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Differences in performance of Aedes aegypti larvae raised at different densities in tires and ovitraps under field conditions in Argentina
Arnaldo Maci1
Instituto de Limnologa Dr. Raul A. Ringuelet, ILPLA, C. C. 712, 1900 La Plata, Buenos Aires, Argentina Received 21 February 2006; Accepted 3 August 2006 ABSTRACT: Alteration of fitness components was assessed in the yellow fever mosquito, Aedes aegypti, in automobile tires and vases (ovitraps) under field conditions. Larval numbers were manipulated in both kinds of containers to compare low, high, and control (natural) densities. Densities were set from a census of a wild population, then doubling and reducing to half the mean crowding, m*. Artificially altered densities were not high or low enough to produce differences among treatments. Tires generated more vigorous larval populations and females with higher fecundity than did small containers, although the mortality was more intense. Journal of Vector Ecology 31 (2): 371-377. 2006. Keyword Index: Mosquito larvae, intraspecific competition, Aedes aegypti, artificial containers, Argentina.

INTRODUCTION Water-filled artificial containers are distinctive habitats for many aquatic insects, and numerous individuals sharing limited space and food can be found in a single container. Mosquitoes are common container-breeding dwellers and often undergo inter- and intraspecific interactions that can generate phenotypical variations among individuals of one population. Such variability, in addition to a genetic component, is a result of the history during preimaginal development (Fish 1985), which is an important force affecting population dynamics. Intraspecific competition during the larval stage alters adult fitness (Christophers 1960, Bradshaw and Holzapfel l986). In immature stages, a shortage of resources generates longer larval development as well as lower success and smaller size at metamorphosis. In adults, body size, survival, fecundity, mating success, and flight capacity are all reduced (Steinwascher 1982, Reisen et al. 1984, Fisher et al. 1990, Bradshaw and Holzapfel 1992, Bradshaw et al. 1993, Hard and Bradshaw 1993). All these demographic parameters are fitness components, and they ultimately impact on population growth. Several studies have demonstrated the importance of using indexes of performance that estimate population growth, such as R0 (Fisher et al. 1990), r (Livdahl 1984, Livdahl and Sugihara 1984), (Pianka 1988) and (Leonard and Juliano 1995, Grill and Juliano 1996), instead of quantifying fitness components. However, such indexes require the knowledge of specific attributes such as survival under natural conditions, preoviposition period, and the relationship between fecundity and adult weight. These attributes have been recorded in the yellow fever mosquito, Aedes aegypti L. from the U.S.A. (Grill and Juliano 1996, Juliano 1998), but they differ in some extent according to the population under study. There is a lack of knowledge about such attributes for strains of Ae. aegypti from Argentina. Although there are papers about phenology (Campos and Maci 1996,

Micieli and Campos 2003, Vezzani et al. 2004, Carbajo et al. 2004), habitat characteristics (Vezzani et al. 2001), and biological control (Mart et al. 2004) of Ae. aegypti from Argentina, there is a lack of experimental studies on wild populations. The species was considered eradicated from Argentina after a countrywide campaign in 1963, but reinfestation was detected in the north of the country by 1986 (OPS 1990). Aedes aegypti spread as far south as Buenos Aires Province (Campos 1993), and the mosquito distribution includes northern and central Argentina above 35 S (Curto et al. 2002). The species is an effective vector of dengue, the risk of which has increased in recent years in Argentina (Avils et al. 1999), mainly due to travelers from neighboring, epidemic countries (Seijo et al. 2000). In fact, there were reports of dengue outbreaks in Argentina (Avils et al. 2000), including several cases in Buenos Aires (Seijo et al. 2000). This paper focuses on the effects of either increasing or reducing the larval density of Ae. aegypti in a field population in Argentina. Two kinds of artificial containers (tires and vases) were compared because they are common breeding habitats for Ae. aegypti in urban areas and are usually used in outdoor tests. In this experiment, a natural substrate was used as a source of nutrients for the larvae that allows a more realistic approach than using artificial food (such as aquarium fish food, liver powder, yeast or ground dog chow), commonly employed in other studies. The objectives were to determine (1) to what extent fitness components (pupal weight, total biomass, survivorship, development time, and female fecundity) are affected if larval density is artificially increased or diminished, and (2) if these parameters differ between two common breeding places for Ae. aegypti. Research assistant, Cientficas, CIC.
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MATERIALS AND METHODS Study area This work was conducted at Florencio Varela, 24 km south of Buenos Aires City (34 50 S, 58 06 W), Argentina. The study area was located at Instituto de Limnologa (ILPLA, University of La Plata), in an 11-hectare campus surrounded by a dense urban area at least 100 m away from the nearest houses, with scattered large buildings and tree clusters. The artificial containers used in this study were set under a 30 m-long line of cypresses (Cupressus sp.) separated from each other by approximately 0.5 m. The line of cypresses separated a large building from a grassy open land measuring 60 x 40 m. Weather was temperate to hot (ranging 15-32 C) and humid during spring and summer, with year-round rains. Artificial containers As breeding habitats, 46 small containers (ovitraps) and 46 automobile tires were used. Ovitraps were black plastic vases (13 cm high x 6 cm diameter) lined with filter paper, numbered and fixed with wire to the trees at 80 cm height, and separated from each other by 1 m. Automobile tires were cut in half, numbered, and placed with the opening up, leaning on the ground, and separated from each other by 0.5 m. Ovitraps and tires were set in October 2000 and partially filled with rainwater (100 ml in the ovitraps and 1 liter in the tires) to facilitate natural colonization by mosquitoes. Fluctuations of water level in both types of traps relied on weather conditions, but water was added when the volume was about 50 ml (ovitraps) or 500 ml (tires). Containers were checked daily with a flashlight to detect the presence of mosquitoes and to measure water levels. To impede interspecific competition, all Culex egg rafts were discarded using a teaspoon. Mosquito eggs, larvae, and pupae were not counted to avoid excessive disturbance due to handling. Mosquito colony Larvae were obtained from a colony maintained at ILPLA for more than ten generations under a L:D 16:8 photoperiod, 80% relative humidity and 26 2C. Adults were maintained in a cage with free access to a sugar solution. Females were fed with human blood and supplied with two 500 ml black jars filled with dechlorinated water to a depth of 1 cm and lined with paper towels. Eggs were collected, air-dried, and stored in plastic bags until experiments started. Submerging eggs in water with powdered yeast stimulated artificial hatching. Resulting larvae were rinsed once in dechlorinated water before being used in any treatment. Mosquito larvae used at the start of each experiment were less than one day old. Estimating larval crowding in nature In order to estimate an index of larval density under natural conditions, a destructive census of containers was carried out and Lloyds mean crowding, m*, was estimated during February 2000. After discarding containers without larvae, five tires and five ovitraps were selected using a table of random numbers, recording water volume, number, and

stage of larvae and pupae. None of these containers were used again. Lloyds mean crowding for tires and ovitraps was calculated (Lloyd 1967, Hulbert 1978) as: m* = [xi(xi-1)/vi]/xi with xi the number of larvae and vi the volume in liters, in the container i with Ae. aegypti. Manipulation of populations Immatures were manipulated to establish how changes in their number might affect population performance. Fifteen tires and 15 ovitraps were selected at random, and all water was extracted from each container and the water volume measured. To avoid new hatchings, mosquito eggs were eliminated from the walls of containers. This was achieved in ovitraps by removing the filter paper and in tires by rubbing the interior walls vigorously with a sponge and cloth. Tire and ovitrap contents (liquid and solid) were transferred to the laboratory. Larvae, pupae, and mosquito eggs were discarded. Water and detritus from tires and ovitraps were mixed separately in two large recipients, one for each container type. Water and organic debris were then restored in each ovitrap and tire, replacing the original volume previously found in each container. This procedure ensured homogeneous mosquito food among containers. One of the following treatments (five ovitraps and five tires/treatment) was assigned to each ovitrap and tire at random - Treatment 1: add 1st instar larvae (LI) up to m* estimated from the census (control); Treatment 2: add LI up to 2m* (high density); Treatment 3: add LI up to m*/2 (low density). High and low densities (double and half of natural density) were chosen arbitrarily because of the availability of experimental larvae. LI were obtained from eggs artificially hatched during the previous day. On the following days, the water level in each treatment was maintained constant, adding rainwater when necessary. To avoid new ovipositions, the experimental containers were covered with a mesh fitted with rubber bands in order to impede the access of gravid female mosquitoes. Containers were checked daily. At the end of the immature development, pupae from ovitraps and tires were removed, counted, sexed, and individually weighed in vivo to the nearest 0.01 mg with a Sartorius BP-21 microbalance. After that, they were placed in pots inside cages (one cage/treatment/container type), and allowed to reach adulthood. Adult mosquitoes Adult insects emerged inside cages (30 x 30 x 30 cm) supplied with raisins. Males had permanent access to females. A human blood meal was offered to females ad libitum. All engorged females were individually isolated in tubes and labeled with the feeding date, treatment, and container type. Cages and live females in tubes were kept under 16:8 (L:D) photoperiod, around 80% relative humidity, and 26 2 C, until eggs were fully developed (assessed by eye). Gravid females were dissected and eggs

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Analysis Differences in weight at metamorphosis, development time, biomass, survival, and fecundity among experimental treatments were subjected to parametric ANOVA. The main factors were Container Type and Treatment, blocked by Replicates (=containers). For development time and weight at metamorphosis, independent ANOVAs were performed for each sex. Biomass productivity (pooled sexes) was compared among experimental treatments (Density) considering replicates as blocks in a randomized block design. Survival data (pooled sexes) were transformed using y= arch sin px, with px= proportion of survivals in cohort x = experimental replicate x, and then subjected to ANOVA with Replicates as blocks, Container Type, and Treatment as main effects. Differences among treatments in female fecundity were analyzed by ANOVA with Container Type and Treatment as main effects. Procedures ANOVA or GLM of SAS (SAS Institute 1999) were used for analysis of balanced and unbalanced data, respectively, using type III sums of squares, followed by Tukeys test (HSD) for comparisons between means, with a level of significance = 0.05. RESULTS Estimating larval crowding As a result of the larval census performed before the experiment, average water volume in ovitraps was 0.15 liters and 4.01 liters in tires, with higher variability in tires (coefficient of variation, CV: 23%), than in ovitraps (CV: 15%). Population densities differed markedly between the two types of breeding containers, the density of larvae in ovitraps being higher than in tires (mean density in ovitraps= 193.46 larvae/liter; in tires= 68.98 larvae/liter). Experimental densities were calculated based on the mean crowding recorded in containers colonized by wild populations of Ae. aegypti: m*ovitraps= 301 larvae/liter; m*tires= 93 larvae/liter. Pupal weight Weight at metamorphosis in the three treatments showed different patterns for ovitraps and tires (Figures 1a, 1b, respectively). Tires produced heavier pupae of both males and females. Male pupae were lighter than female pupae in all combinations container type/treatment. Fiftytwo percent of the pupae were male and 48% females, showing a 1:1 sex rate (2= 1.85, df= 1, p= 0.17). Weight of male pupae ranged between 0.61 and 3.02 mg (meanSE= 1.710.018, n= 595), while female pupae ranged between 0.98 and 4.38 mg (meanSE= 2.750.031, n= 549). Both in males and females, the interaction of Container Type x Treatment affected pupal weight significantly (males: F= 37.09, df: 2, p<0.0001; females: F= 18.15, df= 2, p<0.0001), indicating that response through weight at metamorphosis depends on the combination between both factors. Also, the effects of Type of Container (males: F= 399.42, df:

1, p<0.0001; females: F= 117.28, df= 1, p<0.0001) and Treatment (males: F= 9.56, df: 2, p<0.0001; females: F= 15.73, df= 2, p<0.0001) were significant. Development time Larval development time varied between four and 42 days for both sexes, although on average males (mean days to pupationSE= 8.330.14, n= 595) emerged before females (mean days to pupationSE= 10.510.18, n= 549). In the ovitraps, larvae pupated faster than in tires (mean days to pupationovitrapsSE= 8.220.21, n= 538; mean days to pupationtiresSE= 10.400.09, n= 606). Mean development time in ovitraps (pooled sexes) showed the following pattern: Control < High density < Low density (Figure 2a); and in tires: Control < Low density < High density (Figure 2b). Container Type (F= 81.83, df= 1, p<0.0001) and Treatment (F= 4.84, df= 2, p=0.0082) had additive effects for males, although only 14% of variability was explained by the statistical model. For females, main factors were not significant and neither was the interaction (p>0.05), with just 8% of the variation attributable to the model. Biomass Tires produced almost twice the biomass (1570.10 mg) than did ovitraps (851.21 mg) (Figure 3). ANOVA demonstrated significant differences between ovitraps and tires (F= 8.43, df= 1, p=0.008), but neither the effect of treatments nor the interaction Container Type x Treatment were significant (p>0.05). Survival Ovitraps produced 538 pupae and tires produced 606 pupae, which represent 16% of larvae added at the start of the experiment under natural conditions, so there was approximately 84% overall mortality. In both types of containers, survival was higher at low density than at high density and at control (Figure 4). Survival was higher in ovitraps (67%) than in tires (15%). There was a significant effect of Type of Container (F= 38.47, df= 1, p<0.0001) on survival of Ae. aegypti populations, but the effects of Replicates, Treatments, and their interaction were nonsignificant (p>0.05). Tukeys test demonstrated a higher percentage of surviving larvae in ovitraps (Figure 4). Adult mosquitoes Eggs from 245 Ae. aegypti females fed with blood and raised from pupae grown in natural conditions were counted. Fecundity averaged 59.83 eggs/female (range 13-117). Females raised from tires (meanfecunditySE= 69.831.48, n= 129) had higher fecundity that females from ovitraps (meanfecunditySE= 50.841.54, n= 116). Again, the trend among treatments showed that female mosquitoes grown as larvae at low densities had higher fecundity (Figure 5). ANOVA showed significant differences between tires and ovitraps (F= 70.59, df= 1, p<0.0001) and non-significant differences among Treatments and the interaction (p>0.05).

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Figure 1. Weight at metamorphosis in ovitraps(a) and tires (b) . Points show mean 2 SE. Means sharing the same letter are not significantly different after Tukeys test (p>0.05).

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Figure 2. Development time in ovitraps (a) and tires (b). Points show mean 2 SE. Means sharing the same letter are not significantly different after Tukeys test (p>0.05).

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Figure 3. Biomass productivity in ovitraps and tires. Differences among treatments are not significant after Tukeys test (p>0.05).

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80 60 40 20 0 Control High density Treatment Low density

Figure 4. Survival in ovitraps and tires. Points show mean 2 SE. Comparisons between means among ovitraps and tires are significantly different, but differences among treatments are not significant (Tukeys test, p>0.05). DISCUSSION This study showed that fitness components in Ae. aegypti depend largely on the type of container in which larvae develop. The alteration of larval densities was not high or low enough to promote significant effects in production of final biomass, female development time, and fecundity. Regarding pupal weight, the variation due to treatments effects depended on the kind of breeding habitat. Male development time was significantly affected by density and type of container. Tires generated heavier pupae, slower larval development, higher total biomass, and females with higher fecundity than did ovitraps. Differences in immature performance among both types of container habitats were motivated by intraspecific competition, which would be more intense in smaller habitats, such as the ovitraps used in this study. Since the index m* is an estimate of encounters or collisions between individuals, it is appropriate for showing a degree of competition through mechanical interference among larvae during feeding activity. Under this assumption, a higher m* could reflect a stronger competition. This was shown by differences in population performances from tires and ovitraps. At high densities, feeding activity can be hindered by the presence of other individuals, resulting in a reduction in the efficiency of nutrient exploitation, which is the microscopic biofilm attached to plant debris and sediments for many Aedes species (Clements 1992). Another explanation for negative effects of larval crowding is lowered food availability per individual. Aedes aegypti is very sensitive to low per capita food availability (Juliano 1998). Density-dependent events in Ae. aegypti can be caused by both mechanisms acting complementarily. However, under the present experimental conditions, it cannot be inferred if this competition acts through selective resource exploitation for food or space, through interference competition sensu Schoener (1983), through the existence of growth retardants, or combined effects of those mechanisms. Previous studies on factors altering responses in Ae. aegypti fitness reached different conclusions. Dye (1982) found

Figure 5. Female fecundity in ovitraps and tires. Points show means 2 SE. Comparisons between means among ovitraps and tires are significantly different, but differences among treatments are not significant (Tukeys test, p>0.05). that intraspecific competition in this species is asymmetric, with chemical competition (via metabolites released into water) being less important than physical (via hampering among individuals). In turn, Bedhomme et al. (2005) showed that both depletion of resources and the presence of excreted substances by larvae are factors that contribute to competition. Different responses of interference were detected in two strains of Ae. aegypti by Dye (1984), so populations from different localities can also exhibit subtle variations in fitness based on geographical origin. Mortality was higher in tires (85%) than in ovitraps (33%). At least a part of such mortality can be attributed to some features of the larval environment. It was observed that water in tires had high turbidity soon after the start of experiment, while ovitraps remained with clean water longer. A scarce quantity of leaves was observed in the containers, suggesting a shortage of the physical support for microorganisms. Such bacteria and protozoa constitute a major nutrient source for larval populations and are a limiting resource for container-breeding mosquitoes (Lounibos et al. 1993, Leonard and Juliano 1995, Maci and Bradshaw 2000). Other studies also showed high mortality rates in Ae. aegypti under field conditions (Southwood et al. 1972, Micieli et al. in press). Regardless of the kind of container, competence resulted in similar trends in males and females in pupal weight and development time. A cost due to competition for food is not sex-related, as reported by Bedhomme et al. (2005). Data obtained from the previous census showed a great variability in mosquito numbers among recipients, a characteristic observed in tree-holes commonly used by other Aedes species (Bradshaw and Holzapfel 1988, Lonard and Juliano 1995). The great variability among habitat units masked clear trends related to crowding, which would explain the low R2 in some of the statistical tests. All of the above suggests that the Ae. aegypti population under study was subjected to intraspecific competition, the intensity of which is related to quality of the container habitat. The average potential fecundity (59.8 eggs/female) is similar to records from Coless and Chellapah (1960) of 51.8 eggs/female. The significant difference between fecundity from mosquitoes breeding in tires and ovitraps confirms that

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tires generate adult populations with superior reproductive potential, although this effect could be compensated by a greater preimaginal mortality. Events alleviating intraspecific competition, such as the elimination of a fraction of the population in each container, can increase the amount of resources available for the remaining larvae. As a consequence, the surviving pupae will be bigger and heavier, and the emerging females will have higher fecundity. In small water receptacles with low nutrients, removal of larvae can increase numbers of emerging adults (Agudelo-Silva and Spielman 1984). The final effect would be an increased vectorial capacity in the population of adults. Thus, it is important to carefully evaluate control measures before they are implemented and to consider if total elimination of all individuals in each container is feasible. A similar conclusion was suggested by Arrivillaga and Barrera (2004) after studying resistance to starvation in Ae. aegypti larvae from Venezuela. This experiment is the first report on differences in the fitness of larvae raised both in tires and ovitraps. The general outcome was the emergence of healthier, although less numerous, individuals in larger containers. Reducing the number of larval mosquitoes per container would lead to a fast recovery of the whole population because female survivors would be able to lay more eggs. Acknowledgments I thank Ral E. Campos for assistance during laboratory tasks, logistic support, and critical review of the manuscript. Rodolfo L. De La Sota assisted in performing statistical tests and providing SAS. Ann Montemayor-Borsinger reviewed a draft of the manuscript. To Alberto Rodrgues Captulo and Gustavo Spinelli for providing working space. Thanks to an anonymous reviewer for improving the paper through valuable suggestions. This research was supported by Fundacin Antorchas (Buenos Aires) through Grant 13783/1-19. REFERENCES CITED Agudelo-Silva, F. and A. Spielman. 1984. Paradoxical effects of simulated larviciding on production of adult mosquitoes. Am. J. Trop. Med. Hyg. 33: 1267-1269. Arrivillaga, J. and R. Barrera. 2004. Food as a limiting factor for Aedes aegypti in water-storage containers. J. Vector Ecol. 29: 11-20. Avils, G., G. Rangen, P. Baroni, V. Paz, M. Monteros, J. L. Sartini, and D. Enria. 2000. Outbreak of dengue-2 in Salta, Argentina, 1998. Medicina (Buenos Aires) 60: 875-879. Avils, G., G. Rangen, V. Vorndam, A. Briones, P. Baroni, D. Enria, and M.S. Sabattini. 1999. Dengue reemergence in Argentina. Emerg. Infect. Dis. 5: 575578. Bedhomme, S., P. Agnew, C. Sidobre, and Y. Michalakis. 2005. Pollution by conspecifics as a component of intraspecific competition among Aedes aegypti larvae.

Ecol. Entomol. 30: 1-7. Bradshaw, W.E. and C.M. Holzapfel. 1986. Geography of density-dependent selection in pitcher-plant mosquitoes. In: F. Taylor and R. Karban (eds.). The Evolution of Insect Life Cycles. pp. 48-65. SpringerVerlag, Berlin. Bradshaw, W.E. and C.M. Holzapfel. 1988. Drought and the organization of tree-hole mosquito communities. Oecologia 74: 507-514. Bradshaw, W.E. and C.M. Holzapfel. 1992. Reproductive consequences of density-dependent size variation in the pitcher plant mosquito, Wyeomyia smithii (Diptera: Culicidae). Ann. Entomol. Soc. Am. 85: 274-281. Bradshaw, W.E., C.M. Holzapfel, and T. ONeill. 1993. Egg size and reproductive allocation in the pitcherplant mosquito, Wyeomyia smithii (Diptera: Culicidae). J. Med. Entomol. 30: 384-390. Campos, R.E. 1993. Presencia de Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) en la localidad de Quilmes (Buenos Aires, Argentina). Rev. Soc. Entomol. Argent. 52: 36. Campos, R.E. and A. Maci. 1996. Observaciones biolgicas de una poblacin natural de Aedes aegypti (Diptera: Culicidae) en la provincia de Buenos Aires, Argentina. Rev. Soc. Entomol. Argent. 55: 67-72. Carbajo, A.E., A.M. Gmez, S.I. Curto, and N.J. Schweigmann. 2004. Variacin espacio-temporal del riesgo de transmisin de dengue en la ciudad de Buenos Aires. Medicina (Buenos Aires) 64: 231-234. Christophers, S.R. 1960. Aedes aegypti (L.) The Yellow Fever Mosquito. Its Life History, Bionomics and Structure. Cambridge Univ. Press, London, 739 pp. Clements, A.N. 1992. The Biology of Mosquitoes. Volume 1. Development, Nutrition and Reproduction. Chapman and Hall, London, 509 pp. Coless, D.H. and W.T. Chellapah.1960. Effects of body weight and size of blood-meal upon egg production in Aedes aegypti (Linnaeus) (Diptera: Culicidae). Ann. Trop. Med. Parasitol. 54: 475-482. Curto, S.I., R. Boffi, A.E. Carbajo, R. Plastina, and N.J. Schweigmann. 2002. Reinfestacin del territorio argentino por Aedes aegypti. Distribucin geogrfica (1994-1999). In: O.D. Salomn, (ed.) Actualizaciones en Artropodologa Sanitaria Argentina. pp. 127-137. Publicacin Monogrfica 2, Buenos Aires. Dye, C. 1982. Intraspecific competition amongst larval Aedes aegypti: food exploitation or chemical interference? Ecol. Entomol. 7: 39-46. Dye, C. 1984. Competition amongst larval Aedes aegypti: the role of interference. Ecol. Entomol. 9: 355-357. Fish, D. 1985. An analysis of adult size variation within natural mosquito populations. In: L.P. Lounibos, J.R. Rey, and J.H. Frank (eds.) Ecology of Mosquitoes: Proceedings of a Workshop. pp. 419-429. Florida Medical Entomology Laboratory, Vero Beach. Fisher, I.J., W.E. Bradshaw, and C. Kammeyer. 1990. Fitness and its correlates assessed by intra- and interspecific interactions among tree-hole mosquitoes. J. Anim.

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Ecol. 59: 819-829. Grill, C.P. and S.A. Juliano. 1996. Predicting species interaction based on behaviour: predation and competition in container-dwelling mosquitoes. J. Anim. Ecol. 65: 63-76. Hard, J.J. and W.E. Bradshaw. 1993. Reproductive allocation in the western tree-hole mosquito, Aedes sierrensis. Oikos 66: 55-65. Hulbert, S.H. 1978. The measurement of niche overlap and some relatives. Ecology 59: 67-77. Juliano, S.A. 1998. Species introduction and replacement among mosquitoes: interspecific resource or apparent competition? Ecology 79: 255-268. Lonard, P.M. and S.A. Juliano. 1995. Effect of leaf litter and density on fitness and population performance of the hole mosquito Aedes triseriatus. Ecol. Entomol 20: 125-136. Livdahl, T.P. 1984. Interspecific interactions and the r-k continuum: laboratory comparisions of geographic strains of Aedes triseriatus. Oikos 42: 193-202. Livdahl, T.P. and G. Sugihara. 1984. Non-linear interactions of populations and the importance of estimating per capita rates of change. J. Anim. Ecol. 53: 573-580. Lloyd, M. 1967. Mean crowding. J. Animal Ecol. 36: 1-30. Lounibos, L.P., N. Nishimura, and R.L. Escher. 1993. Fitness of a treehole mosquito: influences of food type and predation. Oikos 66: 114-118. Maci. A and W.E. Bradshaw. 2000. Seasonal availability of resources and habitat degradation for the western tree-hole mosquito, Aedes sierrensis. Oecologia 125: 55-65. Mart, G.A., M.V. Micieli, A.C. Scorsetti, and G. Liljesthrm. 2004. Evaluation of Mesocyclops annulatus (Copepoda: Cyclopoidea) as a control agent of Aedes aegypti (Diptera: Culicidae) in Argentina. Mem. Inst. Oswaldo Cruz 99: 535-540.

Micieli, M.V. and R.E. Campos. 2003. Oviposition activity and seasonal pattern of a population of Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) in subtropical Argentina. Mem. Inst. Oswaldo Cruz 98: 659-663. OPS 1990. Las condiciones de la salud en las Amricas, Vol. 1. Organizacin Panamericana de la Salud, Geneve, 110 pp. Pianka, E.R. 1988. Evolutionary Ecology. 4th edition. Harper and Row, New York, 486 pp. Reisen, W.K., M.M. Milby, and M.E. Bock. 1984. The effects of immature stress on selected events in the life history of Culex tarsalis. Mosq. News 44: 385-395. SAS Institute. 1999. SAS users guide: statistics. Version 8.0. SAS Institute, Cary, NC. Schoener, T.W. 1983. Field experiments on interspecific competition. Am. Nat. 122: 240-285. Seijo, A., D. Curcio, G. Avils, B. Cernigoi, B. Deodato, and S. Lloveras. 2000. Imported dengue in Buenos Aires, Argentina. Emerg. Infect. Dis. 6: 655-656. Southwood, T.R., G. Murdie, M. Yasuno, R.J. Tonn, and P.M. Reader. 1972. Studies on the life budget of Aedes aegypti in Wat Samphaya, Bangkok, Thailand. Bull. Wld. Hlth. Org. 46: 211-226. Steinwascher, K. 1982. Relation between pupal mass and adult survivorship and fecundity for Aedes aegypti. Environ. Entomol. 11: 150-153. Vezzani. D., S.M. Velzquez, S. Soto, and N.J. Schweigmann. 2001. Environmental characteristics of the cemeteries of Buenos Aires City (Argentina) and infestation levels of Aedes aegypti (Diptera: Culicidae). Mem. Inst. Oswaldo Cruz 96: 467-471. Vezzani, D., S.M. Velzquez, and N.J. Schweigmann. 2004. Seasonal pattern of abundance of Aedes aegypti (Diptera: Culicidae) in Buenos Aires City, Argentina. Mem. Inst. Oswaldo Cruz 99: 351-356.