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Update on the distribution of Aedes albopictus (Diptera: Culicidae) in Misiones, Argentina

ABSTRACT The Asian tiger mosquito ( Aedes albopictus, Diptera: Culicidae) has spread rapidly in the last 30 years from its native region in Southeast Asia. In Argentina, studies on its potential distribution suggest that this species could be found in temperate zones of the province of Buenos Aires. However, since its initial detection in 1998 Ae. albopictus is bounded to the subtropical province of Misiones. To evaluate the presence and abundance of Ae. albopictus in the Northeast of Argentina, we preliminarily evaluated the presence of this vector by analyzing its presence in tires of 20 cities belonging to the province of Misiones and four cities in Northern Corrientes, and then performed an evaluation of the vector in the towns where the vector was detected. Aedes albopictus was present only in two cities of Misiones: Eldorado and Colonia Aurora. Aedes aegypti and Ae . albopictus accounted for 86% of the individuals collected in the domiciles of both towns. In Colonia Aurora both species were in similar abundances suggesting a co-dominance. The present study extends the austral distribution of Ae. albopictus in Argentina to the city of Colonia Aurora where the highest abundance recorded in Argentina was detected. Nevertheless, the reasons of its bounded distribution in the region are not known.


INTRODUCTION
In the world, the Asian tiger mosquito Aedes albopictus (Skuse, 1894) (Diptera: Culicidae) is a competent vector for at least 22 arboviruses. These include chikungunya and the four serotypes of dengue virus, mainly transmitted by Ae. aegypti 1 .
This species has spread rapidly in the last 30 years from its native region in Southeast Asia. It is an invasive mosquito whose dry-resistant eggs have facilitated its transport to other continents through used tires and other artificial containers 1,2 . At present, this mosquito is well established in different parts of North and South America, Southern Europe, and islands of Oceania and part of Africa 3 .
Aedes albopictus was discovered almost simultaneously in the United States and Brazil in the mid 1980s 2 . This species had previously been detected in tire shipments in North American ports and in a Tennessee cemetery. The Northern limit of this species corresponds roughly to the -5 °C isotherm, as predicted by cold-resistance studies 3 . The species entered Florida in 1986, from the temperate North and had already colonized all Florida counties by 1994, coinciding with a decline in Ae. aegypti's population 2 .
In Central and South America, this species was reported in 16 countries or islands 4,5 . In Brazil, 24 out of 27 States are already colonized 6 , but in Argentina, Ae. albopictus is bounded to the province of Misiones since its discovery in 1998 7 . Adult, pupae and larvae were recorded at Iguazu National Park and in the cities of San Antonio, Eldorado, Puerto Iguazu and Comandante Andresito 7-9 ( Figure 1). In 1998, the only ecological study to date reported a relative low abundance when compared to Ae. aegypti (≈1: 10) in the municipality of Eldorado, and described that larvae and pupae of both species were collected in the same artificial breeding sites 8 .
To date, studies have described that Ae. albopictus predominates in rural areas and Ae. aegypti in highly urbanized habitats, while both are present in suburban areas 10 . This pattern was proposed as a promoter of the coexistence between species, avoiding direct competition.
Since its dispersion across the world occurred passively through transport and commercial exchange of used tires with presence of preimaginal stages and eggs, the present work has preliminarily evaluated the presence of this vector in tires of different districts of the province of Misiones and Northern of Corrientes; then evaluated the vector in the houses of the cities where its presence was detected.

Study area
We sampled 20 cities of the province of Misiones and four in the North of the province of Corrientes ( Figure 1, Table 1). The study area has a subtropical climate, with

Collection and analysis of data
In January 2014, we collected preimaginal stages present in the study area ( Figure 1, Table 1). We evaluated 5-30 tires per tire deposit in each city (1 to 5 deposits/ city). Up to a total of five deposits were examined in each city, providing that no larvae were found at the first deposit (in some of the cities all deposits available were sampled) 12 .
Pre-imaginal stages were collected and fixed in situ with alcohol 70%.
In March 2015, samples were collected from 50 houses with breeding sites in the towns of Colonia Aurora (20) and Eldorado (30) (Misiones, Figure 1). In Colonia Aurora, sampling was carried out throughout the village. As Eldorado has a greater extension (Table 1), the city was stratified into two areas: one urban and one peri-urban. In each zone, samples were taken from 13 and 17 houses, respectively. All the houses were selected according to the criteria of accessibility and the informed consent of all the owners of the houses was obtained with the approved consent of the Ethics Commission.   Only artificial containers with water in the peridomicile of the houses were examined. Each artificial breeding site was classified according to the type of container. Mosquito larvae were collected by filtration and fixation in alcohol 70%. All specimens collected were transported to the laboratory, examined under a stereoscopic microscope and specimens were identified at the species level 13 .
To evaluate the existence of independence between towns, a Pearson's Chi-squared test was performed using Infostat with the relative abundances of Ae. aegypti (Linnaeus,1762), Ae. albopictus and the rest of the collected species.

Comparison with Eldorado in 1998
In order to compare this study with the situation of Ae. albopictus in Eldorado observed in 1998 8 , we compared the number of positive breeding sites for Ae. albopictus, the number of larvae of Ae. albopictus per artificial breeding site, the percentage of breeding sites for Ae. albopictus with Ae. aegypti, the ratio between Ae. albopictus and Ae. aegypti and the occurrence of other species sharing the breeding site with Ae. albopictus.

RESULTS
In January 2014, Aedes albopictus was present only in two cities out of the 24 sampled ( Figure 1, Table 1). Taking the total number of culicine collected in tire deposits (1772), Ae. aegypti, Cx. quinquefasciatus (Say, 1823) and Ae. fluviatilis (Lutz, 1904) were the most abundant species, corresponding to 89.6% of the sample (Table 1) Regarding the comparison between the 1998 study and ours, in 2015, one can observe that although in the first study the number of inspected containers with water was of 2,237 in comparison with 90 in 2015; in the latter the percentage of containers with Ae. albopictus and the number of individuals of this species was higher (Table 2).

DISCUSSION
The present study extends the austral distribution of Ae. albopictus for Argentina to the municipality of Colonia  7,9 . In the only ecological report, Schweigmann et al. 8 found low abundances in Eldorado, homogeneously distributed. After 20 years, Ae. aegypti remains the most abundant species in this city and occurs simultaneously with the Asian tiger mosquito in all breeding sites. Aedes albopictus continues to be found in a smaller proportion, but in Colonia Aurora both species are the most abundant and they present similar abundances. Regarding the species that share breeding sites with Ae. albopictus, Ae. aegypti remains the species with which it coexists mostly, and to a lesser extent with Li. durhamii, Ae. fluviatilis and Culex species.
Based on the present study and other reports that have also monitored the same study area 9 , the bounded distribution of Ae. albopictus in the province of Misiones is striking, considering that the vector occurs at higher latitudes in Uruguay 4 and is widely distributed in Southern Brazil, more precisely in Rio Grande do Sul State 14 that borders the provinces of Corrientes and Misiones. In this context, it is possible that a greater sampling effort on tires is required to detect Ae. albopictus in the cities of Misiones.
Brazilian studies indicate that it is the most frequent and dominant species in this type of breeding site 15,16 so that searching for Ae. albopictus in tires is expected to retrieve positive results.
Despite this, the reported distribution of Ae. albopictus differs from other studies that predicted that this species could be potentially found to the South of the province of Buenos Aires (≈38° S Latitude) 5,17 . According to Kreamer et al. 5 , one of the most important predictors is the temperature that limits the gonadotrophic cycles of the vector. These studies were based on bibliographic data that investigated the vector survival, information that is unknown for local populations.
Due to the lack of diapause in the South American populations, several studies indicate that the local population of Ae. albopictus could have a tropical origin 18 . It is possible that this attribute influences the distribution of this mosquito. Since warm area insects exhibit a high minimum development threshold temperature 19 , an evaluation of larval development and survival rates of Latin American populations at low temperatures would be necessary to indicate whether temperate zones can affect survival and development. However, the lack of diapause was not a limitation for the expansion of Ae. aegypti, species that also lacks diapause and whose range reaches temperate regions such as the province of Buenos Aires 12 . In this context, it would also be interesting to evaluate other factors that could affect the distribution of the vector in the region, such as eggs mortality, a situation observed in the United States, where eggs of Ae. albopictus have a higher mortality than the ones of Ae. aegypti 20 .
According to the Provincial Institute of Statistics and Census, Province of Misiones 21 , Eldorado is characterized as an urban area, while Colonia Aurora is rural. However, the results of this study differ in part from those observed in the American continent where spatial segregation of both vectors is observed 10 . In our study, in Colonia Aurora, both species were in similar abundances characterizing a co-dominance. According to Braks et al. 10 , Ae. aegypti generally predominates in highly urbanized habitats and Ae. albopictus in rural areas. In that study, they attributed the high abundances of this species in rural environments to a greater competitive advantage compared to Ae. aegypti.
After 20 years since its discovery in the province of Misiones, the reasons for the limited distribution of Ae. albopictus in Argentina remain unknown, considering that it has a great dispersive capacity in other regions of the world 2 . This study demonstrated that Ae. albopictus has not increased its distribution with the speed that was registered in others regions of the world. However, the high abundances in Colonia Aurora and the fact that this species went South in neighboring countries, is warning that this vector has still the potential to expand its geographic niche, posing a risk to the public health in Argentina. For this reason, evaluating the population attributes and interaction with other species and parasites in the region will contribute to understand what factors are affecting the presence and abundance of the vector in the country and to determine the control measures of this vector.

ACKNOWLEDGMENTS
We thank the staff of the Ministry of Ecology and Natural Resources of Misiones for the quick management in granting the sampling permits in the province, and the municipality of Eldorado for their help.

AUTHORS' CONTRIBUTIONS
Arturo A. Lizuain participated on the conception, planning, sampling, analysis, interpretation and writing of the work; Marina Leporace participated on the planning, sampling, interpretation and writing of the work; Maria Soledad Santini and María E. Utgés participated on the planning, interpretation and writing of the work; and Nicolás Schweigmann participated on the planning, sampling, analysis, interpretation and writing of the work. All authors approve the final submitted version.