Assessment of climate change impact on the malaria vector Anopheles hyrcanus, West Nile disease, and incidence of melanoma in the Vojvodina Province (Serbia) using data from a regional climate model

of sites invaded (positive places where it was looked for, but was not found in the preceding sampling period, and the number of sites where was observed in both periods, i.e. established); and iii) the average number of specimens sampled in one trap during single sampling period from the afternoon of the starting day to the morning of the next day (Fig 2A and 2C). Here, we used data from 1,073 mosquito samples (1985–6, 2004–5 and 2014–5), obtained at 54 loca- tion over six years (142 unique location-year combinations) (S2 Table). For Cx. pipiens, the period starting with the first detection of WNV in mosquitoes in Serbia, in 2010 [26], to 2015 was considered. To investigate the impact of microclimate on the com- plex interaction between Cx. pipiens and WNV, we used the following climatic parameters from the EBU-POM model outputs (covering the period 2006–2015) for 11 sites (GPS coordi- nates–S3 Table) in the VPS with different histories of WNV circulation: (i) mean annual tem- perature (Ta); (ii) overwintering temperature (TOA) for the period October–April; (iii) seasonal temperature (TMS) for the period May–September; and (iv) relative humidity (RMS) for the period May–September. For these sites, we examined the correlation between the fre- quency of WNV detections in Cx. pipiens at each site (from 2010 to 2015) and the correspond- ing period averages of climate time series for the same site. For detection of WNV, specimens were sampled, anaesthetised by dry ice, identified to species level [27] on dry ice-cooled paper,

pooled according to date, location, sex, and species, transported on dry ice to the laboratory, and stored at -70˚C before virus detection. Pool size did not exceed 50 mosquito specimens per pool. Mosquito pools were tested for WNV RNA presence by TaqMan-based one-step reverse transcription real-time PCR (RT-qPCR) that amplified both lineage 1 and 2 strains. Viral RNA was extracted using the commercial ISOLATE II RNA Mini Kit (Bioline, The Neth- erlands) according to the manufacturer’s instruction. One-step RT-qPCR was conducted Fig 2. (a) The regional climate model EBU-POM projection of the mean annual air temperature (Ta) for the period 1985–2030 and: i) number of specimens sampled in one trap during single sampling period (blue columns); ii) the number of sites invaded by An. hyrcanus (light blue columns); and iii) relative number of positive samplings per year (dark blue columns), (b) projected increase in the number of sites invaded by An. hyrcanus (the period 2001–2030 ±S.E.), and (c) projected increase in the number of the specimens sampled in one trap during single sampling period (2001–2030 ±S.E.). https://doi.org/10.1371/journal.pone.0227679.g002 Impact of climate change on malaria vector, West Nile disease, and incidence of melanoma PLOS ONE | https://doi.org/10.1371/journal.pone.0227679 January 15, 2020 5 / 17