have negative or neutral effects on another Aedes species, was less abundant in NW than in W. The bacterial community of W-eggs was the richest in species, while dominance and diversity did not differ among groups. Our data show that the diet based on Black soldier fly powder and fish food KOI influences the microbiota of NW tiger mosquito immature stages, but not in a way that may suggest a negative impact on their quality in SIT programs.

In the last decades, the colonization of Mediterranean Europe and of other temperate regions by Aedes albopictus created an unprecedented nuisance problem in highly infested areas and new public health threats due to the vector competence of the species. The Sterile Insect Technique (SIT) and the Incompatible Insect Technique (IIT) are insecticide-free mosquito-control methods, relying on mass release of irradiated/manipulated males, able to complement existing and only partially effective control tools. The validation of these approaches in the field requires appropriate experimental settings, possibly isolated to avoid mosquito immigration from other infested areas, and preliminary ecological and entomological data. We carried out a 4-year study in the island of Procida (Gulf of Naples, Italy) in strict collaboration with local administrators and citizens to estimate the temporal dynamics, spatial distribution, and population size of Ae. albopictus and the dispersal and survival of irradiated males. We applied ovitrap monitoring, geo-spatial analyses, mark-release-recapture technique, and a citizen-science approach. Results allow to predict the seasonal (from April to October, with peaks of 928-9,757 males/ha) and spatial distribution of the species, highlighting the capacity of Ae. albopictus population of Procida to colonize and maintain high frequencies in urban as well as in sylvatic inhabited environments. Irradiated males shown limited ability to disperse (mean daily distance travelled

BACKGROUND:Diflubenzuron (DFB) is one of the most used insecticides in mosquito larval control including that of Culex pipiens, the proven vector of the recent West Nile Virus epidemics in Europe. Two mutations (I1043L and I1043M) in the chitin synthase (CHS) putative binding site of DFB have been previously reported in Cx. pipiens from Italy and associated with high levels of resistance against this larvicide. METHODOLOGY/PRINCIPAL FINDINGS:Here we report the identification of a third mutation at the same I1043 position of the CHS gene resulting in the substitution of Isoleucine to Phenylalanine (I1043F). This mutation has also been found in agricultural pests and has been functionally validated with genome editing in Drosophila, showing to confer striking levels (>15,000 fold) of DFB resistance. The frequency of the I1043F mutation was found to be substantially higher in Cx. pipiens mosquitoes surviving DFB doses largely exceeding the recommended field dose, raising concerns about the future efficient use of this insecticide. We monitored the presence and frequency of DFB mutations in Cx. pipiens mosquitoes from several Mediterranean countries, including Italy, France, Greece, Portugal and Israel. Among the Cx. pipiens populations collected in Northern Italy all but one had at least one of the three DFB mutations at allele frequencies reaching 93.3% for the I1043M, 64.8% for the I1043L and 10% for the I1043F. The newly reported I1043F mutation was also identified in two heterozygote individuals from France (4.2% allelic frequency). In contrast to Italy and France, no DFB resistant mutations were identified in the Cx. pipiens mosquitoes sampled from Greece, Portugal and Israel. CONCLUSIONS/SIGNIFICANCE:The findings of our study are of major concern for mosquito control programs in Europe, that rely on the use of a limited number of available larvicides, and highlight the necessity for the development of appropriate Insecticide Resistance Management (IRM) programs, to ensure the sustainable use of DFB.

BackgroundWest Nile virus (WNV) transmission was much greater in 2018 than in previous seasons in Europe. Focusing on Emilia-Romagna region (northern Italy), we analyzed detailed entomological and epidemiological data collected in 2013-2018 to quantitatively assess environmental drivers of transmission and explore hypotheses to better understand why the 2018 epidemiological season was substantially different than the previous seasons. In particular, in 2018 WNV was detected at least two weeks before the observed circulation in 2013-2017 and in a larger number of mosquito pools. Transmission resulted in 100 neuroinvasive human cases in the region, more than the total number of cases recorded between 2013 and 2017.MethodologyWe used temperature-driven mathematical models calibrated through a Bayesian approach to simulate mosquito population dynamics and WNV infection rates in the avian population. We then estimated the human transmission risk as the probability, for a person living in the study area, of being bitten by an infectious mosquito in a given week. Finally, we translated such risk into reported WNV human infections.Principal findingsThe estimated prevalence of WNV in the mosquito and avian populations were significantly higher in 2018 with respect to 2013-2017 seasons, especially in the eastern part of the region. Furthermore, peak avian prevalence was estimated to have occurred earlier, corresponding to a steeper decline towards the end of summer. The high mosquito prevalence resulted in a much greater predicted risk for human transmission in 2018, which was estimated to be up to eight times higher than previous seasons. We hypothesized, on the basis of our modelling results, that such greater WNV circulation might be partially explained by exceptionally high spring temperatures, which have likely helped to amplify WNV transmission at the beginning of the 2018 season.

Cannibalism is a commonly observed phenomenon in arthropod species having relevant consequences for population dynamics and individual fitness. It is a context-dependent behaviour and an understanding of the factors affecting cannibalism rate is crucial to highlight its ecological relevance. In mosquitoes, cannibalism between larval stages has been widely documented, and the role of density, food availability and length of contact between individuals also ascertained. However, although mosquitoes can develop in temporary water habitats with very heterogeneous topologies, the role of the site shape where cannibals and victims co-occur has been instead overlooked. In this paper, we investigated this issue by using a simulation approach and laboratory cannibalism experiments between old (third- and fourth-instars) and young (first-instar) larvae of the tiger mosquito Aedes albopictus. Three virtual spaces with different shapes were simulated and the number of larval encounters was estimated in each one to assess whether the spatial shape affected the number of encounters between cannibal and victims. Then, experimental trials in containers with similar shapes to those used in the simulations were performed, and the cannibalism rate was estimated at 24 and 48h. Our results showed that the spatial shape plays a role on cannibalism interactions, affecting the number of encounters between individuals. Indeed, in the experimental trials performed, we observed the highest cannibalism rate in the container with the highest number of encounters predicted by the simulations. Interestingly, we found also that spatial shape can affect cannibalism not only by affecting the number of encounters, but also the number of encounters "favorable" for cannibalistic events. Temporary waters are inhabited by several species other than mosquitoes. Our results, showing an influence of the spatial shape on cannibalism in Ae. albopictus larvae, add a new critical factor to those affecting ecological interactions in these habitats.

The capacity of the released sterile males to survive, disperse, compete with wild males and inseminate wild females is an essential prerequisite to be evaluated in any area-wide integrated pest management (AW-IPM) programs including a sterile insect release method. Adequate quality control tests supported by standardized procedures need to be developed to measure these parameters and to identify and correct potential inappropriate rearing or handling methods affecting the overall male quality. In this study, we report results on the creation and validation of the first quality control devices designed to infer the survival and mating capacity of radio-sterilized Aedes albopictus males through the observation of their flight capacity under restricted conditions (flight organ device) and after stress treatment (aspirator device). Results obtained consistently indicate comparable flight capacity and quality parameters between untreated and 35 Gy irradiated males while a negative impact was observed with higher radiation doses at all observation time performed. The male flight capacity registered with the proposed quality control devices can be successfully employed, with different predictive capacities and response time, to infer the adult male quality. These simple and cost-effective tools provide a valuable method to detect and amend potentially sub-standard procedures in the sterile male production line and hence contribute to maintaining optimal quality and field performance of the mosquitoes being released.

Pathogens transmitted to humans by phlebotomine sand flies are neglected, as they cause infectious diseases that are not on the priority list of national and international public health systems. However, the infections caused by protozoa of the Leishmania genus and viruses belonging to the Phlebovirus genus (family Phenuiviridae)-the most significant group of viruses transmitted by sand flies-have a relevant role for human pathology. These infections are emerging in the Mediterranean region and will likely spread in forthcoming decades, posing a complex threat to human health. Four species and 2 hybrid strains of Leishmania are pathogenic for humans in the Mediterranean Basin, with an estimated annual incidence of 239,500-393,600 cases of cutaneous leishmaniasis and 1,200-2,000 cases of visceral leishmaniasis. Among the phleboviruses, Toscana virus can cause neuroinvasive infections, while other phleboviruses are responsible for a typical "3-day fever"; the actual incidence of Phlebovirus infections in the Mediterranean area is unknown, although at least 250 million people are exposed. Here, we reviewed the current literature on epidemiology of sand fly-borne infections in the Mediterranean Basin, with a focus on humans. Our analysis indicates the need for increased public health activities directed to determine the disease burden of these infections as well as to improve their surveillance. Among the emerging challenges concerning sand fly-borne pathogens, the relationships between sand fly-borne protozoa and viruses should be considered in future studies, including epidemiological links between Leishmania and phleboviruses as well as the conditional capacity for these pathogens to be involved in interactions that may evolve towards increased virulence.

Since 2013 in Emilia-Romagna, Italy, surveillance information generated in the public health and in the animal health sectors has been shared and used to guide public health interventions to mitigate the risk of West Nile virus (WNV) transmission via blood transfusion. The objective of the current study was to identify and estimate the costs and benefits associated with this One Health surveillance approach, and to compare it to an approach that does not integrate animal health information in blood donations safety policy (uni-sectoral scenario). Costs of human, animal, and entomological surveillance, sharing of information, and triggered interventions were estimated. Benefits were quantified as the averted costs of potential human cases of WNV neuroinvasive disease associated to infected blood transfusion. In the 2009-2015 period, the One Health approach was estimated to represent a cost saving of €160,921 compared to the uni-sectoral scenario. Blood donation screening was the main cost for both scenarios. The One Health approach further allowed savings of €1.21 million in terms of avoided tests on blood units. Benefits of the One Health approach due to short-term costs of hospitalization and compensation for transfusion-associated disease potentially avoided, were estimated to range from €0 to €2.98 million according to the probability of developing WNV neuroinvasive disease after receiving an infected blood transfusion.

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.

The mosquito larval rearing unit developed at the Insect Pest Control Laboratory (IPCL) of the FAO/IAEA Joint Division was evaluated for its potential use for Aedes albopictus (Skuse, 1895) mass rearing in support of the development of a sterile insect technique (SIT) package for this species. The use of the mass rearing trays and rack did not adversely affect larval development, pupation and survival rates and allowed the management of large larval rearing colonies with reduced space requirements in comparison with classical individual trays. The effects of larval density, water temperature and diet composition on pupal production and size differentiation for sex separation efficacy were analyzed for individual mass rearing trays as well as multiple trays stacked within the dedicated rack unit. Best results were obtained using eighteen thousand larvae per tray at a density of 3 larvae per ml of deionized water at a temperature of 28°C on a diet consisting of 50% tuna meal, 36% bovine liver powder, 14% brewer's yeast and, as an additive, 0.2 gr of Vitamin Mix per 100 ml of diet solution. Pupae were harvested on the sixth day from larval introduction at L1 stage and males were separated out by the use of a 1400 µm sieve with 99.0% accuracy with a recovery rate of ca. 25% of the total available males. With the use of this larval rearing unit, an average production of 100,000 male pupae per week can be achieved in just 2 square meter of laboratory space. Compared to previous laboratory rearing method, the same pupal production and sex separation efficacy could only be achieved by use of ca. 200 plastic trays which required the space of two 5 square meter climatic-controlled rooms.

BACKGROUND: The circulation of West Nile virus and Usutu virus was detected in the Emilia-Romagna region in 2008 and 2009. To evaluate the extent of circulation of both viruses, environmental surveillance, based on bird and mosquito testing, was conducted in 2008 and gradually improved over the years. METHODS: In February-March 2009-2011, 5,993 hibernating mosquitoes were manually sampled, out of which 80.1% were Culex pipiens; none tested positive for the viruses. From 2008 to 2011, 946,213 mosquitoes, sampled between May and October, were tested; 86.5% were Cx. pipiens. West Nile virus was detected in 32 Cx. pipiens pools, and Usutu virus was detected in 229 mosquito pools (217 Cx. pipiens, 10 Aedes albopictus, one Anopheles maculipennis s.l., and one Aedes caspius). From 2009 to 2011, of 4,546 birds collected, 42 tested positive for West Nile virus and 48 for Usutu virus. West Nile virus and Usutu virus showed different patterns of activity during the 2008-2011 surveillance period. West Nile virus was detected in 2008, 2009, and 2010, but not in 2011. Usutu virus, however, was continuously active throughout 2009, 2010, and 2011. CONCLUSIONS: The data strongly suggest that both viruses overwinter in the surveyed area rather than being continually reintroduced every season. The lack of hibernating mosquitoes testing positive for the viruses and the presence of positive birds sampled early in the season support the hypothesis that the viruses overwinter in birds rather than in mosquitoes. Herd immunity in key bird species could explain the decline of West Nile virus observed in 2011, while the persistence of Usutu virus may be explained by not yet identified reservoirs. Reported results are comparable with a peri-Mediterranean circulation of the West Nile virus lineage 1 related strain, which became undetectable in the environment after two to three years of obvious circulation.

BackgroundThe tiger mosquito, Aedes albopictus, is one of the 100 most invasive species in the world and a vector of human diseases. In the last 30 years, it has spread from its native range in East Asia to Africa, Europe, and the Americas. Although this modern invasion has been the focus of many studies, the history of the species' native populations remains poorly understood. Here, we aimed to assess the role of Pleistocene climatic changes in shaping the current distribution of the species in its native range.Methodology/principal findingsWe investigated the phylogeography, historical demography, and species distribution of Ae. albopictus native populations at the Last Glacial Maximum (LGM). Individuals from 16 localities from East Asia were analyzed for sequence variation at two mitochondrial genes. No phylogeographic structure was observed across the study area. Demographic analyses showed a signature of population expansion that started roughly 70,000 years BP. The occurrence of a continuous and climatically suitable area comprising Southeast China, Indochinese Peninsula, and Sundaland during LGM was indicated by species distribution modelling.Conclusions/significanceOur results suggest an evolutionary scenario in which, during the last glacial phase, Ae. albopictus did not experience a fragmentation phase but rather persisted in interconnected populations and experienced demographic growth. The wide ecological flexibility of the species probably played a crucial role in its response to glacial-induced environmental changes. Currently, there is little information on the impact of Pleistocene climatic changes on animal species in East Asia. Most of the studies focused on forest-associated species and suggested cycles of glacial fragmentation and post-glacial expansion. The case of Ae. albopictus, which exhibits a pattern not previously observed in the study area, adds an important piece to our understanding of the Pleistocene history of East Asian biota.

We investigated the impact of CHIKV strains on some Aedes albopictus (Skuse) reproductive parameters and the possibility of vertical transmission. Two strains were collected in the area where the epidemic occurred in 2007, one isolated from mosquitoes, the other one isolated from a viraemic patient. Different types of blood meals, either infected or non-infected, were offered to Ae. albopictus females, that were then analyzed at increasing time post infection. The virus titre, measured by two RT-PCR methods in the blood meals, influenced the rate of infection and the rate of dissemination of CHIKV in Ae. albopictus body. We found individual variability with respect to the infection/dissemination rates and their latency both considering the female's body and appendages. The hatching rate was significantly lower for the eggs laid by the infected females than for the control eggs, while the mortality during the larval development (from first instar larva to adult emergence) was similar among the progeny of infected and non-infected female groups. Our findings seem to support the hypothesis that the vertical transmission is a rare event under our conditions, and that a certain time period is required in order to get the ovarioles infected. Field observations conducted during the Spring 2008 showed no evidence of the presence of infected overwintering progeny produced by Ae. albopictus females infected during the 2007 outbreak.

In 2008, after the first West Nile virus (WNV) detection in the Emilia-Romagna region, a surveillance system, including mosquito- and bird-based surveillance, was established to evaluate the virus presence. Surveillance was improved in following years by extending the monitoring to larger areas and increasing the numbers of mosquitoes and birds tested.A network of mosquito traps, evenly distributed and regularly activated, was set up within the surveyed area. A total of 438,558 mosquitoes, grouped in 3,111 pools and 1,276 birds (1,130 actively sampled and 146 from passive surveillance), were tested by biomolecular analysis. The survey detected WNV in 3 Culex pipiens pools while Usutu virus (USUV) was found in 89 Cx. pipiens pools and in 2 Aedes albopictus pools. Two birds were WNV-positive and 12 were USUV-positive. Furthermore, 30 human cases of acute meningoencephalitis, possibly caused by WNV or USUV, were evaluated for both viruses and 1,053 blood bags were tested for WNV, without any positive result.Despite not finding symptomatic human WNV infections during 2010, the persistence of the virus, probably due to overwintering, was confirmed through viral circulation in mosquitoes and birds, as well as for USUV. In 2010, circulation of the two viruses was lower and more delayed than in 2009, but this decrease was not explained by the relative abundance of Cx. pipiens mosquito, which was greater in 2010. The USUV detection in mosquito species confirms the role of Cx. pipiens as the main vector and the possible involvement of Ae. albopictus in the virus cycle. The effects of meteorological conditions on the presence of USUV-positive mosquito pools were considered finding an association with drought conditions and a wide temperature range. The output produced by the surveillance system demonstrated its usefulness and reliability in terms of planning public health policies.

BACKGROUND: In recent years human diseases due to mosquito-borne viruses were increasingly reported in Emilia-Romagna region (Italy), from the chikungunya virus in 2007 to the West Nile virus (WNV) in 2008. An extensive entomological survey was performed in 2009 to establish the presence and distribution of mosquito arboviruses in this region, with particular reference to flaviviruses. METHODOLOGY/PRINCIPAL FINDINGS: From May 6 to October 31, a total of 190,516 mosquitoes were sampled in georeferenced stations, grouped in 1,789 pools according date of collection, location, and species, and analyzed by reverse transcription polymerase chain reaction (RT-PCR) to detect the presence of RNA belong to Flavivirus genus. WNV was detected in 27 mosquito pools, producing sequences similar to those of birds and human strains obtained in 2008 outbreak, pointed out the probable virus overwintering. Isolation of WNV was achieved from one of these pools. Moreover 56 pools of mosquitoes tested positive for Usutu virus (USUV). Most PCR positive pools consisted of Culex pipiens, which also was the most analyzed mosquito species (81.4% of specimens); interestingly, USUV RNA was also found in two Aedes albopictus mosquito pools. Simultaneous circulation of WNV and USUV in the survey area was highlighted by occurrence of 8 mosquito WNV- and USUV-positive pools and by the overlaying of the viruses "hot spots", obtained by kernel density estimation (KDE) analysis. Land use of sampled stations pointed out a higher proportion of WNV-positive Cx. pipiens pool in rural environments respect the provenience of total sampled pool, while the USUV-positive pools were uniformly captured in the different environments. CONCLUSIONS/SIGNIFICANCE: Obtained data highlighting the possible role of Cx. pipiens mosquito as the main vector for WNV and USUV in Northern Italy, and the possible involvement of Ae. albopictus mosquito in USUV cycle. The described mosquito-based surveillance could constitute the foundation for a public health alert system targeting mosquito borne arboviruses.