Aedes albopictus in a recently invaded area in Spain: effects of trap type, locality, and season on mosquito captures

Mosquitoes are primary vectors of pathogens impacting humans, wildlife, and livestock. Among them, the Asian tiger mosquito, Aedes albopictus, stands out as an invasive species with a global distribution, having established populations on every continent except Antarctica. Recent findings incriminate Ae. albopictus in the local transmission of several pathogens causing human diseases, including dengue, chikungunya, and Zika viruses and worm parasites as Dirofilaria. In Spain, the establishment of Ae. albopictus occurred in 2004 and it rapidly expanded, currently reaching southern provinces and creating novel epidemiological scenarios in recently invaded areas. In this study, we conducted captures of Ae. albopictus from May to November 2022 in two provinces, Granada and Malaga, situated near the current edge of the species' expanding range in Spain. The objective was to identify the primary factors influencing their captures in these regions. Mosquitoes were captured using BG-Sentinel traps baited with CO2 and BG-Lure, and miniature CDC-UV traps in five different localities. Our findings underscore the influence of both extrinsic factors, such as locality, and intrinsic factors, including mosquito sex, on the abundance of captured Ae. albopictus. A higher abundance of Ae. albopictus was observed in the Malaga province compared to localities in the Granada province. Furthermore, similar numbers of Ae. albopictus mosquitoes were captured in more urbanized areas of Granada, while the lowest counts were recorded in the less urbanized area. These results were compared to captures of another common species in the area, specifically Culex pipiens. Overall, these results represent the first monitoring of invasive Ae. albopictus in the area and are discussed in the light of the potential importance of the species as a nuisance for humans and vectors of pathogens of public health relevance.


Comparison of the capture efficiency using BG-Sentinel and CDC-UV traps
Overall, out of 100 capture attempts using the two trap types in the 5 localities throughout the capture season (see methods), BG-Sentinel traps captured Ae. albopictus in 46% of occasions, while in CDC-UV traps this percentage dropped to 5%.This resulted in significant differences between trap types (Pearson's Chi-squared test with Yates' continuity correction: χ 2 = 42.11,p < 0.001).Likewise, BG-Sentinel traps captured Cx. pipiens 93% of the occasions, whereas CDC-UV traps achieved a rate of 52% (χ 2 = 40.13,p < 0.001).Differences in trap performance persist when analyzing the sexes of each species separately (all p < 0.05), except for Cx.pipiens males, which were trapped in similar percentages in both trap types (χ 2 = 0.76, p-value = 0.38).

Discussion
This study is a field investigation aimed to unveil the role of major factors potentially affecting the captures of the invasive Asian tiger mosquito, Ae. albopictus, in an area of recent invasion in the provinces of Granada and Malaga (southern Spain) 23 .To that end, we regularly sampled Ae. albopictus and other autochthonous mosquito species between May and November 2022, at five different localities categorized as natural, urban, and periurban sites.These results provide valuable information for the monitoring and control of invasive mosquitoes with public health relevance.

Effects of trap type and sex on mosquito captures
Our study emphasizes the higher efficacy of BG-Sentinel traps over CDC-UV traps in capturing mosquitoes in our study area.The BG-Sentinel traps effectively captured Ae. albopictus in nearly 50% of the trapping events, while CDC traps achieved a success rate of 5%.Although both traps had a higher overall efficiency in capturing Cx. pipiens than Ae.albopictus, significant differences between trap types persisted.Indeed, the higher effectiveness of BG-Sentinel traps compared to other trapping methods has been extensively demonstrated in various environments and for different mosquito species [26][27][28][29][30] .Certain widely used mosquito traps (e.g., CDC miniature light traps, gravid traps, or New Jersey light traps) have been found to be inefficient in capturing Ae. albopictus 26,27,29 .This may be because this invasive species is predominantly diurnal and seeks hosts near the ground surface 31,32 .Here, CDC-UV traps were placed at approximately 1.5 m above the ground (but see, for example 30,33 , for alternative settings), that may enhance their efficacy in capturing mosquitoes with more crepuscular or nocturnal activity patterns, such as Cx.pipiens 30 .Additionally, the use of visual and olfactory attractants, such as the BG-Lure and CO 2 , employed in BG-Sentinel traps, may further improve the capture of female mosquitoes of both species 26,29 .So, these results support the necessity to consider the capture method in order to compare mosquito captures between different studies.Interestingly, both types of traps demonstrated comparable performance in capturing Cx. pipiens males, whereas male Ae.albopictus were more commonly captured using BG-Sentinel traps.This difference can be attributed to the specific behavior of Ae. albopictus males, who, in order to increase their mating chances, adeptly differentiate the chemical and olfactory signals emitted by hosts, anticipating the presence of females 34 .Lastly, our findings support the general pattern previously reported in various mosquito species, where female mosquitoes outnumber males in captures [26][27][28][29]35 .

Effects of locality and seasonality on mosquito BG-captures
The abundance of both Ae.albopictus and Cx.pipiens mosquitoes was determined by the sampling locality, with varying effects observed between sexes of the latter species (Fig. 2, Table 1).The Bioparc zoological garden, located in the urban area of Fuengirola (Malaga province), had the highest abundance of Ae. albopictus.Zoological gardens may provide suitable habitats for mosquitoes, especially for invasive species like Ae. albopictus.This is www.nature.com/scientificreports/due to the availability of breeding sites (e.g., puddles on the ground or small water holes on plant surfaces, such as in the cut stems of lucky bamboo), the presence of exotic plants that mimic the southeast Asian landscapes where the species is native, and the abundance of potential hosts.These factors enable continuity and proliferation of the mosquito populations within the region 36,37 .Aedes albopictus is capable of blood-feeding on various organisms, including fish, reptiles, and birds.However, the majority of its blood meals come from mammals, with humans recognized as a common host of this species 38,39 .This fact supports the role of Ae. albopictus as an important human nuisance but also a potential vector playing a role in the transmission of pathogens, including those affecting wildlife and humans such as Dirofilaria 13,14 .Moreover, differences in the abundance of Ae. albopictus mosquitoes between Fuengirola (11 m.a.s.l.; Table 3), situated by the sea, and localities in Granada (ranging between 642 and 762 m.a.s.l.; Table 3), could be attributed to climatic differences.The former features milder environmental conditions in contrast to the more extreme and variable conditions present in the localities of Granada 31,[40][41][42] .Considering that the period of mosquito captures in the zoological garden was shorter (traps operated less hours than in the other localities due to the presence of visitors; see methods), much higher differences could be expected if mosquitoes were captured over a 24-h period.Finally, we recorded the presence of Ae. albopictus in all localities of Granada, with significantly higher abundances in the peri-urban site of Gójar compared to the natural site of La Vega.
A previous study developed in Andalusia (Spain) demonstrated that, although Cx. pipiens is the predominant mosquito species in urbanized areas, it also exhibits higher abundance in rural and natural areas 43 .We found significant differences in the abundance of female Cx.pipiens between the natural site of La Vega, the locality with the highest number of captures of this species, with respect to the peri-urbans sites of Cartuja and Gójar (Fig. 2, Table 1).La Vega, a waste-water treatment plant, is surrounded by agriculture fields providing suitable habitats for the breeding of this mosquito species.In urban sites such as Fuengirola and Fuentenueva, the abundance of Cx. pipiens was similar to that in peri-urban sites.
For males, the lowest abundance of Cx. pipiens was also detected in Cartuja, an open and highly exposed sampling point in the peri-urban area of Granada.The lack of correlation among localities in male and female Cx.pipiens abundances may be explained by the different ecological requirements and behavior of these sexes.Male Cx. pipiens, tending to remain near breeding sites, exhibit lower dispersal than females, which actively seek out hosts 44 .Nevertheless, the results obtained for Cx.pipiens, together with those found for Ae.albopictus, suggest that local-scale environmental characteristics, such as microhabitat characteristics suitable for mosquito breeding and temperature or rainfall [40][41][42]45,46 , may strongly determine the presence and abundance of these mosquito species. WhileCx. pipiens is a common species in urban sites, it reaches its maximum abundance in natural habitats 43,[45][46][47] , whereas Ae. albopictus shows a higher preference for urban sites with milder winters and water infrastructures 31,[40][41][42] .
Finally, we did not find significant differences in the captures of Ae. albopictus mosquitoes between trapping sessions.However, the abundance of this species reached its maximum from mid-July to late September (sessions 4-7; Fig. 2), representing almost 75% (100/135) of females and 72% (46/64) of males captured.This seasonality is similar to those previously reported in field surveys in other areas of southern Europe 29,35,48 .For instance, a study conducted in 2019 within the Portuguese Algarve, geographically proximate to our study area, observed that the peak abundance of adult mosquitoes was reached between mid-July and mid-September 35 .In such study, two short peaks were detected at early October and early November 2019, which were not registered in our field samplings.The differences in experimental designs and the extreme weather conditions observed in October and November 2022 in the provinces of Malaga and Granada, where they were the driest and hottest on record, may account for these findings 49 .The relatively small number of Ae. albopictus mosquitoes caught during the study period, especially in the first and last sampling sessions, might account, to some extent, for the absence of variations in overall mosquito captures.
Culex pipiens seasonality has been extensively documented.We observed that population dynamics align with prior field surveys conducted in Spain and other countries within the Mediterranean basin [45][46][47] .Compared to Ae. albopictus, we identified a more consistent and stable seasonal pattern on Cx. pipiens, with more subtle variations along the seasons.Two peaks in abundance were identified: one in May through early June (sessions 1-2; Fig. 2) and a slightly larger peak from September through the end of November (sessions 7-10; Fig. 2), excluding the hottest summer months.Thus, although Cx. pipiens displays broad tolerance to environmental factors, our findings suggest that its populations may thrive in the wetter and warmer months, but face constraints under extreme heat conditions [45][46][47] .www.nature.com/scientificreports/ The observed temporal dynamics for both species show some differences (Fig. 2), with their abundance peaks occurring at different times.These differences in temporal shifts may be attributed to niche differentiations related to climate and the availability of breeding sites in the area 50 .Yet, these results could be due to other factors such as interspecific competition between these species as Ae.albopictus may effectively compete against other species, including Cx. pipiens, during the larval stage 51 .A prior study of two mosquito species in natural environments of northern Italy provide support for the significant impact of interspecific competition and the temporal niche effect on the abundance patterns of both species 50 .This asymmetrical interspecific competition could lead to temporal changes in the dynamics of both species 50 .Nevertheless, complementary field surveys are necessary to identify the relative contributions of niche differentiation and interspecific competition to the temporal dynamics observed in our study.
In conclusion, Ae. albopictus is an important nuisance for human populations in the invaded area where it may also play a role as a potential vector of locally circulating and imported pathogens 40 .The species was introduced in southern Spain in recent decades and is currently experiencing a population increase.The native Cx.pipiens also represents a significant concern for wildlife and public health, as it is a proficient vector for pathogens such as the West Nile virus 47,52 .This virus has caused outbreaks in several Mediterranean countries in recent years, including the southern Iberian Peninsula.Our findings demonstrate that trap type, sex, and locality are significant factors that influence the captures of both invasive and native mosquito species.These findings have implications for monitoring and surveillance of local populations of the recently established Ae. albopictus and the autochthonous Cx. pipiens and, subsequently, to prevent their potential contribution to the transmission of locally circulating and imported pathogens in southern Spain.

Study area
Mosquito sampling was conducted from early May to late November 2022 in five locations throughout southern Spain, including one natural site, two peri-urban sites, and two urban sites (Table 3).One urban location, the Bioparc Zoological Garden, is located in the Malaga province, and the four additional sampling sites are in the Granada province.The study sites in Granada included a natural location near a sewage station surrounded by agricultural fields (the natural site of La Vega), an urban area situated on the Fuentenueva campus of the University of Granada (UGR), and two settings with an intermediate degree of urbanization: the periurban sites of Cartuja campus of the UGR and Gójar (Table 3; Fig. 2a).Localities were classified as urban, peri-urban, or natural sites based on population density and the percentage of natural/urban areas.In brief, land use and population density were obtained from http:// www.junta deand alucia.es/ insti tutod eesta disti cayca rtogr afia/ DERA/ and processed with QGIS v3.18.1 53 .First, we set up buffers of 500 m radius around each sampling point.To quantify the land use in each buffer, we used the ' disolve' and 'intersect' geoprocessing tools, and then the 'statistics by categories' tool, obtaining the total area and percentage of each variable per buffer.For the land use, we obtained a total of 8 categories for the 5 sampling points, which we grouped into 2 classes: natural areas, grouping the "permanently irrigated land", "mainly agricultural land, but including natural vegetation", "olive groves", "crop mosaic", and "natural grasslands" categories, and urban areas, grouping the "industrial or commercial zones", "continuous urban fabric", and "discontinuous urban fabric" categories.In the case of human population density, the data used was estimated as the number of people living in a grid of 250 × 250 m by the Institute of Statistics and Cartography of Andalusia from the latest local census from 2021.We used the 'intersect' geoprocessing tool to determine the percentage of each grid within each buffer and used the 'statistics by categories' to estimate the total population in these grids.

Mosquito sampling
We conducted 10 sessions of mosquito capture in each of the five localities.At each sampling location and trapping session, two Blacklight (UV)-CDC Miniature traps (Centers for Disease Control and Prevention, Atlanta, USA) and two Biogents (BG)-sentinel-2 traps (Biogents, Regensburg, Germany) were set up.Consequently, each trap type was set 100 times throughout the sampling period.BG-Sentinel traps were supplemented with dry ice, as a source of CO 2 , and BG-Lure.Two sampling points were established at each locality, each with one of the two trap types.To minimize bias, sampling locations within localities were placed approximately 10-50 m apart, depending on the possibilities found in each locality.The selection criteria for these locations were determined by maintaining consistent environmental conditions.Ideally, traps were located in shaded and humid areas near water sources and vegetation and representative of the locality being sampled.Additionally, we assigned an individual number to each trap for individual identification and traps were alternated between the two sampling points within each locality to avoid potential bias associated to the trap identity.The sampling order of localities within each trapping session was randomized for the same purpose.Trapping sessions were conducted every 2-3 weeks, avoiding days with adverse conditions for mosquito sampling (e.g., rainy and windy days).Traps operated during 24 h in each trapping locality.However, due to the affluence of human visitors to the Fuengirola sampling locality (i.e., Bioparc zoological garden; Malaga province), in this site traps only operated from 19:00 pm to 10:00 am (local time).
Collected mosquitoes were transported to the laboratory on dry ice and maintained frozen (− 80 °C) until further analysis.Subsequently, mosquitoes were sexed and identified using morphological keys 54,55 .

Statistical analyses
We focus this study on the invasive species Aedes albopictus and the common house mosquito Culex pipiens.Analyses were restricted to these species due to the limited number of captured mosquitoes from other species and in order to address the main focus of the study.Firstly, we performed a Pearson's chi-square test to evaluate

Figure 2 .
Figure 2. Population dynamics, estimated as the total number (abundance) of Aedes albopictus females (A) and males (B) and Culex pipiens females (C) and males (D) trapped using BG-Biogents (BG)-Sentinel-2 traps supplemented with dry ice as a source of CO 2 and BG-Lure.

Table 1 .
Total number of Aedes albopictus and Culex pipiens captured at each sampling locality, categorized by trap type and sex.The number of females (♀) and males (♂) captured are shown in parentheses.M.

Table 3 .
Characteristics of the sampling localities included in this study in the provinces of Granada (four sampling localities) and Malaga (one locality).m.a.s.l.: meters above sea level.