Does temperature affect the outcome of larval competition between Aedes aegypti and Aedes albopictus?

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1 86 Journal of Vector Ecology June, 2002 Does temperature affect the outcome of larval competition between Aedes aegypti and Aedes albopictus? L.P. Lounibos 1, S. Suárez 2, Z. Menéndez 2, N. Nishimura 1, R.L. Escher 1, S.M. O Connell 1, and J.R. Rey 1 1 University of Florida, Florida Medical Entomology Laboratory, th St. SE, Vero Beach FL USA 2 Departamento de Control de Vectores, Instituto de Medicina Tropical Pedro Kouri Apartado 601, Mariano 13, La Lisa, La Habana, Cuba Received 18 September 2001; Accepted 14 November 2001 ABSTRACT: The superior larval competitive ability of Aedes albopictus has been proposed to explain the recent displacement of Aedes aegypti by the former species in parts of the southeastern U.S. Ae. aegypti persists, however, in sympatry with Ae. albopictus in urban areas of southern Louisiana, Florida, and Texas, and the impact of larval competition between these species has not been investigated at higher temperatures that may be characteristic of these urban environments. We compared growth and survivorship of the two species at controlled temperatures of 24 and 30 C in water-containing tires under conditions of intra- and interspecific competition and with or without leaf litter. When other variables were controlled statistically, the estimated finite rate of increase (λ ) was significantly higher for both species at the higher temperature, and the proportional increases in λ did not differ between species. Therefore, our experiment predicts that by itself, temperatures between 24 and 30 C would not alter the outcome of larval competition. Overall, response measures of Ae. albopictus were more sensitive than those of Ae. aegypti to the litter and species/density variables, although the development of Ae. aegypti females was uniquely retarded by a high density of its own species. Journal of Vector Ecology 27(1): Keyword Index: Competition, finite rate of increase, invasive mosquitoes, temperature, tires. INTRODUCTION The establishment and spread of Aedes albopictus (Skuse) in the U.S. that began in the 1980s (Hawley 1988, Craig 1993) was associated with a reduction in abundance and range of the yellow fever mosquito Aedes aegypti (L.). The decline and local disappearance of Ae. aegypti that followed the spread of Ae. albopictus in the southeastern states has been well documented (Hobbs et al. 1991, Mekuria and Hyatt 1995, Nasci et al. 1989, O Meara et al. 1995). In Florida, the yellow fever mosquito remains common only in urban sections of the southern portion of the state (O Meara et al. 1995; O Meara, personal communication). Various mechanisms have been proposed to explain the invader-mediated demise of Ae. aegypti in the U.S., including differential effects of a gregarine parasite introduced with Ae. albopictus (Craig 1993, Blackmore et al. 1995); a satyr effect (Ribeiro 1988), whereby asymmetric cross-mating was proposed as causing female sterility of Ae. aegypti (Nasci et al. 1989); egg hatching inhibition by Ae. albopictus larvae that differentially impacts Ae. aegypti (Edgerly et al. 1993); and larval resource competition (Juliano 1998). This last hypothesis is based on compelling evidence that larvae of Ae. albopictus are superior to those of Ae. aegypti in growth and survivorship under conditions of intra- or interspecific competition in the presence of limiting, litter-based resources (Barrera 1996, Juliano 1998, Daugherty et al. 2000). The laboratory studies that concluded Ae. albopictus is the superior competitor were run at C (Barrera 1996) or 27 C (Daugherty et al. 2000). Field experiments by Juliano (1998) that demonstrated the superior competitive ability of larval Ae. albopictus were conducted outdoors in shaded tires in southern Florida where the average daytime temperature, measured mornings, was 24.8 C (Juliano, personal communication). Although natural populations of Ae. aegypti have been largely extinguished from such wooded habitats of southern Florida, this species persists in nearby urban sites (O Meara, personal

2 June, 2002 Journal of Vector Ecology 87 communication). In unshaded urban tires occupied only by Ae. aegypti in West Palm Beach, we have routinely recorded summertime water temperatures of C (Lounibos & Rey, unpublished data). Many experimental studies have demonstrated that a change in temperature can reverse the outcome of interspecific competition between insects that occupy the same habitat. Classic research by Birch (1953) showed that the outcome of competition between grain beetles was reversed by a 3.2 C temperature change, Rhizopertha dominica predominating at 32.3 C and Calandra oryzae at 29.1 C. Between competing flour beetles, a temperature of 34 C and relative humidity of 70% favored Tribolium castaneum, but at 24 C and 30% RH, Tribolium confusum dominated T. castaneum (Park 1954). The predominant species in interspecific competition between closely related species of Drosophila was determined by whether experiments were conducted at 19 or 25 C (Ayala 1970). Russell (1986) reported that the native Australian mosquito Aedes notoscriptus had a slight competitive advantage over Ae. aegypti at 22 C, but the latter introduced species predominated at 28 C. Alto and Juliano (2001) showed experimentally that population growth of Ae. albopictus is directly proportional to temperatures from 22 to 26 o C, although the carrying capacity (which may be more relevant to competitive ability) of this species was lower at the higher temperature. To date there have been no published reports comparing the outcome of interspecific competition between Ae. albopictus and Ae. aegypti at different temperatures, although the emergence rate of Ae. aegypti was claimed to be higher than that of Ae. albopictus at 30 o C but not different at a lower (unspecified) temperature (Huang 1997). The current study was designed specifically to test the effects of two temperatures experienced by larvae and pupae on correlates of population growth of Ae. aegypti and Ae. albopictus and to evaluate whether the former species might be a superior competitor at 30 o C compared to 24 o C. Species/density treatments and the presence of food, in the form of decaying leaf litter, were also manipulated in our experiments. These variables have been shown to interact with temperature in the population responses of other insect species (Davis et al. 1998). MATERIALS AND METHODS F1 progeny of Ae. aegypti and Ae. albopictus from 1999 collections of these species from discarded tires and cemetery vases in south Florida were used. Discarded golf cart tires (outer diameter = 40.6 cm, inner diameter = 19.7 cm, width = 20.3 cm) obtained from nearby golf courses were used as experimental containers. Before use, these tires were immersed in scalding water, scrubbed with a brush dipped in a dilute bleach solution, and thoroughly rinsed. Water for experiments (= pre-treated tire water) was suctioned from mosquito-containing automobile tires on the grounds of the Florida Medical Entomology Laboratory (FMEL). This water was sieved to remove most macrobiota and detritus and frozen at -20 C to kill remaining organisms. Fallen oak (Quercus virginiana) leaves were collected, oven-dried at 80 C for 48 hours, and weighed into one-gram aliquots for plus-litter treatments. Forty cleaned tires were rested vertically against the walls of each of two walk-in insectaries, one maintained at 24+1 C and the other at 30+1 C. These experimental temperatures were chosen based on the preliminary data, cited above, from wooded and urban habitats in southern Florida as well as the report of Huang (1997). Daily light:dark regimens were 12L:12D in both insectaries. One day before hatch of larvae, each tire received one liter of pre-treated tire water, and 20 tires at each temperature received one gram of dried oak leaves. The pre-treated tire water was judged to contain sufficient organic resources for the completion of larval development in the 20 tires that received no litter. Eggs of each Aedes species were hatched in deoxygenated water. First-instar larvae <24 h old were counted under a dissecting microscope, and aliquots were delivered into tires, assigned to the following experimental combinations of Ae. albopictus:ae. aegypti 80:0, 40:0, 40:40, 0:40, 0:80. Each species/density treatment was replicated four times at the two food levels (0 vs. 1 g litter), providing a total of forty (5x4x2) containers at each temperature. Beginning six days after the start of the experiment, each tire was checked daily for the presence of pupae, which were removed with a pipette and placed in individual eclosion vials covered with a screened cap. The eclosion vials were maintained at the same ambient temperature as the larvae. Records were kept of dates of pupation and adult emergence, species, and sex for each eclosed adult. Adult mosquitoes were killed by freezing within 24 h after emergence, oven-dried at 80 o C for 48 h, and weighed individually to the nearest g on an electrobalance. The experiment was terminated when all mosquitoes had either emerged or died. For each tire, survivorship to adulthood and mean female and male dry mass were calculated. Survivorship was calculated as the proportion of adults that emerged from the initial cohort of first-instar larvae. Medians of developmental times were calculated for each tire owing

3 88 Journal of Vector Ecology June, 2002 Table 1. ANOVA results for λ and survivorship of both species in response to the independent variables of temperature (Tem), litter (Lit), and species/density treatments (Tre). λ Survivorship Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypti Source df F P F P F P F P Tem Lit Tre Tem X Lit Tem X Tre Lit X Tre Tem X Lit X Tre Error 36 to skewed distributions of this variable among cohorts. These univariate measures were included in calculations of a composite index of performance, λ, which is an analog of the finite rate of increase defined as in Juliano (1998): ln No λ' = exp xaxf D + x ( 1 ) Axf ( wx) x ( wx) Axf x ( ) wx where N 0 is the initial number of females (assumed to be 50% of a cohort), A x is the number of females eclosing on day x, w x is the mean mass of females eclosing on day x, and f(w x ) is a function relating number of eggs to female mass. D is the time from adult eclosion to reproduction, assumed to be 12 d for Ae. aegypti (Grill and Juliano 1996) and 14 d for Ae. albopictus (Livdahl and Willey 1991). A regression relating adult dry mass to fecundity for Ae. aegypti was derived by Juliano (1998) from data of Colless and Chellapah (1960) and Grill and Juliano (1996): f(w x ) = w x (r 2 = 0.10, P<0.05) As a comparable relationship between these two variables was not available for Ae. albopictus, larval collections of this species from FMEL were reared to adulthood in the laboratory. Two cohorts of 100 larvae each, were provided either one or three grams dried oak leaves at o C to obtain a broad spectrum of adult sizes. From emerged females (n=115) sampled from both cohorts, one wing was removed and measured under a dissecting microscope with an ocular micrometer and the remainder of the body was dried at 80 o C for 48 h and weighed to the nearest g on an electrobalance. A second sample of females (n=91) from both cohorts was given a replete blood meal from a restrained chicken and allowed to oviposit on wet germination paper in individual vials. Laid eggs were counted and females were subsequently killed and dissected for microscopic examination of unlaid (stage five) eggs, which were included in total fecundity estimates. A wing of each female was measured, as for the previous group. Linear regressions of number of eggs vs. wing length and wing length vs. female dry mass were both highly significant (Figure 1). These two regression equations were combined to obtain the following relationship between fecundity and female dry mass for Ae. albopictus: f(w x ) = w x where r 2 = (sqrt[r 2 1 ] x sqrt[r2 2 ])2 = For each species, three-way ANOVAs with interactions were run with PROC GLM (SAS Institute 1985) for the independent variables of temperature, litter, and species/density treatments on the means or medians of the univariate performance measures as well as on survivorship and λ from each tire. Survivorship values were arcsine-transformed prior to ANOVA. Leastsquares (LS) means were generated for λ and for each of the univariate measures of performance with the LS MEANS option of PROC GLM and compared between and among litter, temperature, and species/density variables by a posteriori Tukey-Kramer tests (SAS Institute 1985). The LS means are estimates for a variable while other variables are held constant.

4 Table 2. Least squares means (+SE) of λ, median developmental time, adult dry mass, and survivorship of Ae. albopictus exposed to three independent variables. Temperature a Litter a Treatments b Response 24 o 30 o 0 gram 1 gram 80:0 40:0 40:40 λ ±006 * ** a b c Median (d) *** ** Development Time Median (d) *** *** Development Time Mean ** ** a b a Mass (mg) Mean *** b a ab Mass (mg) Survivorship a Significant differences (Tukey-Kramer test) between means indicated by asterisks: * = P <0.05, ** = P <0.01, *** = P < b Means followed by letters that are not commonly shared are significantly different (P <0.05, Tukey-Kramer test) June, 2002 Journal of Vector Ecology 89

5 90 Journal of Vector Ecology June, 2002 RESULTS Survivorship to adulthood Survival to adulthood of both species was unaffected by the three independent variables (Table 1). LS means of proportion surviving varied little among experimental conditions and were remarkably similar for Ae. albopictus ( ) and Ae. aegypti ( ) (Tables 2 and 3). Developmental Time Median number of days from hatch to adulthood of both sexes of both species was strongly affected by temperature (Table 4). The presence or absence of litter also significantly influenced developmental times of both species and sexes, albeit with a stronger effect on Ae. albopictus. The species/density treatment was significant only on the developmental times of Ae. aegypti females (Table 4) whose LS mean of median days to adulthood was significantly longer in the 0:80 treatment (Table 3). A significant litter x species/density treatment interaction (Table 4) was attributable to a difference between intra- and interspecific competition in the relative developmental time of Ae. aegypti females in the presence and absence of litter. Adult Mass Species/density treatments significantly affected adult mass of males and females of both species (Table 5). The presence or absence of litter affected adult mass of Ae. albopictus more prominently than that of Ae. aegypti, for which species the significant effect was limited to females (Table 5). Temperature significantly influenced adult mass of only Ae. albopictus males. Significant interactions among experimental variables were observed only for Ae. aegypti females (Table 5). Litter affected the temperature-treatment relationship on Ae. aegypti females differently in the 0:40 treatment, contributing to a significant three-way interaction (Table 5). Comparisons of LS means for Ae. albopictus showed that significantly larger males were produced at the higher temperature and in the presence of litter (Table 2). Mean female mass of both species was significantly higher in the presence of litter (Tables 2, 3). Among species/density treatments, the lower larval density yielded significantly larger adults of both sexes and species. Estimated Finite Rate of Increase (λ ) The estimated finite rate of increase of both species was significantly affected by both temperature and species/density treatments (Table 1). However, the presence or absence of litter was significant only for Ae. albopictus. All interactions of manipulated variables were non-significant. Comparisons of LS means showed that λ was significantly higher at 30 o than 24 o for both species and at one gram compared to no litter for Ae. albopictus only (Tables 2,3). The species/density treatments had a more pronounced effect on λ of Ae. albopictus, which in the presence of litter at 30 o was significantly higher than the mean of this variable at the lower intraspecific density and significantly higher than mean λ under interspecific competition (Table 2). For Ae. aegypti, only one comparison of LS means of λ was statistically significant (Table 3). DISCUSSION Given our results, the outcome of larval competition between Ae. albopictus and Ae. aegypti in the field would not be expected to change between 30 o and 24 o C. The mean finite rate of increase, λ, was significantly higher at 30 o for both species, the proportionate increase of mean λ being nearly identical for Ae. albopictus and Ae. aegypti (Tables 2, 3). Thus, there is no evidence for a reversal in competitive abilities, as reported for grain beetles and Drosophila, across a similar or narrower temperature differential (Birch 1953, Ayala 1970). Also, our results do not indicate that larval temperatures play a role in the urban-rural habitat segregation observed for Ae. aegypti and Ae. albopictus in south Florida. Although the two species reacted similarly to temperature, the same cannot be said for the litter and species/density treatments. Mean λ of Ae. albopictus was uniquely sensitive to the presence or absence of litter and more affected by species/density treatments than that of Ae. aegypti (Table 1). Median developmental times and mean adult masses of Ae. albopictus were more strongly influenced by the presence or absence of litter than those of Ae. aegypti (Tables 2, 3). The effects of competition treatments were apparent in changes in body mass, but not developmental times, of Ae. albopictus (Tables 2, 4). By contrast, competition treatments strongly influenced the developmental times of Ae. aegypti females (Table 4), whose median developmental time was especially retarded at the higher intraspecific density (Table 3). Interactions between experimental variables were significant only for Ae. aegypti, whose female mass and developmental time were significantly affected by interactions of temperature and/or litter with the species/ density treatment variable. These interactions underscore the complexity of responses and the need for caution in

6 Table 3. Least squares means (+SE) of λ, median developmental time, adult dry mass, and survivorship of Ae. aegypti exposed to three independent variables. Temperature a Litter a Treatmentsb Response 24 o 30 o 0 gram 1 gram 0:80 0:40 40:40 λ * a b ab Median (d) *** * Developmental Time Median (d) *** * a b b Developmental Time Mean a b a Mass (mg) Mean * a b a Mass (mg) Survivorship a Significant differences (Tukey-Kramer test) between means indicated by asterisks: * = P <0.05, *** = P < b Means followed by letters that are not commonly shared are significantly different (P <0.05, Tukey-Kramer test) June, 2002 Journal of Vector Ecology 91

7 92 Journal of Vector Ecology June, 2002 Table 4. ANOVA results for median developmental time of both species in response to the independent variables of temperature (Tem), Litter (Lit), and species/density treatments (Tre). Ae. albopictus Ae. aegypti Females Males Females Males Source df F P F P F P F P Tem < < < < Lit Tre Tem X Lit Tem X Tre Lit X Tre Tem X Lit X Tre Error 34 Table 5. ANOVA results for mass at adulthood of both species in response to the independent variables of temperature (Tem), Litter (Lit), and species/density treatments (Tre). Ae. albopictus Ae. aegypti Females Males Females Males Source df F P F P F P F P Tem Lit < Tre < Tem X Lit Tem X Tre Lit X Tre Tem X Lit X Tre Error 34

8 June, 2002 Journal of Vector Ecology 93 Y = (78.02*X) r 2 = P < n = 91 Y = (1.96*X) r 2 = P < n = 115 Figure 1. Relationships between (A) number of eggs and wing length and (B) wing length and female dry weight for Ae. albopictus. Linear regression estimates (insets) were combined to derive an expression relating fecundity to dry weight (see text).

9 94 Journal of Vector Ecology June, 2002 extrapolating from the experimental conditions. Researchers working in the native range of Ae. albopictus doubted that biological interactions with Ae. aegypti could influence the patterns of distribution of these two species in the Oriental Region (e.g. Macdonald 1956, Chan et al. 1971). However, the rapid disappearance of Ae. aegypti from the southeastern U.S. shortly after the spread of Ae. albopictus through this region put to rest most doubts about interactive explanations of displacement in the New World (Barrera 1996). Laboratory experiments conducted in the wake of reduction in distribution and abundance of Ae. aegypti in the U.S. failed to identify a clear-cut superior competitor or support larval competition as a displacement mechanism (Black et al. 1989, Ho et al. 1989). However, when nutrient-rich laboratory media such as liver powder or yeast were replaced by leaf detritus as a larval resource base, Ae. albopictus had significantly higher pupation success, longer adult survivorship, and shorter developmental times than Ae. aegypti (Barrera 1996). In a Florida field study with leaf litter rationed in automobile tires, Ae. albopictus outperformed Ae. aegypti under all experimental conditions except when the two species were exposed to low-density intraspecific competition (Juliano 1998). Values of λ estimated by Juliano (1998) were generally below replacement values for Ae. aegypti and lower than recorded in our indoor, controlled-temperature experiment. Juliano s results supported the hypothesis that larval competition was an important factor in the displacement of Ae. aegypti by Ae. albopictus, a phenomenon observed throughout most of rural and suburban Florida in the late 1980s and 1990s (O Meara et al.1995). The results of the current study differ from Juliano s (1998) in the higher mean survivorship of both competing species, but especially of Ae. aegypti, and the higher mean masses of both species. However, larval densities per unit volume in the small (100 ml) cages used by Juliano (1998) were much higher than those of the current study. More stressful larval conditions, as imposed by the experiments of Barrera (1996), Juliano (1998) and Daugherty et al. (2000) but not the present study, affect Ae. aegypti more detrimentally than Ae. albopictus. The present, more equivocal results, underscore (again) the importance of experimental conditions and suggest that multiple factors may be important in determining the current patterns of distribution and abundance of Ae. aegypti and Ae. albopictus in Florida. For example, the higher sensitivity to desiccation of Ae. albopictus eggs may result in greater mortality of this species in warmer, drier areas of Florida, and this greater mortality of Ae. albopictus may reduce the impact on Ae. aegypti of larval competition with Ae. albopictus in those areas (Juliano et al. 2001). Acknowledgments We thank B. Alto, S. Juliano, G. O Meara, and two anonymous reviewers for helpful comments on a draft of this paper. Research was supported, in part, by grants from the American Council of Learned Societies/ Social Sciences Research Council Working Group on Cuba and from the National Institutes of Health (AI 47793). This is Florida Agricultural Experiment Station Journal Series No. R REFERENCES CITED Alto, B.W. and S.A. Juliano Temperature effects on the dynamics of Aedes albopictus (Diptera: Culicidae) populations in the laboratory. J. Med. Entomol. 38: Ayala, F.J Competition, coexistence, and evolution. Pp In: M.K. Hecht and W.C. Steere (eds.) Essays in Evolution and Genetics, Appleton, Century Crofts, NY. Barrera, R Competition and resistance to starvation in larvae of container-inhabiting mosquitoes. Ecol. Entomol. 21: Birch, L.C Experimental background to the study of the distribution and abundance of insects. III. The relation between innate capacity for increase and survival of different species of beetles living together on the same food. Evolution 7: Black, W.C., K.S. Rai, B.J. Turco and D.C. Arroyo Laboratory study of competition between United States strains of Aedes albopictus and Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 26: Blackmore, M.S., G.A. Scoles and G.B. Craig Parasitism of Aedes aegypti and Ae. albopictus (Diptera: Culicidae) by Ascogregarina spp. (Apicomplexa: Lecudinidae) in Florida. J. Med. Entomol. 32: Chan, K.L., Y.C. Chan and B.C. Ho Aedes aegypti (L.) and Aedes albopictus (Skuse) in Singapore City. 4. Competition between species. Bull. Wld. Hlth. Org. 44: Colless, D.H. and W.T. Chellapah Effects of body weight and size of blood-meal upon egg production in Aedes aegypti (Linnaeus) (Diptera: Culicidae). Ann. Trop. Med. Parasitol. 54: Craig, G.B The diaspora of the Asian Tiger Mosquito. Pp In: Biological pollution: the

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