article: Aedes albopictus is a tropical invasive species which in the last decades spread worldwide, also colonizing temperate regions of Europe and US, where it has become a public health concern due to its ability to transmit exotic arboviruses, as well as severe nuisance problems due to its aggressive daytime outdoor biting behaviour. While several studies have been carried out in order to predict the potential limits of the species expansions based on eco-climatic parameters, few studies have so far focused on the specific effects of these variables in shaping its micro-geographic abundance and dynamics. The present study investigated eco-climatic factors affecting Ae. albopictus abundance and dynamics in metropolitan and sub-urban/rural sites in Rome (Italy), which was colonized in 1997 and is nowadays one of the most infested metropolitan areas in Southern Europe. To this aim, longitudinal adult monitoring was carried out along a 70 km-transect across and beyond the most urbanized and densely populated metropolitan area. Two fine scale spatiotemporal datasets (one with reference to a 20m circular buffer around sticky traps used to collect mosquitoes and the second to a 300m circular buffer within each sampling site) were exploited to analyze the effect of climatic and socio-environmental variables on Ae. albopictus abundance and dynamics along the transect. Results showed an association between highly anthropized habitats and high adult abundance both in metropolitan and sub-urban/rural areas, with “small green islands” corresponding to hot spots of abundance in the metropolitan areas only, and a bimodal seasonal dynamics with a second peak of abundance in autumn, due to heavy rains occurring in the preceding weeks in association with permissive temperatures. The results provide useful indications to prioritize public mosquito control measures in temperate urban areas where nuisance, human-mosquito contact and risk of local arbovirus transmission are likely higher, and highlight potential public health risks also after the summer months typically associated with high mosquito densities.
  The mosquito Aedes (Stegomyia) albopictus (Skuse) is classified among the 100 worst invasive species in the Global Invasive Species Database (http: //www. issg. org/database/species) including all species of micro-organisms, fungi, plants and animals globally recognized as major threats to biodiversity and/or human activities. In the last 40 years, the species has been able to spread from its native range of distribution in rural tropical South-East Asia worldwide, largely through the transportation of its relatively cold-hardy and long-lived eggs via the international trade in used tires [1,2] and to the capacity to colonize temperate regions by photoperiodic egg diapause [1,3, 4]. Another key element favouring Ae. albopictus expansion particularly to urban environments has been its ability to shift from natural larval habitats in forest edges (e. g. tree holes, bamboo stumps, and bromeliads) to anthropogenic containers (e. g. rain catch basins, tires, cemetery urns, vases, water storage containers) [4]. The first introductions of Ae. albopictus in Europe were documented in Albania in 1979 [5] and 10 years later in Italy [6], where it has become a permanent pest in most regions [7]. In recent years the species gradually spread into other Mediterranean countries, including France, Spain, Slovenia, Croatia, Bosnia and Herzegovina, Montenegro, and Greece [3]. Due to its aggressive daytime outdoor biting behaviour and to its ability to transmit a large variety of arboviruses [3], the species represents an important nuisance as well as a major public health concern in all non-native countries where it has established [8]. Aedes albopictus has been responsible for large chikungunya virus (CHIKV) epidemics in the Indian Ocean in 2005–2007 [9,10] and of a CHIKV outbreak in northern Italy in 2007 [11]. Since then it has been associated with autochthonous transmission of CHIKV and dengue virus (DENV) in France [12–15] and of DENV in Croatia [16]. Notably, these were the first autochthonous DENV cases reported in Europe since the outbreak in Greece in 1927–1928 caused by temporary establishment of a population of the tropical vector Aedes aegypti [17,18]. Models estimating climate change impact on spatio-temporal trends for risk exposure and season of transmission of CHIKV in Europe predict that Mediterranean regions will become increasingly climatically suitable for transmission, with highest risk of transmission by the end of the 21st century in France, Northern Italy and the Pannonian Basin (East-Central Europe) [19]. Moreover, a recent epidemic of Zika virus, with 440 000–1 300 000 estimated human cases in Brazil in 2015, is raising concerns about the risk of its introduction and local transmission by Ae. albopictus in Europe [20]. Finally, its opportunistic biting behavior [21,22] could involve Ae. albopictus in the transmission to humans of zoonotic pathogens such as West-Nile virus [23] and Dirofilaria canine nematodes [24,25]. Several authors used eco-climatic factors to predict the potential spatial distributions of Ae. albopictus and public health related threats. Most of these studies have focused on temperature to identify potential limits of the species range, indicating thresholds of minimum temperature in the coldest months and of heat accumulation [26–29] and producing maps to identify areas suitable for stable colonization [30–32]. Other studies exploited models to predict the species distribution using a broader range of climatic variables, including rainfall[1,31,33–38]. Recently, Kraemer et al. [39] developed an improved model combining climatic, environmental, land-cover and anthropogenic variables to predict the species probability of occurrence. Finally, Roche et al. [40] showed that human activities are particularly important for the species dispersion, while land use is a major factor for its establishment. Given the scale at which these studies were carried out, they are useful to predict Ae. albopictus future expansion and to improve surveillance programs by detecting the species introduction at its earliest stages when it is still possible to prevent its establishment [41]. However, in areas permanently colonized by Ae. albopictus it is crucial to identify potential spatial and temporal hot-spots of abundance which could be associated with higher nuisance biting and risk of disease transmission in order to prioritize mosquito control interventions. In fact, it has been demonstrated that treatment of hot-spot can be incorporated successfully into existing integrated mosquito management programs to increase their cost-effectiveness [42]. In general, availability of suitable breeding and resting sites along with the presence and abundance of competing species and of potential hosts (which in turn vary in relation to landscape composition, climatic conditions and host demography) are known to shape mosquito abundance at a local scale. In the absence of competition with Aedes aegypti, urbanization has been shown to favor high Ae. albopictus abundance [43,44] and landscape and human activities have been found to be crucial to predict its actual local distribution and relative abundance [45]. To date, the specific effects of these variables in shaping Ae. albopictus micro-geographic abundance and dynamics at temperate latitude in Southern Europe are poorly understood. The few studies carried out so far showed a positive association between host-seeking female abundance and temperatures and a negative one with rainfall in north-east Italy [46]. Additionally, a positive association between number of eggs and vegetation around ovitraps was detected in a small highly urbanized site within Rome [47]. The present study aims to investigate eco-climatic factors affecting Ae. albopictus abundance and dynamics in metropolitan versus sub-urban/rural sites in Rome (Italy), which was colonized by Ae. albopictus in 1997 and became one of the most infested metropolitan areas in Southern European temperate regions [48,49].
  Twenty-one study sites (hereafter referred as stations) were selected along a 70 km-transect across and beyond the most urbanized and densely populated metropolitan area of Rome (Italy), corresponding to the train route from the coast to Appennino mountains (Fig 1). All sites were below 300m asl, with the exception of station 20 (330m asl) and 21 (460m asl). Groups of four sticky traps (STs, [50]) were located in a 300 m-radius area within each station (for a total of 84 STs), positioned on site and geo-referenced using GPS. The 300 m-radius was calculated from the centroid of the convex hull generated from groups of four neighbouring sampling points (i. e. STs) and corresponds to Ae. albopictus maximum dispersal range (i. e. 300 m; [51]). A 20 m circular buffer was calculated around each ST. This buffer at ST level corresponds to the largest one used in a similar study that showed an association between land cover variables and mosquito abundance [47]. Moreover, a 3 km-circular buffer was calculated from the centroid of the convex hull generated from groups of four neighbouring STs. Information obtained from the 3 km-circular buffer was used to assign each station to either metropolitan or suburban/rural area. Sticky traps were monitored for 18 weeks from July 10th to November 8th 2012 on a weekly basis by substituting sticky panels with freshly glued ones and recording water leftovers. Trapped mosquitoes were morphologically identified and counted on site. A fine scale spatio-temporal dataset was built to analyze the effect of a set of climatic and socio-environmental variables on Ae. albopictus abundances in the 21 stations. Open source software were used for the construction of the spatial dataset: GRASS GIS [52] for data processing and spatial analysis and QGIS [53] for spatial analysis and layout generation. The spatial dataset included time-dependent climatic variables and time-independent socio-environmental variables, as follows: The relationship between socio-environmental and climatic variables and Ae. albopictus abundance was investigated through generalized linear mixed models (GLMMs) and generalized additive mixed models (GAMMs). Response variable was the weekly number of Ae. albopictus adult females collected in each STs. Time-dependent (climatic) and time-independent (socio-environmental) variables were included in the model as predictors. In addition, the variable Day of Year (DoY) was also considered to investigate the temporal pattern of the population dynamic during the sampling period. Scatterplots, conditional boxplots, variance inflation factor (VIF) and concurvity [57] were used to assess non-linear relationships and collinearity among variables.
  Results of GAMMs showed that Ae. albopictus population dynamics was better modelled (lowest AIC with significant coefficients) by the Day of the Year (DoY) smoother than by time-dependent climatic predictors (i. e. temperature and rainfall) computed in different temporal windows (S2 Table). Therefore, DoY was taken as time-dependent variable in the full GAMM carried out to assess the effect of time-independent environmental predictors (i. e. Vegetation Cover) on Ae. albopictus abundance, and its role in Metropolitan vs Sub-Urban/Rural Environments. No multicollinearity among linear predictors was found (Variance Inflation Factor values < 2). On average, weekly Ae. albopictus abundance did not differ between Metropolitan and Sub-Urban/Rural environment (Table 1). However, a significant interaction term between Vegetation Cover and Environment was found in the 20 m-radius buffer, meaning that an increase of the proportion of Vegetation Cover was positively associated with Ae. albopictus abundance in the Metropolitan Environment, but not in the Sub-Urban/Rural one (Table 1, Fig 2). On the other hand, an increase of the proportion of Vegetation Cover in the 300 m-radius buffer was negatively associated with mosquito abundance in both Environments (Table 1). Fig 3A shows temperature and rainfall during the 18 week-sampling period. Fig 3B shows the observed Ae. albopictus temporal dynamics in Metropolitan and Sub-Urban/Rural Environments. The shape of the estimated smoother (Fig 3C) shows bimodal mosquito population dynamics in both environments (no differences in the shape of the estimated smoothers in the two Environments was found), characterized by a first peak during the last weeks of August (weeks 34 and 35) and a second peak during the first week of October (week 40). The two phases (Phase-1 and -2) of highest mosquito abundance (in grey in Fig 3C) were analyzed separately in order to assess their respective climatic drivers. Results of univariate models—carried out in the preliminary analysis to assess which time-dependent variables would be the best predictor of mosquito abundance and to identify their lagged effect (different temporal windows) —showed that the accumulated LST at Lag 1 and the accumulated rainfall at Lag 3 were the most informative climatic predictors related to Phase-1 (S3 Table). However, when included in the full model with time independent variables, only LST at Lag 1 remained significant (Table 2). On the other hand, the accumulated rainfall at Lag 4 and the mean LST at the week of sampling (i. e. Lag 0) were the most informative climatic predictors in the preliminary analysis for Phase-2 (S3 Table). However, when they were included in the full model, only the accumulated rainfall at Lag 4 remained significant (Table 2). Socio-environmental time-independent variables in both models carried out for the two high abundance phases showed the same effects observed in the model carried out for the entire sampling season (Table 1). Specifically, a significant interaction term was detected between Vegetation Cover and Environment at 20 m-radius buffer, meaning that an increase of the proportion of Vegetation Cover was positively associated with Ae. albopictus abundance in the Metropolitan Environment, but not in the Sub-Urban/Rural one (Table 2). On the other hand, in both high-abundance phases an increase of the proportion of Vegetation Cover in the 300 m-radius buffer was negatively associated with mosquito abundance in both Environments (Table 2).
  The results of the analysis of the spatial distribution and relative abundance of Ae. albopictus along the 70 km-long transect encompassing the metropolitan area of Rome highlighted a complex relationship between landscape composition and mosquito abundance. When focussing the analysis to the scale of the estimated flight range of the species [51]—corresponding in the experimental set up to a 300 m-buffer within each sampling station—results showed that high adult abundance was on average associated with highly anthropized habitats (rather than with highly vegetated ones), in both the Metropolitan and the Sub-Urban/Rural areas. This is consistent with characteristics of highly anthropized habitats which favour the mosquito life-cycle, such as high human population density providing more opportunities for blood-feeding and larger numbers of artificial water containers (such as flowerpots, rain catch basins, abandoned tires, and disposable tins) suitable for container-breeding mosquitoes. Moreover, especially in temperate regions, the replacement of natural soil and vegetation with artificial surfaces is known to elevate temperatures and alter rainfall regimes with respect to surrounding regions [65–67], favoring mosquito development and gonotrophic cycle [36]. Indeed, Ae. albopictus has been shown to reach very high densities in highly anthropized areas, in particular in the absence of sympatric competing Ae. aegypti populations [43,44,68]. On the other hand, when the analysis was restricted to a 20 m-scale in order to focus on the landscape factors at sticky trap level (as in [47]), a different pattern of mosquito abundance was observed depending on the location of the traps in Metropolitan or in Sub-Urban/Rural areas. In fact, while in the latter, the vegetation coverage at 20 m-scale did not affect Ae. albopictus abundance, in the Metropolitan area, sticky traps positioned within highly vegetated 20 m-buffers collected higher number of mosquitoes, especially when located in highly anthropized stations. Overall, results suggest that hotspots of Ae. albopictus abundance within the highly anthropized Metropolitan stations are associated with “small green islands”. Since the sticky traps used for mosquito sampling mostly collect ovipositing females as well as resting adults [22], these “small green islands” may represent ideal sites in which the mosquito founds optimal conditions to lay eggs and rest. Moreover, it can be speculated that “small green islands” are associated with higher abundance of potential resting and larval sites characterized by more suitable temperatures for larval development (as shown in New Jersey, US [69]). Finally, “small green islands” in highly anthropized Metropolitan areas (such as children playgrounds, elderly people meeting places, small private gardens and condominium gardens) may be also attractive for host-seeking females, as they are largely exploited by people for outdoor activities especially during late summer afternoons when the species is most active. In fact, from an epidemiological perspective, it would be relevant in the future to extend the analysis in order to represent spatial heterogeneities not only in mosquito abundance but also in mosquito-to-host ratio [45]. The results of the analysis of the Ae. albopictus seasonal dynamics showed a bimodal pattern (with a peak of abundance in August and one in October) in most sampling stations, revealing that the species may unpredictably reach very high abundance also after the summer season. The first peak in August was clearly temperature-driven, while rainfall accumulated in September (average value of 103 mm) following a month with very low rainfalls (average value in August = 7. 4 mm) seems to be the major driver of the second peak unexpectedly observed in mid-October. Afterwards, when temperatures and photoperiod became sub-optimal for the species life cycle, the mosquito abundance rapidly decreased despite frequent rainfall. The first peak in August followed by a decrease in mosquito abundance in the following weeks is consistent with data from the early stage of the species colonization of Rome [70,71], as well as from subsequent years when the species had already become an established urban pest [50,72]. The second peak observed in the present work is less consistent with data reported in the past, although a second increase in the population abundance was frequently shown to occur in October, probably due to relatively high temperatures and rainfall accumulating after a few dry summer weeks, a common climatic pattern in Rome at least in the last 10 years (S1 Fig). Moreover, subsequent data from human-landing catches carried out in Rome confirm a bimodal seasonal dynamics with very high host-seeking mosquito abundance in the final part of the reproductive season (Manica et al. in preparation). Some aspects of the experimental design deserve discussion. First, it may be argued that the seasonal-long trapping effort influenced mosquito abundance in the study sites. However, results from previous work clearly showed that only a small fraction of wild Ae. albopictus females is collected by STs even in the frame of a much more intense sampling scheme than the present one [51]. Second, it is conceivable that competition of sticky traps with other potential oviposition sites likely non-homogeneously distributed in space and time may have created a bias in the comparison of mosquito abundance among sampling sites and among different phases of the season. Unfortunately, every collection approach may suffer of some kind of bias. Given appropriate resources, it would have been beneficial to monitor potential breeding sites during the field activities. Third, the results refer to ovipositing/resting females and may not directly reflect mosquito/human ratio, which is a more relevant epidemiological parameter to be assessed by the recording abundance of host-seeking females. Finally, inferences on the landscape determinants of the spatial and temporal distribution of Ae. albopictus abundance here presented are aggregated (i. e. vegetation cover includes trees, grass, bushes, etc.) to increase statistical power and do not take into account the land use associated with human activities. Despite these study limitations, the results allow relevant speculations from a public health perspective. First, the analysis of the climatic determinants of Ae. albopictus seasonal dynamics highlights how the association of permissive photoperiod and temperatures associated with rainfall at the end of the summer period may result in a second phase of high Ae. albopictus abundance. This is likely to occur not only in the Rome area, but also in other Mediterranean regions colonized by the species and showing a similar climatic pattern. Roiz et al. [55] showed that an extreme rainfall event increased and extended the species abundance in Montpellier (coastal France) leading to at least 11 cases of autochthonous CHIKV transmission. This led the authors to propose that mosquito control campaigns must be implemented after such heavy rainfall events. Our results extend this concept, as they suggest that also less extreme and repeated rainfall after a relatively long dry period (which characterized Rome in the past years and is predicted to became a typical scenario in Italy in future years due to climate changes [73]), may cause the replenishment of peridomestic containers where desiccated eggs of Ae. albopictus are present, giving rise to increased mosquito abundance a few weeks later. This implies that in South European areas, characterized now or in the future by a similar climatic pattern, monitoring and control campaigns should be planned also after the end of the summer season to prevent a possible second peak of the mosquito population abundance and its associated health threats. Second, our spatial analysis emphasizes the need to prioritize public mosquito control activities in “small green islands” within highly anthropized metropolitan settings (such as children playgrounds or elderly people meeting places) where nuisance, human-mosquito contact and risk of local arbovirus transmission are likely to be higher [74,75]. On the other hand, the study suggests that such a prioritization strategy might be ineffective outside the metropolitan areas, where no hot-spots of mosquito abundance have been identified.
summary: The tiger mosquito (Aedes albopictus) originates from tropical Asia, but over the last decades has invaded many regions worldwide. In several Southern Europe and US urban areas it has become the mosquito species creating the highest nuisance to the citizens, spoiling recreational outdoor activities by aggressively biting during daytime hours. Moreover, it has also become a public health concern due to its capability of transmitting to humans several exotic arboviruses. For an effective control strategy against these mosquitoes a profound knowledge of their preferences in terms of environment is mandatory in order to better understand what affects and determines their distribution. We analyzed some of these factors in the city of Rome and in its outskirts by collecting mosquitoes during an entire summer season. Results show that Ae. albopictus presence is more abundant in strongly urbanized areas compared to more green ones, but that within metropolitan Rome the highest densities are observed in small green areas within heavily urbanized settings, such as private gardens or public recreational areas, which should thus be the first target of anti-mosquito activities. Results also show that very high densities are not only observed in the warmest summer months as expected, but also in autumn, when people are less prepared to defend themselves from mosquito attacks.
keywords: invertebrates
  species colonization
  medicine and health sciences
  ecology and environmental sciences
  invasive species
  geographical locations
  animals
  seasons
  summer
  population biology
  insect vectors
  public and occupational health
  epidemiology
  disease vectors
  insects
  arthropoda
  people and places
  italy
  population metrics
  mosquitoes
  earth sciences
  biology and life sciences
  population density
  europe
  organisms
title: Spatial and Temporal Hot Spots of Aedes albopictus Abundance inside and outside a South European Metropolitan Area