Abundance and Updated Distribution of Aedes aegypti (Diptera: Culicidae) in Cabo Verde Archipelago: A Neglected Threat to Public Health

Background: Mosquito-borne viruses, such as Zika, dengue, yellow fever, and chikungunya, are important causes of human diseases nearly worldwide. The greatest health risk for arboviral disease outbreaks is the presence of the most competent and highly invasive domestic mosquito, Aedes aegypti. In Cabo Verde, two recent arbovirus outbreaks were reported, a dengue outbreak in 2009, followed by a Zika outbreak in 2015. This study is the first entomological survey for Ae. aegypti that includes all islands of Cabo Verde archipelago, in which we aim to evaluate the actual risk of vector-borne arboviruses as a continuous update of the geographical distribution of this species. Methods: In order to assess its current distribution and abundance, we undertook a mosquito larval survey in the nine inhabited islands of Cabo Verde from November 2018 to May 2019. Entomological larval survey indices were calculated, and the abundance analyzed. We collected and identified 4045 Ae. aegypti mosquitoes from 264 positive breeding sites in 22 municipalities and confirmed the presence of Ae. aegypti in every inhabited island. Results: Water drums were found to be the most prevalent containers (n = 3843; 62.9%), but puddles (n = 27; 0.4%) were the most productive habitats found. The overall average of the House, Container, and Breteau larval indices were 8.4%, 4.4%, and 10.9, respectively. However, 15 out of the 22 municipalities showed that the Breteau Index was above the epidemic risk threshold. Conclusion: These results suggest that if no vector control measures are considered to be in place, the risk of new arboviral outbreaks in Cabo Verde is high. The vector control strategy adopted must include measures of public health directed to domestic water storage and management.

One of the tools used in Ae. aegypti surveillance is the determination of Stegomyia indices, namely the House Index (HI), Container Index (CI), and Breteau Index (BI). These indices measure the abundance, spatial distribution, and provide information about areas or periods of mosquito population growth [27][28][29][30][31].
Most of the studies on Ae. aegypti based on Stegomyia indices support a significant association between these and the transmission risk of arboviruses [32][33][34][35][36]. In studies that did not observe this association, mosquito and human migration were considered as possible factors that affected the lack of association [37][38][39]. Hence, knowledge of these indices allowed for the timely application of control measures and strategies [40].
In this context, we aimed to evaluate the actual risk of vector-borne arboviruses in Cabo Verde based on the Stegomyia indices, as a continuous update of the geographic distribution of Ae. aegypti. To our knowledge, this study represents the first entomological survey for this species that includes all islands of Cabo Verde archipelago.

Study Area
We collected mosquito larvae in the 22 municipalities of Cabo Verde, a volcanic archipelago with an area of 4033 km 2 located about 550 km off the coast of Senegal. The archipelago consists of 10 islands, nine of which are inhabited with approximately 537,660 inhabitants ( Figure 1). It has an arid and semi-arid climate, warm and dry, with an average annual temperature of around 25 • C, and low rainfall. Two seasons can be identified: the dry season, from December to June, and the rainy season, from August to October [41,42]. from Santiago Island during the dengue outbreak in 2009 [23,25]. However, the situation changed in 2012 and 2014, with the first reports of resistance to these insecticides [26]. One of the tools used in Ae. aegypti surveillance is the determination of Stegomyia indices, namely the House Index (HI), Container Index (CI), and Breteau Index (BI). These indices measure the abundance, spatial distribution, and provide information about areas or periods of mosquito population growth [27][28][29][30][31].
Most of the studies on Ae. aegypti based on Stegomyia indices support a significant association between these and the transmission risk of arboviruses [32][33][34][35][36]. In studies that did not observe this association, mosquito and human migration were considered as possible factors that affected the lack of association [37][38][39]. Hence, knowledge of these indices allowed for the timely application of control measures and strategies [40].
In this context, we aimed to evaluate the actual risk of vector-borne arboviruses in Cabo Verde based on the Stegomyia indices, as a continuous update of the geographic distribution of Ae. aegypti. To our knowledge, this study represents the first entomological survey for this species that includes all islands of Cabo Verde archipelago.

Study Area
We collected mosquito larvae in the 22 municipalities of Cabo Verde, a volcanic archipelago with an area of 4033 km 2 located about 550 km off the coast of Senegal. The archipelago consists of 10 islands, nine of which are inhabited with approximately 537,660 inhabitants ( Figure 1). It has an arid and semi-arid climate, warm and dry, with an average annual temperature of around 25 °C, and low rainfall. Two seasons can be identified: the dry season, from December to June, and the rainy season, from August to October [41,42].  In 2010, 141,762 accommodations, including 114,469 buildings, were registered in the country. Of those, 94,894 (82.9%) have one division/room, 10,646 (9.3%) have two divisions/rooms, and 6983 (6.1%) are buildings with three or more rooms. Of the total buildings, 74,404 (65%) are finished, while the remaining are under construction. Regarding the type of habitat, 44,185 (38.6%) of the houses are in urban areas and 30,449 (26.6%) in rural areas [43].
More than 95% of the population use conventional material for construction of their houses, 3.9% use non-conventional material, and 1.3% use a thatched roof, brass, drum plates, or others for cover [44,45].
The most common pavement types (99.4%) are cement and mosaic, and only 0.6% are clay or other [46]. In terms of wall cladding, 66.7% are plastered and painted, while just over 16% do not have any type of coating. Regarding the ceilings, most (79.3%) use reinforced concrete terraces [42].
One-third of Cape Verdeans do not have access to public water [45] and for those who do have access, the distribution is irregular, leading to water storage inside and outside of the homes.
In rural and semi-rural areas, pig pens or henneries are found around the houses, from which additional income is obtained [47].

Entomological Collections and Sampling Methodology
From November 2018 to May 2019, mosquito larvae were collected in all municipalities of Cabo Verde. We selected the sampling area with each municipal health delegation team, according to the high incidence history of mosquito-borne diseases, Ae. aegypti densities, and the human population. The houses were selected randomly, both in rural and urban areas. In urban areas with two parallel rows of houses, the selection was made by choosing a first house and then skipping four houses, counting a zigzag pattern. All containers, or potential breeding sites with water for larvae, were inspected and recorded (container type, position, vegetation, and sun exposure). The collected larvae were transported to the National Institute of Public Health (INSP) Medical Entomology Laboratory for morphological identification.

Morphological Identification
Larvae and reared adult mosquitoes were morphologically identified as Ae. aegypti under a stereomicroscope, according to the identification keys of Ribeiro et al. [19,[48][49][50]. The larvae were mounted on slides with 2% glycerinated Hoyer's medium, and adult reared mosquitoes, and stored at −20 • C for further molecular and genetic analysis.

Statistical Analysis
We compiled the data into a Microsoft Excel database. For the data analysis, the continuous variables were expressed in measures of central tendency and dispersion, and the categorical ones in simple frequency. The chi-square test was used to determine the association between the presence of Ae. aegypti and the type of breeder/container (type, position, and physical characteristics). We considered positive breeding sites for Ae. aegypti where there was at least one larva. The level of significance for statistical analysis was 0.05. We used IBM SPSS Statistics 20 (International Business Machines Corporation, New York City, NY, USA) to analyze the data.
Larval indices were calculated, namely, HI, CI, and BI. The maps were drawn using ArcGIS 10.6 (Environmental Systems Research Institute, Redlands, CA, USA).

Results
A total of 2612 houses were surveyed in the 22 municipalities of Cabo Verde, and 6113 containers were inspected. Of these, immature mosquitoes were detected in 7.5% (n = 458) of all inspected containers, the majority (84.3%; n = 386) located outdoors, and the others (15.7%; n = 72) indoors. No significant differences were observed in the distribution of indoor/outdoor and positive/negative breeding sites (p > 0.05 in both cases) ( Table 1). Of the 458 containers with immature mosquitoes, 57.6% (n = 264) were positive for Ae. aegypti, of which 20.8% (n = 55) were found inside dwellings, and 79.2% (n = 209) were found outside.

Entomological Indices
The average HI, CI, and BI were 8.4%, 4.4%, and 10.9, respectively. Fifteen out of the 22 studied municipalities presented a BI above five. The maximum values were in the municipality of Brava (HI = 25%; CI = 9.7% and BI = 30.2) and the minimum values, below 1%, were found in Sal. In the municipality of Santa Catarina (Fogo island), despite no Ae. aegypti larvae being found, adults were recorded during the survey (Table 3).

Discussion
This study represents the first archipelago-wide analysis of the Ae. aegypti breeding sites in Cabo Verde, which are exclusively domestic containers. This domestic mosquito is one of the most important arthropod vectors of arboviruses worldwide, namely dengue, Zika, chikungunya, and yellow fever [51]. Although there has been an ongoing focus on vaccine development for prevention of these diseases, vector control has been the key strategy to control or prevent the transmission of mosquito-borne arbovirus infections [52]. In previous studies, Ae. aegypti populations infected with DENV-2 and DENV-4 were found in Cabo Verde, with high vector competence to transmit DENV-2 and DENV-3, and to be infected with and transmit chikungunya and yellow fever [53][54][55].
In Cabo Verde, recent entomological data on the mosquito species distribution in the nine inhabited islands are missing, and data regarding Ae. aegypti are scarce. Although several factors influence a breeding site's availability and mosquito distribution, in this study, Ae. aegypti was mostly found in water drums used in water storage by the population, which corroborates previous results [56][57][58][59][60].
High vector density and susceptible human population are key factors to arbovirus disease outbreaks. Between these two, the first one is the major contributor and can be estimated by Aedes indices, such as CI, HI, and BI [61]. These larval indices provide useful information to plan, evaluate, and monitor the efficacy of vector control interventions. The BI is the most used, considering the number of positive containers and searched houses. We noticed variation in the indices among the municipalities, with some municipalities showing BI as high as 31.9 and others 0. This variation is highly dependent on a container's availability, which can be affected by numerous factors, such as seasonality (rainy or dry season), local population habits, customs, and traditions, and local microclimate. The irregularities in the water distribution force people to store water in containers; thus, this factor plays an important role in the ecology of larval mosquito habitats [59]. Positive containers found outside of dwellings were associated with domestic animals and agriculture (not statistically tested), but further studies should be done to approve or disprove this claim.
In this study, the fieldwork occurred during the dry season and we observed that Ae. aegypti is extremely adapted to domestic habitats. It is also important to note that no correlation was found between indoor and outdoor containers in this study.
Thirteen municipalities presented a BI above the epidemic risk threshold [40,62]. Our results suggest that Ae. aegypti is well established in all archipelago islands, and several municipalities in Cabo Verde are at risk of arboviral disease outbreaks.

Conclusions
Aedes aegypti is a major threat to public health in Cabo Verde, considering the values of larval indices found in this study associated with previous studies showing Ae. aegypti vector competence to diseases registered and not registered in Cabo Verde [54,63], as well as resistance to insecticides in the archipelago [25,26]. Nevertheless, more studies are crucial to evaluate this species' resistance to more insecticides used in the public health context. We also recommend implementation of a countrywide vector control strategy with environmental management and modification, according to general international guidelines [31]. A program for monitoring Ae. aegypti, carried out by each municipality's health delegation, with the support of the Ministry of Health, is also desirable.