Mem Inst Oswaldo Cruz, Rio de Janeiro, 98(2) March 2003
Dispersal of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in an Urban Endemic Dengue Area in the State of Rio de Janeiro, Brazil
Laboratório de Transmissores de Hematozoários, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
IFundação Nacional de Saúde-Distrito Sanitário de Nova Iguaçu, Nova Iguaçu, RJ, Brasil
IIDepartamento de Química, Instituto Nacional de Controle e Qualidade e Saúde-Fiocruz, Rio de Janeiro, RJ, Brasil
IIIFlorida Medical Entomology Laboratory, University of Florida, Florida, USA
Experimental releases of femaleu00a0Aedes (Stegomyia) aegyptiu00a0andu00a0Aedes (Stegomyia) albopictusu00a0were performed in August and September 1999, in an urban area of Nova Iguaçu, State of Rio de Janeiro, Brazil, to estimate their flight range in a circular area of 1,600 m where 1,472 ovitraps were set. Releases of 3,055u00a0Ae. aegyptiu00a0and 2,225u00a0Ae. albopictusu00a0females, fed with rubidium (Rb)-marked blood and surgically prevented from subsequent blood-feeding, were separated by 11 days. Rb was detected in ovitrap-collected eggs by atomic emission spectrophotometry. Rb-marked eggs of both species were detected up to 800 m from the release point. Eggs ofu00a0Ae. albopictusu00a0were more numerous and more heterogeneously distributed in the area than those ofu00a0Ae. aegypti. Eggs positively marked for Rb were found at all borders of the study area, suggesting that egg laying also occurred beyond these limits. Results from this study suggest that females can fly at least 800 m in 6 days and, if infected, potentially spread virus rapidly.
Female mosquitoes may disperse to find mates, food or oviposition sites. Dispersal to seek a host is epidemiologically important as the mechanism whereby female mosquitoes acquire and disseminate pathogens. Additionally, dispersal for oviposition is relevant to disease propagation. Females of the classic dengue vector Aedes aegypti (Linnaeus) distribute their eggs among several oviposition sites (Christophers 1960, Reiter et al. 1995) and frequently take multiple blood meals within a single gonotrophic cycle (McClelland & Conway 1971, Trpis & Hausermann 1986, Scott et al. 1993), which increases dispersal of their progeny. According to Trpis and Hausermann (1986), Ae. aegypti also disperse to search for carbohydrates and a mate.
Studies on the dispersal of Ae. aegypti show females generally fly 100-500 m (McDonald 1977, Trpis & Hausermann 1986, Muir & Kay 1998). This range is short compared to other mosquitoes, such as Aedes tae-niorhynchus (Wiedemann) which disperse up to 10 km (Provost 1957). Some authors believe that Ae. aegypti fly only a short distance from their emergence sites (Reuben et al. 1975, Trpis & Hausermann 1986, Trpis et al. 1995, Service 1997). However, a study conducted in urban San Juan, Puerto Rico, showed that female Ae. aegypti could, in a few days, travel at least 441 m from a releasing point (Reiter et al. 1995). Recapture studies of marked female Aedes albopictus (Skuse), another potential dengue vector (Schatzmayr 2000), showed a maximum dispersal of 400-600 m (Rosen et al. 1976, Niebylski & Craig 1994). This same species flew no more than 200 m in studies in Hawaii and Japan (Bonnet & Worcester 1946, Mori 1979).
Despite its importance to dengue transmission, dispersal of Ae. aegypti and Ae. albopictus in urban Brazil has not been examined. In order to assess and compare their pre-oviposition dispersal, we fed adult females on blood enriched with rubidium chloride (RbCl) a method developed by Kimsey and Kimsey (1984). The release of Rb-marked females was carried out in an endemic dengue area of Southeastern Brazil, and dispersal was measured by the detection of Rb-labeled eggs in ovitraps.
MATERIALS AND METHODS
Study area - Nova Iguaçu is a municipality of the State of Rio de Janeiro, Brazil, located at 22°45'S 43°27'W, 25 m altitude, with an area of 566.6 km2 (Fig. 1). Atlantic forest is still present in 35% of this area, although it is predominantly urban. The climate is hot and humid, with annual temperature and rainfall averages of 22°C and 2,100 mm, respectively. Nova Iguaçu has an approximately population of 850,000 inhabitants, with an average density of 1,414 inhabitants/km2 (Prefeitura Municipal de Nova Iguaçu 1999, Honório 1999, Honório & Lourenço-de-Oliveira 2001). The release point was chosen as the center of a circle with an 800 m radius that encompassed seven districts of Nova Iguaçu: Ambaí, Bela Vista, Boa Esperança, Figueira, Jardim Ocidental, Miguel Couto and Parque Flora (Fig. 1). These districts are essentially residential areas with small shops, have a limited sewage system, and sparse solid waste refuse collection. Figueira is unique among these in having a rural area, with low population density (Fig. 1).
Entomological inspection - Two weeks before the release of Rb-marked mosquitoes, all houses (nearly 5,000) located in the 1,600 m diameter study area were inspected for containers containg immature mosquitoes which were identified and counted. Containers yielded 5,280 immatures identified as 3,055 Ae. aegypti and 2,225 Ae. albopictus. Most of the small containers that represented potential breeding sites forAedes mosquitos were eliminated or removed by collectors, and the others, particularly large ones such as water reservoirs, were treated with Temephos® (20g/1000 l of water) to kill the larvae. These procedures were done to avoid increases in mosquito density attributable to oviposition by released females.
Oviposition traps - 1,472 ovitraps were placed in shaded peridomestic areas located at ground level and protected from the rain. In order to distribute the ovitraps homogeneously, the 800 m radius circular area was divided in to five concentric areas of 0-100 m radius (23 ovitraps), 100-200 m (69 ovitraps), 200-400 m (276), 400-600 m (460) and 600-800 m (644), respectively (Fig. 1). Each ovitrap contained 270 ml of tap water and 30 ml of a 10% by weight aqueous hay infusion that had been previously incubated for 7 days. A 12 cm x 5 cm wooden paddle was placed in each ovitrap. On the third day after setting all paddles were collected and replaced with new ones. Throughout the study, missing ovitraps were replaced with new traps.
Mosquitoes - Ae. aegypti and Ae. albopictus mosquitoes used in the experiments were F3 from laboratory colonies derived from large numbers of eggs collected in Ambaí. Mosquito colonies were maintained in an insectary at 27 ± 1°C and 80 ± 10% humidity. Larvae were fed with fish food (Tetramin®) and reared according to Consoli and Lourenço-de-Oliveira (1994). Adults of both species were provided with sucrose solution from emergence to one day before blood feeding.
Blood meal mixed with RbCl - Four to six day old F3 females were separated from males in cardboard cylindrical cages (9 cm height, 9 cm diameter). These females were offered defibrinated sheep blood containing 0.025 M RbCl (Reiter et al. 1995). Rb is an alkali metal, non-toxic to plants, insects and other animals, that is incorporated into ovarian follicles and accumulates in mosquito eggs if it is present in the blood meal (Kimsey & Kimsey 1984, Anderson et al. 1990). Eggs containing Rb were detected by atomic emission spectrophotometry (Reiter et al. 1995). A Rb-marked blood meal was offered to mosquito females in a feeding apparatus described by Rutledge (1964). Females that fed to repletion were kept in cages (60 x 60 x 60 cm) in the insectary. Twenty-four hours after the Rb-blood meal, the tip of proboscises of all Ae. aegypti and Ae. albopictus females were cut off with a dissection scissors to prevent subsequent feeding. This procedure prior to release was recommended by the institutional ethics committee to avoid the risk of increased virus transmission in the dengue endemic area. Available evidence suggests that proboscis amputation should not affect post-feeding dispersal for oviposition (Shirai et al. 2000).
In order to assess both timing of oviposition and effectiveness of Rb detection, 12 females of each species received an Rb-blood meal and were housed in the laboratory in individual glass cylinders (7 x 2.5 cm) containing filter paper and dechlorinated water at the bottom. Eggs were recovered up to five days after blood feeding and tested for the presence of Rb as described below. Rb was detected in 9/12 (75%) Ae. aegypti and 11/12 (91%) Ae. albopictus. These frequencies were not significantly different from one another (P = 0.771, Fisher exact test).
Release of Rb-marked females - One day after a Rb- blood meal, gravid females were released at dusk (1700-1900 h) at the center of the study area (Fig. 1). Two Rb female releases were performed, one for each species. A total of 3,055 Ae. aegypti and 2,225 Ae. albopictus females were released on August 17 and 28, 1999, respectively. The 11 day interval between releases was considered sufficient to distinguish between the species because the chances of Ae. aegypti laying eggs more than 13 days after a single blood meal, and deprived of additional blood meals, is very small (Christophers 1960).
Ovitrap collections and Rb-marked eggs detection - On the third day after releases, the wooden paddles in each ovitrap were collected and replaced by fresh paddles. The replacement paddles were subsequently collected after an additional three days. The number of eggs collected per paddle was estimated after examination with a stereomicroscope. All eggs from a positive paddle were gently transferred to a 10 ml glass flask to which 2 ml of nitric acid were added. The flasks were heated to 100ºC on a hot plate for 30 min, followed by the addition of 1 ml distilled water. Each sample was analyzed by atomic emission spectrophotometry (Perkin Elmer model 5500). Eggs from laboratory colonies of Ae. aegypti and Ae. albopictus were used as controls for each assay.
Statistical analysis - Data were analyzed by chi-square and Fisher exact tests using SPSS for Windows (8.0). Chi-square tests were used to compare frequencies of Rb positive ovitraps in different portions of the study area.
Egg trap collection for Rb detection - Of the 1,472 ovitraps placed in the study area, 1,347 were recovered on the third day after the release of Rb-marked Ae. aegypti females. On the sixth day 1,411 out of 1,472 ovitraps were recovered. The remaining 186 paddles (6.3%) were missing. For Ae. albopictus, on the third day after release, 1,433 of 1,472 ovitraps were recovered and on the sixth day, 1,381 of 1,472 ovitraps. In total, 120 paddles (4.1%) were missing. For both species, more Rb-eggs were detected on the sixth day (Tables I, II).
Dispersal of Ae. aegypti - Fifty-one ovitraps (17 on day 3 and 34 on day 6) were found with Rb-marked Ae. aegypti eggs (Table I, Fig. 2). Ambaí and Jardim Ocidental were the districts with the highest number of Rb-positive eggs (28 positive ovitraps out of 1,400 set), followed by Miguel Couto (8), Figueira (6), Boa Esperança (5) and Bela Vista and Parque Flora (4). Ae. aegypti Rb-marked eggs were found up to 800 m from the release point. None of the 23 ovitraps placed up to 100 m from the release point was positive for Rb- marked Ae. aegypti eggs. In the 100-200 m area, where 118 paddles were recovered, only 2 (1.7%) were positive (Table III). In the 200-400 m area, 12 had Rb-marked Ae. aegypti eggs (or 2.3% of the 515 recovered). Out of the 850 paddles collected in the 400-600 m, 20 (2.3%) were positive while in the 600-800 m area there were 17 (1.3%). The percentages of positive paddles were not significantly different among areas divided either as concentric circles (c2 = 3.3, df = 4, P = 0.503; Table III), pie-shaped wedges of equivalent size (c2 = 4.76, df = 7, P = 0.688; Table IV) or by districts (c2 = 2.37, df = 4, P > 0.05; Table I).
Dispersal of Ae. albopictus - A total of 304 ovitraps were recovered with Rb-marked Ae. albopictus eggs: 95 on day 3 and 209 on day 6 after release of Rb-marked females (Table II, Fig. 2). As for Ae. aegypti, Ambaí and Jardim Ocidental were the districts with the most Rb-marked eggs (127 positive ovitraps), followed by Miguel Couto (79), Figueira (47), Bela Vista and Parque Flora (28) and Boa Esperança (23).Ae. albopictus Rb-marked eggs were also found up to 800 m from the release point. A total of 8 ovitraps placed up to 100 m from the release point were positive for Rb-marked eggs (18.2%). In the 100-200 m area, where 127 paddles were recovered, 21 (16.5%) were positive. In the 200-400 m area, 50 had Rb-marked eggs (9.5% of the 525). Out of the 875 paddles collected in the 400-600 m area, 82 (9.3%) were positive and in the 600-800 m area there were 143 (11.4%) positive. Ae. albopictus Rb positive eggs were found in all blocks in the experimental area, even in those located as far as 800 m for the release point. The percentages of positive paddles were significantly different among areas divided either as concentric circles (c2 = 10.08, df = 4, P = 0.039; Table III), pie-shaped wedges of equivalent size (c2= 32.24, df = 7, P< 0.001) or by districts (c2= 10.43, df = 4, P = 0.034; Table II).
In the preliminary laboratory experiment in which females were kept individually in glass vials, 75% of Ae. aegypti and 91% of Ae. albopictus females laid Rb-marked eggs. Although this difference between species was not statistically significant, the trend corresponds to the poorer recovery of Rb-positive Ae. aegypti in the field experiment. Although more Ae. aegypti females were released in the field than Ae. albopictus, more Rb-positive paddles of the latter were collected.
The flight of mosquitoes is influenced by factors such as species-specific traits, oviposition site availability, climate (e.g., wind, humidity, temperature, rainfall), terrain, vegetation, housing characteristics (for synanthropic species) and blood source (Forattini 1962). Our experiment changed the number and location of available oviposition sites in the study area, and we cannot discount the possibility that these alterations influenced the dispersal patterns of the released mosquitoes.
Rb-marked Ae. aegypti eggs were found as far as 800 m from the release point (Table I, Fig. 2). These results indicate a greater flight range for Ae. aegypti than those estimated by other authors who suggest £ 200 m (Morlan & Hayes 1958, Sheppard et al. 1969, Reuben et al. 1975, Trpis & Hausermann 1986).
In Brazil, female Ae. aegypti were released inside a house in an urban area of the State of Bahia and the maximum recovery distance after 24 h was 120 m (Shannon et al. 1930). In a second experiment, the maximum dispersal distance was 300 m for two individuals (Shannon & Davis 1930). Because of the difficulties in recapture of Ae. aegypti, owing to its escape ability and behavior when approaching for blood (Christophers 1960), both values should be considered underestimates of the true flight range of the species. In a third experiment, 12,000 females were marked with methylene blue and released in a boat anchored in the sea 1 km from the coast (Shannon & Davis 1930). Only eight specimens were recovered on land, indicating that, dispersal of up to 1 km is possible for Ae. aegypti. Conditions were also unnatural for gravid Ae. aegypti females released in the desert of Israel (Wolfinshon & Galun 1953), a zone free ofAe. aegypti with no houses that could act as adult attractants. Under these conditions, oviposition of Ae. aegypti was recorded as far as 2,500 m from the release point.
In our field study, paddles were collected on days 3 and 6 after release, that correspond to 4 and 7 days after the blood meal, respectively. Paddles with Rb-positive eggs were more numerous on day 6. BecauseAe. aegypti generally lays eggs 3 to 5 days after a blood meal (Christophers 1960), some females may not have been ready to oviposit before the first paddle-collection.
The number of ovitraps with Rb-positive eggs was highest for Ae. aegypti at Ambaí and Jardim Ocidental (Table I), but the number of ovitraps set there was also greater, as these districts encompass most of the study area (Fig. 1). Positive ovitraps were also common along the railroad that crosses Parque Flora and Figueira, which are mostly poor neighborhoods of rudimentary housing in an undulating terrain. In these districts the water supply is irregular, which leads inhabitants to store water in containers. It is likely that these containers are used as oviposition sites by container dwelling mosquitoes.
The recovery of marked Ae. aegypti eggs was not significantly different among 5 concentric circles up to 800 m from the release point. The overall low recovery of Rb-positive paddles for Ae. aegypti contributed to the absence of detectable differences in dispersal of this species among districts or sectors of the study area.
Studies on Ae. albopictus dispersal have not been previously done in Brazil. In Missouri, USA, non-blood fed Ae. albopictus females marked with fluorescent dyes dispersed up to 525 m (female) and 225 m (male) (Niebylski & Craig 1994). We observed a dispersal range of at least 800 m for this species. In the 600-800 m area 143 ovitraps were positive for Rb-marked eggs, (11.4% of 1,253 paddles).
Egg distribution in the study area was significantly heterogeneous for Ae. albopictus but not for Ae. aegypti(Tables III, IV, Fig. 2). When Ae. albopictus were released, winds were blowing to the east, which could have contributed to biased dispersal. Although the number of Ae. albopictus released females (2,225) was lower than that of Ae. aegypti (3,055), more marked Ae. albopictus eggs were recovered in ovitraps (Table II). The highest frequency of Rb-marked eggs occurred in the 100-200 m area (Fig. 2). Frequencies of Rb-marked eggs of Ae. albopictus were significantly different among the districts, among concentric circles, and among pie-shaped wedges of equivalent size. `Hot spots'of recovery were concentrated in Figueira and Miguel Couto, mainly at the 600-800 m area. The recovery of Rb-positive ovitraps was significantly higher in sectors 1 and 2 than in sectors 7 and 8 (Table IV). Unlike sectors 7 and 8, the terrain of sectors 1 and 2 is hillier, the houses have trees in the yards, and are separated by patches of pasture. This result agrees with known preferences of this species for rural and vegetated habitats (Hawley 1988).
The dispersal of Ae. aegypti and Ae. albopictus at least 800 m occurred in a dengue endemic area within a 6 day period. It is known that the extrinsic incubation period of dengue virus, the interval of time from acquisition of infectious blood to subsequent transmission, is approximately 11-14 days for Ae. aegypti(Siler et al. 1926). If a female feeding once on a viremic inhabitant can fly at least 800 m in 6 days, it can spread the virus very rapidly. This extensive flight range must be taken into account in focal insecticidal treatment in response to yellow fever or dengue outbreaks. A new dengue case could appear at 1 km distance from another in few days. This has been observed in previous dengue epidemics, when time for control action measures was minimal between the first confirmed cases and the rapid spread of the disease (Kuno 1995). Rapid dispersal would also apply to Aedes re-infestation of areas considered free of these vectors. Our results suggests that from an initial focus, in 6 days one might expect house infestation of almost 1 km2 due to female dispersal independent of human activies, such as transport and trade. It is clear that female dispersal by flight has the potential to be very important for the spread of dengue vectors, either between adjacent cities or in urban centers such as Rio de Janeiro.
To Dr Pedro Cabello for guidance on statistical procedures, Drs MG Rosa-Freitas, D Valle, François Noireau, BW Alto, and SP Yanoviak for comments and critical review of the manuscript. To Dr Silvana Jacobs for her valuable advice. To Genilton Vieira and James M Newman who assisted the authors with the figures, and to the technicians of Fundação Nacional de Saúde for logistical support in the field.
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