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

Study area

The study area was located in Northeastern Italy (Fig. 2). Specifically, it encompassed 13 municipalities in the Valbelluna (located in Belluno Province), Valsugana, and Cembra valleys (located in Trento Province). The study area has a sub-continental, temperate climate, with cold, often snowy winters and warm, mild summers. Human settlements consist mainly of small villages composed of country houses with private gardens and public parks, all surrounded by forested areas; among the sampled municipalities, only Belluno and Feltre had more than 10,000 inhabitants.

Host survey

The presence and abundance of domestic animal hosts in each site were estimated through a door-to-door census. As the flight range of Ae. koreicus is unknown, a field inspection was performed within a 200-m radius of the sampling site, corresponding to the average flight distance of Ae. albopictus recorded in a study conducted in Italy⁴⁶. The survey was carried out once in 2020. Residents were asked if they owned animals (dogs, cats, farm animals) and how many they had or, where possible, they were counted directly by the study team (visual inspection). The presence of wild ungulates was estimated according to data provided by the Forestry and Fauna Service—Wildlife Office of the Autonomous Province of Trento. The wild ungulate census was carried out in spring by visual inspection along transects, and repeated three times by hunters and personnel of the wildlife management provincial office.⁴⁷. The average number of roe deer, red deer, and chamois in 2020 was considered for the analyses. Collected information was used to qualitatively estimate potential host availability in the sampling areas. Human population density in the areas surrounding the sampling point was estimated using the Global Human Settlement Database (GHS Data)⁴⁸.

Collection of Aedes koreicus and blood meal analysis

Sampling was carried out from 2013 to 2020 (from May to October) with different frequencies in the various years; most collections were made in 2020 (20 collections) and just one in 2019. In total, 23 different sites were sampled where Ae. koreicus were known to be present: 14 in Trento and 9 in Belluno Province, respectively (Table 1 and Supplementary Table S1 online), with altitudes ranging from 234 to 775 m a.s.l.^6,16. Engorged mosquitoes were collected in public and private houses, garden centers, cemeteries, and from periurban dry-stone walls using a home-built handheld aspirator (a modified handheld vacuum) (Fig. 3). Mosquitoes were aspirated from shady areas under vegetation, walls, and catch basins. In addition, all engorged females collected during routine invasive mosquito surveillance were used for the analyses. In this surveillance, BG-sentinel traps (Biogents AG, Regensburg, Germany) baited with a BG-Lure cartridge (Biogents) were activated for 24 h fortnightly. Immediately after collection, each sample was placed in a cooler, transported to the laboratory, and stored at − 80 °C until molecular analysis.

Sampled mosquitoes were identified at species level according to Montarsi et al.²¹ and ECDC guidelines for invasive mosquito surveillance in Europe⁴⁹. Blood-fed females were isolated from collected mosquitoes to identify the blood meal host.

DNA of single blood-fed mosquito samples, collected from 2013 to 2016, was extracted using Microlab Starlet automated liquid-handling workstations (Hamilton), using a MagMAX Pathogen RNA/DNA kit (Applied Biosystems, USA), according to the manufacturer’s instructions. DNA of a single abdomen of blood-fed mosquitoes, collected from 2017 to 2020, was extracted using QIAamp DNA Investigator kit tissues (Qiagen, Germany), following the manufacturer’s protocol. All samples were analyzed using a nested PCR with a specific set of primers targeting the vertebrate mitochondrial cytochrome c oxidase subunit I (COI) gene, as previously described⁵⁰. The first PCR reaction was carried out in a total volume of 50 μl, containing 2 units of AmpliTaq Gold DNA Polymerase (Applied Biosystem, USA), 5 μl of 10X Buffer, 2.5 mM of MgCl2, 0.2 mM of each dNTP, 2.5 μl of DMSO, 0.2 mM of primers M13BCV-FW (5’-TGT AAA ACG ACG GCC AGT HAA YCA YAA RGA YAT YGG-3’) and BCV-RV1 (5’-GCY CAN ACY ATN CCY ATR TA-3’), and 5 μl of extracted DNA. The second PCR reaction was carried out in a total volume of 50 μl containing 2 units of AmpliTaq Gold DNA Polymerase (Applied Biosystem, USA), 5 μl of 10X Buffer, 2.0 mM of MgCl2, 0.2 mM of each dNTP, 2.5 μl of DMSO, 0.4.mM of primers M13 (5’-GTA AAA CGA CGG CCA GTG-3’) and BCV-RV2 (5’-ACY ATN CCY ATR TAN CCR AAN GG-3’), and 1 μl of the PCR products obtained during the first amplification step. The thermal profile of the first PCR consisted of activation at 95 °C for 10 min, followed by 40 cycles at 94 °C for 40 s, 45 °C for 40 s, and 72 °C for 1 min, with a final extension step of 7 min at 72 °C. The thermal profile of the second PCR consisted of activation for 10 min at 95 °C followed by 16 cycles of a touchdown protocol at 94 °C for 40 s, decreasing the annealing temperature from 60 °C to 45 °C for 40 s (1 °C/cycle), followed by 72 °C for 1 min. Then, 30 cycles at 94 °C for 40 s, 45 °C for 40 s, and 72 °C for 1 min, with a final extension step of 7 min at 72 °C. Negative controls were included during the extraction and amplification stages to confirm avoidance of contamination.

The amplicons were sequenced in both directions using a 16-capillary ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, USA). To identify the blood meal host species, nucleotide sequences were compared with representative sequences available in the GenBank database using the Basic Local Alignment Search Tool (BLAST). Positive identification was made when > 97% identity was attained between the query and subject sequence.

Statistical analysis

As most of the identified hosts were either humans or wild ungulates (see Results), we investigated how the probability of feeding on these two host groups was affected by different abiotic factors. Specifically, we considered two binary response variables indicating whether or not the blood meal was acquired from a human/wild ungulate host. We developed univariate (i.e., with only one explanatory variable) generalized linear models (GLMs) with a binomial-distributed error structure, considering in turn, for each response variable, the following four explanatory covariates: (i) the altitude of the sampling point; (ii) the human population density in the area surrounding the sampling point, defined as 250 m square units, as per the Global Human Settlement Database⁴⁸; (iii) the percentage of non-artificial land cover within different buffers (100, 250 and 500 m radius from the sampling point), as per the Corine Land Cover dataset (defined as the sum of the fractions of agricultural and forested areas)⁵¹; the distance associated with the model with the lowest AIC value was then selected; (iv) the minimum distance of the sampling point from the nearest pixel labeled as forest, according to the Corine category. All analyses, including plot creation, was performed using R v4.0.2⁵² and “tidyverse”, “ggplot2”, and “gridExtra” libraries.

Map in Fig. 1 was generated by QGIS 3.22 using Sentinel-2 cloudless as background satellite image and urban places from OpenStreetMap database^53,54,55.

Source: Ecology - nature.com