Aedes aegypti and Aedes albopictus ( Diptera : Culicidae ) : coexistence and susceptibility to temephos , in municipalities with occurrence of dengue and differentiated characteristics of urbanization Aedes aegypti e Aedes albopictus ( Diptera : Culicidae ) : coexistência

Introduction: The aim of the present study was to verify the coexistence between Aedes aegypti and Aedes albopictus populations in municipalities of the States of Paraná and Santa Catarina with different urbanization profiles where dengue occurs and evaluate their susceptibility to the organophosphate temephos. Methods: The number of eggs per ovitrap were counted and incubated for hatching to identify the species. Data analysis of the populations was conducted to determine randomness and aggregation, using the variance-to-mean ratio (index of dispersion). Susceptibility to temephos was evaluated by estimation of the resistance ratios RR50 and RR95. Aedes aegypti samples were compared with the population Rockefeller and Aedes albopictus samples were compared with a population from the State of Santa Catarina and with the Rockefeller population. Results: Coexistence between Aedes aegypti and Aedes albopictus and the aggregation of their eggs were observed at all the sites analyzed in the State of Paraná. Conclusions: All the Aedes aegypti populations from the State of Parana showed alteration in susceptibility status to the organophosphate temephos, revealing incipient resistance. Similarly, all the Aedes albopictus populations (States of Paraná and Santa Catarina) presented survival when exposed to the organophosphate temephos.

Aedes aegypti is the primary vector of viral serotypes that cause dengue and urban yellow fever in the Americas, where the incidence of these arboviruses have increased significantly in the last 25 years 1 .Aedes albopictus is considered a secondary vector of dengue virus in the Old World 2 and in Brazil, its presence was first reported in 1986 3 .Currently, it is widely distributed in the country, particularly in the southern and southeastern regions.However, little is known regarding the susceptibility of Ae. albopictus to insecticides and the influence of coexistence with Ae. aegypti.Currently, Ae. albopictus is not implicated as a transmitter of the dengue virus in Brazil, so there is no control program for this Culicidae.Generally, the occurrence of dengue fever epidemics is directly related to the presence and density of vectors, when viral circulation occurs.These mosquito species can often coexist in artificial containers in urban and periurban localities 4 .However, immature Ae. albopictus may also inhabit natural containers, such as bromeliads, bamboo and holes in the tree trunks 5 .This plasticity of Ae. albopictus to colonize artificial containers and natural breeding sites and to coexist with other species in urban and periurban localities can increase its dispersion to new areas where control is impaired.In addition, since Ae.albopictus can colonize bromeliads, it may also expand its distribution to more protected areas.Most importantly, arbovirus circulation may cause the emergence of diseases within this ecological system 6 .
Due to the ability of both species to coexistent and colonize the same breeding places, it is expected that the pressure exerted by control with insecticides affects these species in very similar manner.Changes in the susceptibility of Ae. albopictus to chemical insecticides, similar to that which has been occurring with Ae. aegypti, could be observed in the near

METHODS
Prophiro JS et al -Aedes aegypti and Aedes albopictus: coexistence and temephos susceptibility future 7,8 .This is a problem that can be detected in advance and resulted in the inclusion of this species in the National Network for the Resistance Monitoring of Ae. aegypti to Insecticides (Rede Nacional de Monitoramento da Resistência de Ae. aegypti, MoReNAa) [9][10][11] .
The emergence of resistant populations has caused serious problems for mosquito control.Changes in susceptibility have been identified for all classes of insecticides, directly affecting the re-emergence of diseases transmitted by vectors 12 .Regardless of important advances in alternative methodologies, chemical insecticides are a powerful tool against vectors and will continue to play an important role in integrated control 13 , at least until the discovery of alternative methods that permit fast, safe, and sustainable control of vectors.
In the process of entomological surveillance, it is very important to monitor the biological behavior of these vectors and the resistance development process 14 .
The objective of this study was to verify the coexistence of Ae. aegypti and Ae.albopictus populations and their aggregation and susceptibility to the insecticide temephos, in municipalities with differentiated urbanization characteristics where dengue occurs.

Area of study and collection of material
In partnership with the Secretary of State for Health of Paraná, oviposition traps (ovitraps) were set with a 500mL of 10% hay solution.The ovitraps (345) were randomly distributed in peridomiciliary areas at various points of the following municipalities: Ubiratã, Santa Helena, Foz do Iguaçu South Sector, and Foz do Iguaçu North Sector.In the City of Ubiratã, two collections were conducted due to low hatching.These municipalities had autochthonous cases of dengue in the summer of 2006-2007.Besides Ae. albopictus populations in the State of Paraná, a population of Ae. albopictus in the town of Tubarão, State of Santa Catarina, was also evaluated, which had no history of temephos application.Monitoring the susceptibility of Aedes spp. the State of Santa Catarina is very important because this state is the only state in Brazil that has no record of autochthonous case of dengue (Figure 1).
The ovitraps were randomly placed per area for 5 days in peridomiciles inside the urban area (in residential neighborhoods and downtown) of the municipalities with confirmed records of dengue and/or Aedes spp.and according to the recommendations of the National Health Foundation 10 .

Study of Aedes populations
In the laboratory, the eggs of each ovitrap were counted (no species distinction) and placed individually for hatching, rearing and subsequent recording of males and females of both species (species distinction).Adults were then placed in cages to obtain the F 1 larvae generation, which was used in the temephos susceptibility bioassays.The whole process, including egg storage and adult breeding, was performed under controlled temperature (25 ± 2°C) and relative humidity (80 ± 10%) under 1h photophase.
Data analysis of the populations to determine randomness and aggregation and the distribution of eggs per ovitrap was calculated using variance-to-mean ratio (index of dispersion), here called equation 1.Values lower than 1 suggest regular or uniform spatial arrangement, values equal to 1 indicate random spatial arrangement, while values significantly higher than 1 show aggregate arrangement 15,16 .
The  The results were analyzed with the program Statistica version 7.0.Only data with p < 0.05 were considered significant.The nonparametric tests Kruskal-Wallis (KW) and Mann-Whitney U (MW) were applied in order to verify statistical differences between the species and municipalities evaluated.

Temephos bioassays
The larvicide used was 90% technical grade temephos, batch 002/2005, manufactured by the Fersol Mairinque Laboratory, City of São Paulo.The bioassays consisted of a dose-response of temephos with 640 late third or early fourth instars of each Ae.aegypti and Ae.albopictus populations.Four replicates of 20 larvae, totaling 80 larvae per concentration were exposed to eight different concentrations of temephos, including the diagnostic dose.This diagnostic concentration was applied to qualitatively detect the presence of individuals resistant to the susceptible strain, 0.0060 mg/L (as previously determined by our laboratory) for this same batch of insecticide, corresponding to twice the CL 99 of the Rockefeller susceptible strain, as recommended elsewhere.Additionally, four replicates of 20 larvae, totaling 80 larvae of each population were exposed to ethanol solvent, as negative control [19][20][21] .
The Rockefeller reference strain was used as control for Ae.aegypti and Ae.albopictus.Due to lack of a reference population of Ae. albopictus for analysis of susceptibility to insecticides, a population of the Rockefeller reference strain was used.
Larval mortality was observed after 24h of exposure to temephos.Larvae were considered to have died when they were unable to reach the water surface when touched.The tests were repeated four times on different days and all tests were performed under controlled temperature (25°C ± 1) and photoperiod (12:12) in a climatic chamber model CDG-347 Fanem® 19,20,22 .

Criteria for evaluation of susceptibility and statistical analysis
The criteria used to detect qualitative changes in susceptibility status of the populations analyzed followed the protocol of Davidson & Zahar 23 : a) mortality above 98% in response to the diagnostic concentration is considered susceptible; b) between 98 and 80% suggests incipient resistance status; and c) less than 80% mortality indicates resistance.
The rate of resistance (RR 50 and RR 95 ) as a quantitative indicator was calculated by dividing the lethal concentrations (LC 50 and LC 95 ) of each population studied by the lethal concentrations (LC 50 , LC 95 ) of the Rockefeller colony, for Ae.aegypti and Ae.albopictus.Resistance levels were classified as low RR < 5.0, medium (5.0 < RR < 10.0) or high (RR > 10.0) 24 .To determine the lethal concentrations (LC 50 , LC 95 ), X 2 test, slope and confidence intervals from the GW-Basic Probit program were used 25 .

Study of Aedes populations
Of the 345 ovitraps installed, 63% were positive for eggs (11,220), which resulted in 6.132 adults (3,222/53% Ae. aegypti and 2.910/47% Ae. albopictus).The positive traps and the percentage of adult males and females in both species, in different regions of collection, are presented in Table 1.
The number of eggs and the number of emerged adults from each of the localities showed significant differences.The relationship between sex ratio was close to the expected, 1:1 in all municipalities for both species (Table 1).
The variance-to-mean ratio (index of dispersion) for eggs of Ae. aegypti and Ae.albopictus exhibited values significantly higher than 1, indicating highly aggregated distribution in all the municipalities studied (independent of the species).Similarly, the values obtained with the k parameter of the negative binomial also indicated aggregate distribution of egg samples in all the municipalities studied, whose values were always positive between zero and eight (Table 2).

Temephos bioassays
Following exposure of the populations evaluated to the diagnostic concentration 0.0060mg/L, analysis verified that the population of Foz do Iguaçu, in both the south and north sectors, presented status resistant.For the municipalities of Ubiratã and Santa Helena, the Ae.aegypti populations were susceptible to temephos.All the Ae.albopictus populations evaluated, from Paraná and the population of Tubarão, Santa Catarina, showed survival when exposed to the diagnostic concentration of the organophosphate temephos (Table 3).
For the populations of Ae. aegypti from municipalities of the State of Paraná submitted to bioassays using different concentrations (CM) of temephos, the RR 95 was: 1.70 for Ubiratã, PR; 1.65 for Santa Helena, PR; 3.62 for Foz do Iguaçu south, PR; and 3.13 for Foz do Iguaçu north, PR (Table 4).For the populations of Ae.Albopictus from municipalities of Santa Catarina and Paraná submitted to bioassays using different concentrations (CM) of temephos, the RR 95 was: 2.01 for Tubarão, SC; 1.97 for Ubiratã, PR; 2.36 for Santa Helena, PR; 2.23 for Foz do Iguaçu south, PR; and 2.58 for Foz do Iguaçu north, PR (Table 4).
In general, the slope values of the Ae.aegypti and Ae.albopictus populations studied were lower compared to those obtained of the Rockefeller strain.This finding confirmed their heterogeneity compared to the reference strain and the differences in their response to the insecticide.The lethal concentrations and resistance rates of populations in all the municipalities are presented for comparison in Table 4.

Study of Aedes populations
In the municipality of Ubiratã, collections 1 and 2, differences occurred between the predominance of species collected for Ae.albopictus (Ubiratã 1: 88% and Ubiratã 2: 84%) and Ae.aegypti (Ubiratã 1: 12% and Ubiratã 2: 16%, respectively).Ae. albopictus predominated where there was higher density of inhabitants in urban centers than in rural areas.The municipality of Ubiratã is a wooded site, which may have favored the presence and/or increased survival of Ae. albopictus in relation to Ae. aegypti.
In this study, coexistence of Ae. aegypti and Ae.albopictus was observed in all the municipalities studied in the State of Paraná, similar to that observed by Gomes et al 26 and Fantinatti et al 27 .Fantinatti et al 27 evaluated the abundance and aggregation of Ae. aegypti and Ae.albopictus eggs in several municipalities of Paraná and described the coexistence of these species in all the sites studied.
It is important to emphasize that the ovitraps in Ubiratã were installed in 2007, in the same period of the dengue outbreak, which registered cases of autochthonous dengue cases.Currently, there is no official record of the dengue virus transmission by Ae. albopictus in Brazil.Nevertheless, only by viral serotype isolation and the confirmation of virus transmission competence of Ae. albopictus

Bioassays with larvicidal
Mortality among larvae using the Rockefeller diagnostic concentration on Ae. aegypti populations in Foz do Iguaçu north and south was less than 80%, indicating that the process of selective resistance has been established.Only the Ae.aegypti populations of Ubiratã and Santa Helena were considered susceptible to temephos.
Although Ae. albopictus is not targeted in the control programs, all species populations from the State of Paraná and Santa Catarina were analyzed and showed survival against the organophosphate temephos when exposed to the diagnostic concentration.These results suggest that in locations where coexistence of Ae. aegypti and Ae.albopictus occurs both species are being exposed to the same process of selective pressure due to the insecticides applied.Thus, the high co-occurrence of Ae. aegypti and Ae.albopictus in Brazil, in areas under intense selective pressure of insecticides, may justify the survival of Ae. albopictus populations tested for temephos.These results also suggest that Ae. albopictus populations could become resistant to temephos in the near future, as is currently occurring with Ae. aegypti in several Brazilian states.In addition, the laboratory sample of Ae. albopictus from Santa Catarina probably originates from an environment with previous use of organophosphates in agriculture and thus, certain mechanisms of persistence or even resistance to this compound may have been previously selected.
Changes in the susceptibility status of Ae. aegypti to temephos have been previously reported in Brazil and in Malaysia 29 , Thailand [30][31][32] , India 33 , Cambodia 34 and Venezuela 24 .For Ae. albopictus, monitorization of the development of resistance to temephos has been reported in Malaysia [35][36][37] and Thailand 32,38 , India 33  The RR 50 and RR 95 values determined by Duque et al 41 for Ae.aegypti populations of Foz do Iguaçu, collected in 2005, were 2.6 and 3.9, respectively.In the present study, the values of RR 50 and RR 95 , determined for the populations of Foz do Iguaçu, collected in 2007, were 2.29 and 3.62 for Foz do Iguaçu south and 2:23 and 3:13 to Foz do Iguaçu north, respectively.In comparisons between the values of RR 50 and RR 95 of Duque et al. 41 with the RR 50 and RR 95 of this study, a decrease was verified; however, to explain the decrease in RR 50 and RR 95 values, long-term monitoring of these populations is required.
The RR 50 and RR 95 values observed in Paraná and Santa Catarina for the populations studied are considered low, similar to those for some municipalities in São Paulo 42,43 .However, in several Brazilian states (Rio de Janeiro 7 , Alagoas 7 , Sergipe 7 and Ceará 8 ), medium and high resistance status was observed 21 .According to Câmara et al 44 and Duque et al 41 , the southern region of Brazil shows lower values for RR 50 and RR 95 compared with other regions.This is probably due to reduced vector densities in the cold seasons, together with lower selective pressure due to less intense use of insecticides.
Although the Ae.aegypti and Ae.albopictus populations tested showed low levels of susceptibility to temephos, constant resistance monitoring of these populations to insecticides is of great importance.Such studies can provide early warning of the problem and alert us to the need for new strategies to control the vectors of dengue in southern Brazil in the coming years.

FIGURE 1 -
FIGURE 1 -Map of Brazil, showing the cities analyzed in the State of Parana and Santa Catarina, Brazil.
aggregation index was indicated by the k parameter of the negative binomial distribution.Negative k values indicate uniform distribution, low and positive values (k < 2) indicate a highly aggregated arrangement, k values ranging from 2 to 8 indicate moderate aggregation and k values above 8 (k > 8) indicate a random arrangement 16-18 .m RESULTS and Italy39 .Following the criteria of Mazzari & Georghiou 24 , all Ae.aegypti (PR) and Ae.albopictus populations (PR and SC) presented RR 50 and RR 95 values at low levels.The same was determined by Duque et al40 and Duque et al41 for Ae.aegypti populations in other municipalities of the State of Paraná, including Curitiba, Foz do Iguaçu, Paranavaí, Maringá, Ibiporã, Cambé and Jacarezinho.

TABLE 1 -Aedes aegypti and Aedes albopictus collected with ovitraps in the municipalities of Ubiratã, Santa Helena, Foz do Iguaçu north and Foz do Iguaçu south during the summer of 2006-2007.
*Ovitraps positive: number of positive ovitraps x 100/total number of ovitraps, n: number total, ♀: female.

TABLE 3 -Bioassays with the diagnostic concentration 0.0060mg/L (calibrated with the Rockefeller strain) in larvae of Aedes aegypti of municipalities of Paraná and Aedes albopictus of Paraná and Santa Catarina States, in the summer of 2006-2007.
SC: State of Santa Catarina, PR: State of Paraná.