1. Roberto Barrera, Manuel Amador, and Gary G. Clark Centers for Disease Control and Prevention, Dengue Branch, San Juan, Puerto Rico Aedes aegypti is regulated by abiotic (rainfall, temperature, evaporation) and biotic factors (competition, food) that interact in a diversity of aquatic container habitats, which have varying internal properties (organic matter, microbial communities, other aquatic insects) depending on their size and shape, location (e.g., under tree canopy, exposed to the sun, etc.), and season (e.g., tree leaf-shedding). Ae. aegypti is also markedly influenced by extensive water storage in the Tropics, where seasonal changes are dampened. Predation and parasitism are not frequent regulatory factors for Ae. aegypti immatures in artificial containers of urban areas. Food limitation, or larval competition, is a common, density-dependent regulatory factor in water storage containers, as suggested by several field studies. However, there is a paucity of studies on regulatory factors of immature Ae. aegypti in the diverse array of artificial containers in urban areas (e.g., disposable containers), including containers managed by humans other than water-storage containers (e.g., animal watering pans, plant pots, etc.). Materials and Methods A total 624 premises was visited from May to July, 2004 in an urban area in southern Puerto Rico (17° 58’ N, 66° 18’ W) with 1,344 buildings We observed in containers: water volume (ml), surface water temperature (ºC), number of stage III and IV larvae, male and female pupae, source of water (added by human intervention, direct rainfall, rain through foliage, drainage rain off the roof), exposure to the sun (full, partial, none), presence of tree canopy, and the tree species over the container To determine if the emerging adults in the field were small or not, we compared their biomass with that of adults reared in the laboratory from first instar larvae across a wide range of feeding levels. We reared forty first-instar larvae in plastic cups with 200 ml of aerated tap water (5 replicates), and one of the following feeding regimes with liver powder: 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mg / larva / day until adult emergence Mean number of male, female pupae (log10 + 1 transformed), or larvae (log10 + 1 transformed) per container was compared within and between levels of sun exposure, source of container water, and presence or absence of tree canopy ( 3-way ANOVA) A stepwise multiple regression analysis was employed to evaluate the simultaneous effects of the following container variables: water volume, water temperature, and number of third, and fourth instar larvae per container (log10 transformed) on the number of male or female pupae per container, or on the mass of female adults emerging from the pupae collected in the field A factor analysis, using principal components analysis for factor extraction (forms uncorrelated linear combinations of the observed variables) was conducted to derive underlying dimensions that, when interpreted and understood, describe the data in a much smaller number of concepts than the original individual variables. The following variables (log10) were explored; some of which were interrelated: adult female body mass, number of female pupae, number of larvae, water temperature and water volume. After significant factors were extracted, we explored which variables were significantly correlated with each factor to facilitate their interpretation A stepwise multiple regression analysis of the number of female pupae per premise (log10 + 1) was performed, based on the following independent variables: number of trees per premise (log10 + 1), premise area (log10), mean water volume in the containers (log10), and mean water temperature (log10). Stats Factor analysis. The first 2 components (latent roots larger than 1) explained 58.5% of the variance. The first factor or component (35.7%) yielded high scores when larvae and pupae were numerous, but pupae were small. The second factor (22.8%) yielded high scores for containers with larger contents of water and number of pupae, and to a lesser degree with adult female mass and lower water temperature (Fig. 2). We inferred two processes in the containers; one in which body mass of emerging females was smaller in containers where there were many larvae and pupae (Factor 1), and another one in which containers with larger volumes of water held more, bigger pupae (Factor 2). Figure 2. Correlations between variables and factors Conclusions Objectives We investigated the multivariate effects of environmental factors and immature density on Aedes aegypti productivity (emerging females and their biomass in artificial containers in an urban area) We explored the hypothesis that immature Ae. aegypti populations were under nutritional stress in the containers, possibly due to food limitation or crowding. We thank Gilberto Felix, Ariana Muñoz, José Aponte, Khristian Pizarro, Abraham Rivera, Alejandro Rodríguez, and José Torres for their laboratory and field assistance, and Marcos Rodríguez for his help with the identification of the tree species. We appreciate the collaboration of the Municipality of Salinas, and Centro de Recaudación de Impuestos Municipales (CRIM) for providing us with maps of the study area. This study was partly supported with funds (OD/TS-03-00523) from the UNICEF/UNDP/WORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases (TDR/WHO) and the Centers for Disease Control and Prevention (CDC). Acknowledgments Figure 1. Number of pupae vs. larvae (A) and adult female mass vs. larvae in containers Results: Environmental variables Mean female pupae of Ae. aegypti per container significantly changed with: source of water and presence of a tree canopy. There were more female pupae in containers with rain water coming through foliage and under a tree canopy. Male pupae and larvae responded similarly. A stepwise regression analysis of female pupae on water temperature, water volume, and number of larvae was significant and positive for larvae and water volume. i.e. more pupae were produced in containers with more larvae (Fig. 1A) and larger water volume. A similar analysis for adult female body mass was significant and negative for number of larvae (Fig. 1B), and positive for water volume. i.e. females were smaller in containers with more larvae, but bigger in containers with a large volume of water. Environmental variables of premises. We analyzed the association between environmental variables and mean number of female pupae of Ae. aegypti per premise. Significant, positive regression coefficients resulted for number of trees and water volume, and negative for water temperature. These results were consistent with our previous analysis for the containers, where water volume, water temperature, and water coming through foliage were significantly associated with the number of female pupae of Ae. aegypti. Evidence for food limitation in artificial containers Mean dry mass of emerging males and females per experimental container gradually increased with the amount of food until a maximum mass was attained (Fig. 3). An inverse function was fitted between adult male dry mass and feeding regime (Male_dry_mass = 0.6031 – 0.0705 / Feeding_regime); R2 = 0.93, F1,34 = 461, P < 0.001), and between adult female dry mass and feeding regime (Female_dry_mass = 1.1407 – 0.1495 /Feeding regime; R2 = 0.95, F1,34 = 617, P < 0.001; Fig. 3). Mean female dry mass varied with feeding rations between 0.395 mg at 0.2 mg/larva/day and 0.992 mg at 1.6 mg/l/d (total mean for females = 0.881 mg), whereas male dry mass changed between 0.238 and 0.536 mg (total mean for males = 0.482). Mean dry mass of males and females emerging from containers in the study area were 0.281 ± 0.010 mg (SE) and 0.425 ± 0.017 mg, respectively. The dry mass distribution of field derived Ae. aegypti adults was shifted to the left (lower body mass) in comparison with the distribution observed in adults reared in the laboratory at varying feeding regimes (Fig. 4). Using the above equations, we calculated that average field males and females had the equivalent feeding rations of larvae reared in the laboratory with 0.20 ± 0.01 mg of liver powder / larva / day and 0.24 ± 0.01 mg / larva / day, respectively; that is, the lowest feeding regime utilized to rear Ae. aegypti in this study (Fig. 3). Figure 3. Aedes aegypti adult dry mass (mg) of emerging females (open dots) and males (closed dots) reared from first instars in the laboratory We found evidence that Aedes aegypti was under nutritional stress (food limitation or competition) in unmanaged, artificial containers subjected to local environmental conditions of household yards in an urban area. These results are concordant with previous studies reporting nutritional stress in preadult Aedes aegypti where water storage containers were the most common aquatic habitats. Containers under a tree canopy, filled with rainfall coming through foliage, lower water temperatures and larger volumes of water produced more Aedes aegypti adults. Yards with more trees also produced more mosquito adults. Figure 4. Distribution of Aedes aegypti adult dry mass (mg) reared in the laboratory (males in upper left, females in upper right) or emerged from artificial containers (males in lower left, females in lower right). Total means for each condition are provided within each graph.