{"paper_id":"20c7caca-1e87-4fa3-b716-da6839e85b6f","body_text":"Winter Survival of Culex pipiens f. pipiens Adults in \nCentral Greece\nCharalampos Ioannou1¶, Stavroula Beleri2¶, Persa Tserkezou3, Antonios Michaelakis4, \nEleni Patsoula2, Christos Hadjichristodoulou3, Nikos T. Papadopoulos1*\n1Department of Agriculture, Crop Production and Rural Environment, University of \nThessaly, Magnisias, Greece\n2Department of Public Health Policy, School of Public Health, University of WestAttica, \nAthens, Greece\n3Laboratory of Hygiene and Epidemiology School of Medicine, University of Thessaly\n4Scientific Directorate of Entomology and Agricultural Zoology, Benaki \nPhytopathological Institute, Attica, Greece\n*Corresponding author\n E-mail: nikopap@uth.gr(NTP)\n¶These authors contributed equally to this work\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nAbstract\nWinter survival consists a major component of insect vectors life history in temperate \nenvironments that is directly related with early and later population growth next \nseason with major consequences in the epidemiology of vectored diseases. The \ncommon European mosquito Culex pipiens is a major vector of the West Nile Virus \n(WNV) in Europe, including Greece. West Nile Virus outbreaks are frequently reported \nin Greece over the last 2 decades and Thessaly, Central Greece, is included in the \naffected areas. Here we report on overwintering trials conducted in three regions of \nThessaly to investigate the overwintering dynamics of the subspecies of the Cx. pipiens \ncomplex, Cx. pipiens f. pipiens. Two overwintering experiments regarding adults of Cx. \npipiens f. pipiens carried out in two coastal areas of Thessaly (Nea Anchialos and Volos) \nand an inland area (Kalamaki). Results demonstrated the successful overwintering of \nCx. pipiens f. pipiens females, as well as the failure of males to survive in all three \nregions considered. Successful overwintering females were capable of initiating egg \nlaying following a blood meal in spring onsetting the first summer generation. \nNonetheless, mortality patterns differ between the coastal and the inland area as well \nas among different cohorts of adults.  \nKeywords: winter survival, longevity, mosquitoes, population dynamics, vector borne \ndiseases\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nIntroduction\nIn regions with a temperate or cold climate, like Greece, mosquitoes have developed \na variety of overwintering mechanisms that, depending on the species, may include \nthe egg, larval, adult or more than one developmental stage [1-4]. Several factors, \nmost importantly low temperatures and precipitation are those that determine the \nduration of the overwintering period and may vary for a given species depending on \nlatitude [5-8]. Usually, winter temperatures in cooler temperate areas do not allow \nbreeding and population growth of mosquitoes. This fact, combined with the high \nmortality rates observed during the cold months may result in a dramatic decline in \npopulations that onset the first spring generation. From an epidemiological point of \nview, the proportion of a vector mosquito population that will successfully overwinter \nis of particular importance, as the initiation and development of the spring generation \nwill depend on it [5]. Moreover, the overwintering of mosquitoes’ vectors is important \nnot only for their population biology but also for the evolvement of associated \ndiseases [3,9]. For example, the persistence of certain pathogens in overwintering \nmosquitoes may contribute in maintaining the transmission cycle each year, rendering \nthe disease endemic [10,11].\nWest Nile virus (WNV, family: Flaviviridae) is currently the most important mosquito-\nborne pathogen spreading in Europe [12,13]. Data on overwintering of WNV in \nmosquitoes are crucial for understanding WNV circulation [14]. In temperate regions, \nmost species of mosquitoes are subject to facultative diapause initiated by a decrease \nin day length and temperature, which results in the interruption of transmission cycles \nof mosquito-borne pathogens during winter [2,9]. The mosquito Culex pipiens \n(Diptera: Culicidae) is of growing concern, as it is considered the main vector of WNV \nin Europe [6,11,15] including Greece [16-18]. This species includes two distinct forms, \nknown as “pipiens” (Linnaeus, 1758) and “ molestus” (Forskål, 1775), which can form \nhybrids [7,19,20]. The two forms are morphologically identical but display important \ndifferences in their behaviour and physiology. One of the major distinctions between \nthe biotypes is their overwintering biology [19,21-23].\nIn cold, temperate regions, the ''pipiens'' form overwinters as adult inseminated \nfemales entering into facultative reproductive diapause [2,6,24,25]. The factors \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nresponsible for the induction of female diapause are the low autumn temperatures \ncombined with reduced photophase affecting the last larval stages (3rd and 4th) and \nthe pupa [26,27]. In contrast, males do not enter diapause and do not survive the \nwinter [23,27].\nDormant females are characterized by the absence of host search for blood meals and \nfeed on “sugary” plant secretions, building up rich fat body reserves just before inhabit \noverwintering sites [27]. Feeding females with a 10% sugar solution for 7 to 10 days is \nsufficient to build up fat reserves [25]. Sites that remain frost-free during the winter \nsuch as caves, barns, underground storage facilities, channels and cracks in the ground \nare selected by adult mosquitoes as hibernating shelters. Although dormant \nindividuals of Cx. pipiens f. pipiens do not exhibit host-seeking behavior, some females \nmay be motivated and receive a blood meal when in close proximity to a host for a \nperiod of time [28], using the blood to build body fat rather than for ovarian \ndevelopment [29,30]. These blood meals are not useful to diapausing females either \nfor body fat production or for ovarian development as they were found to be \nsignificantly inferior in quantity compared to non-diapause counterparts [25]. The \nambient conditions (e.g. temperature, relative humidity) of the overwintering habitats \nmay vary a great deal and determine female survival rates. For example, in cool and \nhumid habitats with low fluctuation, females may remain at the same spot for weeks \nreserving precious stored energetic metabolites. In contrast, in more exposed to \nexternal conditions shelters, during the warmer hours of the day females may forage \nfor sugar food or seek more appropriate overwintering places and activity that can risk \nsurvival and reduce longevity [1,2,6,9,19]. As soon as temperature increases in the \nspring, females abandon overwintering sites and forage for blood meals in appropriate \nhosts that assures egg maturation and oviposition.\nIn contrast to the above, Cx. pipiens f. molestus appears to be a taxon adapted to \nwarmer climates and individuals of this form remain active during winter and can \nreproduce as long as temperatures allow (≥10 ºC) in both surface and groundwater, \nmainly in groundwater [20,31,32]. At present, Cx. pipiens s.s. biotypes are regarded as \ndistinct monophyletic evolutionary units [33-35]. As the biotype “pipiens” is usually \nfound in aboveground habitats, while the biotype “molestus” is exclusively found in \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nurban, below ground habitats, populations of both biotypes were considered \ngenetically isolated in the northern regions [23 24,34,36-38]. However, despite their \necological and behavioural differences, “pipiens” and “molestus” occur sympatrically \nabove ground in many European regions, and may interbreed to produce ‘hybrids’ \nwhere their distributions overlap [32,34,37,39]. \nWe investigated the overwintering capacity of Cx. pipiens f. pipiens in three selected \nregions of Thessaly and in various habitats. We therefore aimed to quantify survival \nthroughout the autumn and winter months, to define when they terminated diapause \nand start blood feeding in the spring and also to determine the impact of \noverwintering on vector competence of emerging Cx. pipiens mosquitoes for WNV.\nMaterials and Methods\nStudy areas and mosquito colonies\nThe overwintering experiments considering Cx. pipiens f. pipiens individuals were \ncarried out in the area of Thessaly, central Greece, where three locations were chosen: \n(a) the village of Kalamaki on the mainland, adjacent to Lake Karla, (b) the town of Nea \nAnchialos, a coastal region and (c) Volos, the coastal port city of Thessaly (Fig. 1). \nExperiment 1 carried out in Nea Anchialos and Kalamaki in 2012-2013, while \nExperiment 2 took place in Volos, aiming to study in addition the possible effect of \norganic matter (as a food source) accumulating in the waters of the shelters during \noverwintering. In particular, overwintering adults were provided with either plain \nwater (Treatment A) or plain water + organic water (a vial containing liquids/juices \ncollected from a composting bin) (Treatment B). \nThe adult mosquitoes used in the overwintering experiments came from egg rafts that \nhad been laid by laboratory-reared adults of Cx. pipiens f. pipiens. Colonization took \nplace within the insectary of the laboratory of Entomology and Agricultural Zoology at \nthe University of Thessaly, Greece.\nThe experimental procedure performed in this study included the following steps: \nhatching of Cx. pipiens eggs, development of immature stages to the adult stage, \noverwintering of adults in cages, which underwent three treatments, provision of \nblood meal to adult females and oviposition.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nExperimental procedures\nThe rafts of eggs (~70) that were collected in the laboratory were transferred to a \nplastic container with 12 L of water and artificial food (Purina Adult cat food, Friskies) \nin a protected from rain outdoor sites of the Department of Agriculture, Crop \nProtection and Rural Environment at University of Thessaly, Volos, Greece. \nDuring the development of the immature stages, both water and food were renewed \nat regular intervals, ensuring that the conditions were suitable until pupation. At the \ncompletion of development, 100 pupae, of both sexes, were transferred to plastic \ncontainers with 250 ml of water and placed in 20x20x20cm, Plexiglass cages until the \nemergence of adults. \nAdults were transferred in a heated storage room (15 ± 2 oC) with natural lighting \nconditions and offered a 10% sugar solution during the first 10-12 days of life in order \nto build up the necessary adipose tissue reserves for overwintering and mating. After \nthis period, the sugar solutions in the cages were replaced with plain water and then \nthe cages were transferred to the overwintering sites. Five cages (500 adults in total) \nwere transported to Kalamaki and Nea Anchialos on 30/12/2012 and 4/1/2013 \nrespectively, and placed in moist and dark storage areas protected from rain and wind. \nTen cages (1000 adults in total) that were placed to Volos on 1/12/2013 were \nrandomly assigned to one of the following twotreatments: (a) five cages were \nprovided with plain water (egg hatching 1/10/2013), (b) five cages with plainwater and \nplain water + organic water (egg hatching 1/10/2013). Treatment C was the same as \nTreatment A, entered later on the experimental procedure (egg hatching 15/10/2013) \nas a control. \nIn both experiments, at the end of the winter period (first two weeks of March), a 10% \nsugar solution was once again placed in the cages and then they transferred back to \nthe semi-outdoor area of the Agricultural School at the University of Thessaly. This \nprocedure took place on 7/3/2013 in Kalamaki, on 14/3/2013 in Nea Anchialos, and \non 19/3/2014 in Volos. \nThe surviving (overwintering) females from Kalamaki and Nea Anchialos, were pooled \nin two cages and offered a two-hour blood meal via a special device for this purpose, \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\non 11/4/2013. From 20/4/20213 to 22/4/2013 plastic containers with 250 ml of water \nwere placed in the two cages (one from each overwintering location) for the females \nto lay their eggs. The overwintering females in Volos were provided with a blood meal \non 23/4/2014 while the oviposition took place on 28/4/20214.\nSurvival of adults in the different treatments was recorded at regular intervals and \ndead individuals were removed from the cages. The deposited egg rafts were \nexamined under the stereoscope to determine hatch.\nMeteorological data\nThe temperatures that prevailed throughout the experimental study, from the \ndevelopment of the immature stages to the oviposition of the females that survived \nin the three locations, were recorded with the help of special electronic devices \n(HOBO, Onset, USA), and are given in the following Figs 2-4. Figures include also \ninformation regarding the exposure period of adults in the overwintering sites as well \nas feeding and oviposition opportunities.\nStatistical analysis\nThe Kaplan-Meier curves and the log-rank test was used to compare the survival times \nbetween males and females. Cox regression was employed to examine whether the \nsex of adults and the overwintering site were significant predictors of adult mortality \nrates. R version 4.3.2 (The R Foundation for Statistical Computing, Vienna, Austria) was \nused for data analysis. P values less than 0.05 were considered statistically significant.\nResults\nWinter survival of Culex pipiens f. pipiens in Kalamaki and Nea \nAnchialos\nThe average daily temperatures that prevailed in Nea Anchialos and Kalamaki during \nthe experiment, from the development of the immature stages to the oviposition of \nthe females that survived, are shown in Figs 2 and 3 respectively. In both areas \ntemperatures were rather high until end of December and the cold period (winter) \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nstarted in the beginning of January. Temperature increased in the beginning of March \nmarking the end of winter period. Although comparable, winter temperatures were \nlower in the continental area of Kalamaki compared to Nea Anchialos. A total of 450 \nadults (90 per cage; 173 females, i.e. 38.95% of exposed individuals) were transported \nand exposed to winter conditions of Kalamaki, as determined at the end of the \nexperimental procedure considering also accidental loses. The corresponding \nparameters for Nea Anchialos were 456 adults (91.2 per cages; 294 females, i.e. \n64.65% of the exposed individuals). In both sites males did not manage to survive until \nthe end of the cold season (early to mid-March) (S1 Fig). Higher survival rates for males \nwere recorded in Kalamaki compared to Nea Anchialos. In Nea Anchialos, the \nmortality of females during the winter was progressive (S1 Fig). On the contrary, the \nsurvival of females in Kalamaki was particularly high until 26/2, followed by a \nsignificant decline after that date (S1 Fig). The addition of sugar solution with the rise \nin temperatures resulted in the stabilization of female mortality in both cases. At the \nstart of oviposition (20/4) the average percentage of females that finally survived in \nKalamaki and Nea Anchialos reached 23.85% (44 individuals in total) and 25.26% (76 \nindividuals in total) respectively. Of these females, 52.27 and 47.37% oviposited \nrespectively, while 95.65% and 91.67% of the laid egg rafts hatched. \nMortality rates of males and females at Kalamaki and Nea Anchialos were compared \nwith the Kaplan Meir curves and the Cox proportion hazard model. Overall, and in \nboth locations, male longevity was shorter than that of females with males failing to \noverwinter (Fig 5; p < 0.01). The hazard ratio of males compared to females was 12.9 \n(95%CI: 9.69, 17.2) in Kalamaki and 78.9 (95%CI: 47.4, 131.0) in Nea Anchialos. \nCox regression analysis considering location of exposure, sex and their interaction as \npredictors revealed that males have a significant higher hazard rate compared to \nfemales (p < 0.001), adjusted for location, and mortality rates were higher in Kalamaki \ncompared to Nea Anchialos (p < 0.001) (Fig 6, S2 Fig). Overall hazard rates considering \nboth males and females were similar between the two locations ( p = 0.425). The \nsignificant interaction between sex and location is associated with higher mortality \nrates for males in Nea Anchialos compared to Kalamaki. Comparing survival patterns \nof females in the two location no significant differences were found (Fig 6; Log-rank \ntest, p = 0.16).  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nWinter survival of Culex pipiens f. pipiens in Volos\nThe average daily temperatures that prevailed in Volos, during the overwintering \nexperiment are shown in Fig 4. The temperature drop in the beginning of December \nmarks the onset of winter while the increase in middle March the end. The total \nnumber of adults exposed to winter conditions was 482 (average per cage 96.4; 371 \nfemales, i.e. 76.9%), 475 (average per cage 95; 349 females, i.e. 73.5%) and 509 \n(average per cage 101.8; 388 females, i.e. 76.2%) for the treatments A, B and C \nrespectively. In all three treatments male survival rates were much lower than that of \nfemales and no male survived after the end of December to reach the end of the \nwinter period. Details of female and male survival patterns in each cage are given in \nS3 Fig. Among cage variation was low in treatment A and B and quite higher in \ntreatment C. Overall, at the end of the winter period 9.42, 15.7 and 12.44% of the \nfemales managed to survive in treatments A, B and C respectively. Among cage \nvariation in male survival was minimal within the same treatment and overall survival \npatterns among treatments negligible as well (S4Fig). Comparing the survival rates of \nthe two sexes of Cx. pipiens, the ability of females (S5a Fig) to overwinter in all three \ntreatments and the inability of males (S5b Fig) to survive was evident. In all three \ntreatments provision of sugar solution on 16/3/2014 reduced mortality rates. At the \nstart of oviposition (28/4) in treatment A, treatment B and treatment C, the number \nof females alive was 35, 53, and 48, of which 13, 25, and 10 oviposited, with the \noviposition rate reaching 37.14, 47.17, and 20.83% respectively.\nKaplan Meir analysis followed by the long-rank test revealed the higher mortality of \nmales compared to that of females within each treatment (Fig 7, p < 0.01). Cox \nregression analysis including treatment, sex and their interaction as predictors \nconfirmed the overall higher hazard rates for males compared to females ( p < 0.001) \nand differences among the three treatments (Fig 8). The hazard ratio of treatment B \nand C compared to baseline A was slightly but significantly lower, and higher \nrespectively (Fig 8; p < 0.05). The interaction between sex and treatment was not \nsignificant. Kaplan Meir analysis comparing survival patterns of the three female \ngroups followed by the log-rank test revealed significant differences among the three \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\ntreatments (p < 0.01, Fig 7). Nevertheless, the proportion of surviving females at the \nend of the exposure period was similar among the three treatments (chi-square test, \np > 0.05).\nDiscussion\nThe overwintering experiments of Cx. pipiens f. pipiens were carried out somewhat \nlate in relation to the development of winter, but they are nevertheless informative \nof the survival of this species during the winter months in Central Greece. The \nsuccessful females overwintering at all three regions, where our experiments were \nconducted, demonstrated that a significant proportion of the population was capable \nof surviving until next spring, constituting a remarkable basis for rapid growth of the \nspecies once temperatures allow. However, of particular interest is the fact that a \nproportion of the survived females managed to oviposit. To the contrary the males \nfailed to survive until the end of the cold season, but they did live longer in Kalamaki \nthan in Nea Anchialos. Given that males do not accumulate fat reserves, these \ndifferences are probably due to reduced metabolism due to the lower temperatures \nin Kalamaki, the mainland village, compared to Nea Anchialos, the coastal area. The \npresence of male Culex mosquitoes in early spring, given that male mosquitoes do not \nhibernate, is an indicator of when the first generation of mosquitoes, produced by \npost-diapause female Culex, achieves reproductive maturity [5].\nThe accurate prediction of WNV seasonal transmission cycles and the evaluation of \nthe effectiveness of mosquito surveillance and control can be achieved by improving \nour understanding and knowledge of the initiation and termination of Culex diapause \n[5]. The two biotypes can hybridize, and hybrids show intermediate behaviour. Due to \ntheir more opportunistic feeding behaviour, hybrids are considered important bridge \nvectors which can transmit WNV from birds to humans [40-42]. In Europe the \nbehavioural differences between the biotypes of Cx. pipiens may have an impact on \ntheir contribution to the WNV transmission cycle, therefore, it is essential to \ndistinguish between biotypes when investigating the role of Cx. pipiens in WNV \ntransmission [13,35]. In Germany, winter survival of WNV in vectors has been \nconfirmed, suggesting its long-term persistence, as it has been detected several times \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nin mosquitoes of the Cx. pipiens complex during the transmission period, and also in \nhibernating females of this complex in winter [38].\nIt is of vital importance for the survival of mosquitoes to select suitable hibernation \nsites, in which the microclimate can clearly have an impact on the winter survival of \nCx. pipiens f. pipiens. The temperatures that are considered optimal for overwintering \nof adults are between 2 and 6°C [6]. Temperatures lower than 0°C can cause death \nafter several days, whereas higher temperatures increase metabolic rates, which can \nresult in the depletion of lipid reserves in female Cx. pipiens f. pipiens. For a better \nunderstanding of the dynamics of mosquito populations after winter and the way in \nwhich arboviruses survive in temperate regions, it would be of interest to explore \nwhich are the preferred hibernation sites and the determinants of winter survival in \nthese sites [9,43]. Temperature plays an important role in whether mosquito vectors \ncan overwinter in a given area, thus facilitating their establishment in new areas [44], \nwhereas temperature variation can also affect disease transmission [45]. On the other \nhand, regarding the longevity of populations, Ciota et al. observed that the longevity \nof field populations tended to be longer than that of laboratory populations [46]. \nUnder simulated field conditions, Abouzied noticed that female Cx. pipiens \nmosquitoes, in the winter/spring season survived for an average of 120 days, while in \nthe summer/autumn season they survived for an average of 80 days, significantly \nexceeding the relative constant temperatures [47]. Data from the study of Spanoudis \net al. indicated that certain biological parameters of Cx. pipiens f. molestus differ when \nmeasured at constant and fluctuating temperatures, highlighting the importance of \ntesting fluctuating temperatures that simulate field conditions [48].\nUnderstanding the environmental determinants of Cx. pipiens diversity is important \nbecause it can help us predict changes under future climate and land use regimes [49]. \nThe longevity of adult Cx. pipiens has a negative correlation with temperature within \nthe upper and lower survival limits. Warmer winters may expand the latitudinal zone \nwithin which “molestus” can survive aboveground, increasing hybridization between \necotypes in northern areas where they currently remain distinct. Such changes would \nbe invisible at the morphological level, but nevertheless have potential consequences \nfor disease transmission [37]. Climate change is considered to be a significant key \nfactor that contributes to the global spread of mosquito-borne diseases. The rise of \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nthe median winter temperatures has been shown to extend host seeking and \noviposition behavior in Cx. pipiens favouring overwintering of the virus in contrast to \nprevious decades. Lower annual average winter and higher spring and summer \ntemperatures have been linked to an increased risk of epidemic outbreaks [50,51]. \nMosquito populations employ a diversity of overwintering mechanisms to better to \nwithstand the consequences of climate change, such as sudden periods of unusually \ncold weather and decreased winter rainfall [51].\nIn the future, WNV and other flaviviruses that have significantly extended their \ndistribution are predicted to become an increasing burden for public health systems \n[50]. The overwintering strategies could be used to design control approaches during \nthe winter, as they would prevent the occurrence of high mosquito population in \nsummer. A new approach to effectively control mosquitoes before they are capable \nof transmitting deadly pathogens to birds, humans and other animals, could \npotentially be provided by targeted pesticide applications in early spring [21,43]. The \nhibernating adults' sites are of high importance and should be seriously examined. \nMicroclimates encountered in urban areas (subways, houses) have often higher and \nmore stable temperatures than outdoor environments [10]. Τhe experiment of Beleri \net al. on the winter survival of adult Ae. albopictus in human made shelters, in Athens, \nGreece, revealed the importance of elaborating more on gaining further insights into \nwinter survival of female mosquitoes [52]. Controlling the mosquito vector is an \nimportant control approach that should be adapted to the local vector ecology, \nconsidering the climatic conditions of microhabitats of overwintering vectors in urban \nareas [53-55].\nThe results of the current study could provide the basis for further research on the \noverwintering of mosquitoes in our country, covering a wider range of areas with \ndifferent climatic conditions. Winter survival studies of native and invasive mosquito \nof medical significance will have specific importance for mitigating the disease and \nnuisance burdens caused by these mosquitoes. In northern temperate climates \nmosquitoes' ability to successfully overwinter is to a large extent due to their ability \nto diapause [36]. A main part of these investigations should focus on areas where \ndisease outbreaks occur repeatedly, with special emphasis on the detection of the \nWNV in overwintering mosquitoes of the genus Culex in order to elucidate the effect \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\nof this parameter on the maintenance of the infection each year and to mitigate the \nspread of the virus and reduce the impact of the upcoming epidemic. \nSupporting information\nS1 Fig. Winter survival of Culex pipiens f. pipiens in Kalamaki (a mainland village) (A) \nand in Nea Anchialos (a coastal area) (B), Thessaly, 2012-2013.\nS2 Fig. Kaplan Meier curves (with 95%CI) including log-rank test (shown p-value) for \nthe survival of females in Nea Anchialos and Kalamaki. Black dashed lines represent \nthe median survival for females in Nea Anchialos (66 days) and females in Kalamaki \n(67 days).\nS3 Fig. Survival rate of Culex pipiens f. pipiens female adults regarding treatment 1 (a), \ntreatment 2 (b), and treatment 3 (c), in each cage, in Volos, Thessaly, 2013-2014.\nS4 Fig. Survival rate of Culex pipiens f. pipiensmale adults regarding treatment 1 (a), \ntreatment 2 (b), and treatment 3 (c), in each cage, in Volos, Thessaly, 2013-2014.\nS5 Fig. Survival rate of Culex pipiens f. pipiens adults (a) females, (b) males, regarding \nthe three treatments in Volos, Thessaly, 2013-2014.\nAcknowledgements\nAuthor Contributions\nC.I., C.H and N.T.P. conceived the study; N.T.P. and C.I. designed the experiments; C.I \nand P.T. collected the data, S.B., C.I. and E.V. analyzed the data; S.B., A.M., E.P., and \nN.T.P. wrote the first draft of the manuscript; N.T.P, C.H., C.I., S.B., E.P. and A.M. edited \nthe manuscript. All authors reviewed the manuscript. C.H., E.P. A.M. and N.T.P \nacquired funding.\nFunding\nThis study was supported by the MALWEST project and Mosquito surveillance project \nsupported by the EO DY. \nReferences\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.06.622242doi: bioRxiv preprint \n\n1. BeckerN, Petrić D, Zgomba M. Mosquitoes-identification, ecology and control, \n3rd edn. Springer, Cham, Switzerland, 2020.\n2. DenlingerDL, Armbruster PA. Molecular physiology of mosquito diapause. Adv. \nInsect Phys. 2016; 51, 329–361.\n3. 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