Differential Survival of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) Larvae Exposed to Low Temperatures in Taiwan

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1 DEVELOPMENT, LIFE HISTORY Differential Survival of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) Larvae Exposed to Low Temperatures in Taiwan LUN-HSIEN CHANG, ERR-LIEH HSU, HWA-JEN TENG, 1 AND CHAU-MEI HO 2 Department of Entomology, National Taiwan University, Taipei, Taiwan 106 J. Med. Entomol. 44(2): 205Ð210 (2007) ABSTRACT Aedes aegypti (L.) and Aedes albopictus (Skuse) differ in their distribution in Taiwan. The former species is distributed in the south of Taiwan, whereas the latter is found throughout the island. One possible explanation proposes that low temperatures in the winter prevent the expansion of Ae. aegypti. Hence, the impact of low temperatures on immatures of both species was studied in the laboratory and in the Þeld. Our study showed that, under most conditions, Ae. aegypti were more sensitive to low temperatures than Ae. albopictus both in the laboratory and in the Þeld. The survival rates at 10 C for the Þrst and fourth instars of Ae. albopictus were signiþcantly better than those of Ae. aegypti. At 2.5 and 5.0 C, the Þrst instars of Ae. albopictus survived better than the same stadium of Ae. aegypti, but the fourth instars of Ae. aegypti survived better. Short exposures to low temperatures did not affect the acclimatization of Ae. aegypti immatures but longer exposures did increase the physiological adaptation to low temperatures. For Ae. albopictus, exposure to low temperatures increases the acclimatization of this species. In Þeld experiments, Ae. aegypti larvae had a signiþcantly higher mortality than Ae. albopictus during exposures to cold fronts in the 2004 winter. We conclude that low temperatures in northern and central Taiwan have a negative impact on the distribution of Ae. aegypti, but this factor alone is not sufþcient to prevent this species from occupying the rest of Taiwan. KEY WORDS Aedes aegypti, Aedes albopictus, survival, low temperature, LT 50 Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae) are the main vectors of dengue virus in Taiwan. Small- or medium-sized local outbreaks of dengue transmitted by these mosquitoes occurred in the summers after the introduction of dengue virus carried by infected travelers from other countries (Shu et al. 2005). Although the research on a potential vaccine seems very promising, a tetravalenced vaccine will not be available in the near future. Therefore, suppression of the vector population remains the most effective preventive strategy for dengue control. Understanding the ecology of vectors is essential for implementing such a strategy. These two vector species have different global distributions, due to different characteristics such as resistance to cold, diapause, and drought tolerance (Fontenille and Rodhain 1989, Lounibos 2002, Juliano and Lounibos 2005). Ae. aegypti does not diapause (Mitchell 1995), and this species is conþned to subtropical and tropical areas within the 10 C cold-month isotherm. Ae. albopictus females can produce diapausing eggs (Hawley et al. 1989, Focks et al. 1994), which 1 Corresponding author: Division of Research and Diagnostics, Center for Disease Control, Taipei, Taiwan 115 ( hjteng@cdc.gov.tw). 2 Department of Parasitology, National Yangming University, Taipei, Taiwan 112. will survive in cold temperatures, such as 5 and 2 C in continental Asia and Japan, respectively (Nawrocki and Hawley 1987, Mitchell 1995, Kobayashi et al. 2002). However, under various conditions of relative humidity at 22 to 26 C, Ae. albopictus eggs always showed a higher mortality than Ae. aegypti eggs (Juliano et al. 2002). This result helps to explain the distribution of these two species in Florida and Madagascar (Fontenille and Rodhain 1989, Juliano et al. 2002). Differential egg survivorship, however, cannot explain the distribution of these two mosquitoes in Taiwan where the climates belong to subtropical and tropical zones. A strain of Ae. albopictus in northern Taiwan was not sensitive to photoperiod, which triggers diapause in this species (Hawley et al. 1987). Ae. aegypti occurs only in the south of Taiwan, whereas Ae. albopictus is found islandwide (Huang and Chen 1986, Hwang 1991). One possible explanation proposed for this distribution difference was that low temperatures in the winter prevent the expansion of Ae. aegypti. The threshold temperatures of larval development in Taiwan (Chen and Huang 1988) were estimated in the laboratory as 13.4 C for Ae. aegypti and 11.2 C for Ae. albopictus. These parameters for both species were similar to estimates derived from other laboratory studies (Bar-Zeev 1958, Tun-Lin et al. 2000, Lee 1994), but they were higher than the esti /07/0205Ð0210$04.00/ Entomological Society of America

2 206 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 2 Fig. 1. Distribution in Taiwan of Ae. aegypti (blackened areas) and January air temperature means and minima from representative cities. Localities positive for Ae. aegypti are based on 2003Ð2005 surveys, and air temperatures are 1971Ð 2000 averages from the Central Weather Bureau. mate ( 10 C) from Þeld experiments in Japan (Tsuda and Takagi 2001). From the Central Weather Bureau of Taiwan, mean temperatures in January during the period of 1971Ð2000, from south to north, were 20.6 C in Hengchun, 18.8 C in Kaohsiung, 19.2 C in Tainan, 16.1 C in Chiayi, 16.2 C in Taichung, and 15.8 C in Taipei (Fig. 1). Moreover, the temperature may temporarily drop below 10 C when a cold front occurs. The mean minimal temperature in January is higher than the threshold of Ae. aegypti (13.4 C) in southern Taiwan where this species is found. In other Ae. aegypti-free areas, these averages (12.1, 12.4, and 13.3 C in Chiayi, Taichung, and Taipei) were lower than this threshold. The impact of this short-term cold temperature on both species has not been studied and compared. Our objective of this current study is to analyze the effects of low temperatures on the survival of immatures of Ae. aegypti and Ae. albopictus both in the laboratory and in the Þeld. Materials and Methods Origin and Maintenance of Mosquitoes. The colonies of Ae. aegypti and Ae. albopictus used in this study originated from collections from Donggang, Pintung County (22 o 47 N, 120 o 45 E) in 1987 and Linku, Taoyuan County (25 o 05 N, 121 o 22 E) in 2002, respectively. Mosquito colonies were maintained in a walk-in growth chamber at 25 C with a photoperiod of 12:12 (L:D) h. Three hundred to 500 larvae were reared in a plastic pan (21 by 14 by 7 cm) containing 450 ml of deionized water. A sufþcient amount of food (yeast powder and pig liver; 1:1 by weight) was provided daily. Adult mosquitoes were kept in an acrylic cage (29 by 20 by 20 cm) and were provided with a 10% sucrose solution. On the fourth day posteclosion, females were blood-fed. Eggs were collected using paper towels (24 by 4 cm) and were stored in a 25 C growth chamber. All of the eggs used in these experiments were 6 wk old. Low Temperature Exposure in the Laboratory. Experiments were conducted to determine the effect of low temperatures on the survival of immatures of two instars of the two species. Exposure times that resulted in survival rates between 2 and 95% were used to Þt linear relationships. In the fourth instar, the selected exposure times for these two species at 2.5, 5.0, and 10 C were 4Ð14, 3Ð18, and 36Ð144 h, respectively. For the Þrst instar of Ae. aegypti, the exposure times at 2.5, 5.0, and 10 C were 6Ð36, 6Ð60, and 48Ð168 h, respectively, whereas for the Þrst instar of Ae. albopictus, the exposure times were 12Ð72, 24Ð144, and 240Ð840 h, respectively. First instars were 5 h old or less at the start of each experiment, whereas the fourth instars of Ae. aegypti and Ae. albopictus were 4 and 5 d old, respectively. Six replicates of each combination were conducted. Forty larvae in a plastic cup (8Ð9 by 6 cm in top to bottom diameters and height) containing 150 ml of water were kept in an incubator (SANYO MIR- 152, SANYO Electric Co., Tokyo, Japan). After exposure to cold temperatures, the larvae were transferred to a plastic pan (19 by 13 by 6 cm) containing 450 ml of water, which were monitored daily and provided with food. The lightðdark regimen during the experiments was set at 12:12 h by using an aquarium light (13 W, 7000K). Cold Adaptation Selection Experiments. The selection was conducted with a repeated measures design in which the generation served as a repeated measurement through time. First instars of Ae. aegypti and Ae. albopictus were exposed to temperatures of 5 C for a varied duration of time over the course of four generations. Based on the previous studies, the exposure times were set at 6, 12, 24, 36, 48, and 60 h and 24, 48, 72, 96,120, and 144 h for Ae. aegypti and Ae. albopictus, respectively. After exposure, the larvae were removed and kept in an incubator at 25 C for the rest of the growth stages similar to the procedures outlined in the temperature exposure experiment. Field Experiments. Our Þeld experiments were carried out on the campus of National Taiwan University (25 o 06 N, 121 o 52 E) in northern Taiwan during winter Two replicates for each species of 40 newly hatched larvae were kept in a plastic pan containing 450 ml of water. A hole (10 by 5 cm) covered with a piece of iron net was cut into the lid for ventilation. The containers were checked daily, and food was added in excess. The number of surviving larvae was counted daily. Pupae were collected in a cup and

3 March 2007 CHANG ET AL.: SURVIVAL OF Ae. aegypti AND Ae. albopictus 207 Fig. 2. Relationship between survivorship rate and exposure time to cold in Þrst instars (AÐC) and fourth instars (DÐF) of Ae. aegypti and Ae. albopictus at 2.5, 5, and 10 C. Open circles and dashed lines represent Ae. aegypti. Closed circles and solid lines represent Ae. albopictus. placed in a cage with openings covered by nylon stocking material. Water temperatures were measured by a Hobo temperature logger (Stowaway XTI, Onset Computer Corporation, Bourne, MA) at intervals of 30 min during the experimental periods. Data Analysis. The lethal time taken to kill 50% of a population (LT 50 ), the most reliable measure of an insectõs capacity to withstand exposure to a given temperature (Andrewartha 1971), was used as the indicator of acclimatization for Ae. aegypti and Ae. albopictus. Survivorship rates were transformed by the arcsine function to Þt a linear relationship between the survival rate and the exposure time to cold temperatures for laboratory exposure experiments. An estimate of LT 50 can be obtained through the regression of survival against exposure time (X), by using the following equation (Snedecor and Cochran 1980): Y arcsine survival rate a bx where a and b are the least square estimates of the intercept and the slope of regression (PROC REG, SAS Institute 2001). Survivorship rates in this study

4 208 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 2 Table 1. LT 50 (hours) estimates for first and fourth instars of Ae. aegypti and Ae. albopictus at temperatures of 2.5, 5.0, and 10.0 C derived from linear regressions Instar Temp ( o C) LT 50 of Ae. aegypti LT 50 of Ae. albopictus Estimate 95% CI Estimate 95% CI First Ð Ð Ð Ð a Ð Ð Fourth Ð Ð Ð Ð Ð Ð a Negative value was replaced by 0. were corrected with corresponding to zero survival. The LT 50 was estimated from Y 50 (0.6614) by using the equation LT 50 ( a)/b (Chow and Liu 1995). The 95% conþdence interval for the estimated LT 50 was calculated by FiellerÕs theorem (Chow and Liu 1995). The survivorship rates from the selection experiment were subjected to multivariate repeated measures analysis (SAS Institute 1991). For both species, differences in survivorship between the Þrst and fourth generations were compared by paired StudentÕs t-tests (PROC TTEST, SAS Institute 2001) to correct for the correlation between generations. The survivorship in each batch categorized either alive or dead (nominal data) was compared for Ae. aegypti and Ae. albopictus with a chi-square test (PROC FREQ, SAS Institute 2001). Results Low Temperature Exposure in the Laboratory. A signiþcant negative linear relationship (P 0.005) was calculated between survivorship and exposure time for each treatment combination (Fig. 2). InterspeciÞc differences were compared by analyzing the slopes and intercepts of the linear regressions. For Þrst instars, the decrease in survivorship over time for Ae albopictus was signiþcantly slower than for Ae. aegypti at 2.5 C(H o : equal slope, F 1, ; P ) and 5 C (H o : equal slope, F 1, ; P ). At 10 C, the decreased survivorship over time for Ae. albopictus was signiþcantly faster than that of Ae. aegypti (H o : equal slope, F 1, ; P ) but was estimated 350 h later. For fourth instars, the decrease in survivorship over time for Ae. albopictus was signiþcantly faster than Ae. aegypti at 5 C (H o : equal slope, F 1, ; P ) but not at 2.5 C (F 1, ; P 0.056) or at 10 C(F 1, ; P ). At 2.5 C, the survivorship of Ae. aegypti was signiþcantly higher than that of Ae. albopictus at various exposure times (H o : equal intercept, F 1, ; P ), whereas the converse was found for Ae. albopictus at 10 C(H o : equal intercept, F 1, ; P ). Additionally, the LT 50 estimates, calculated from linear regressions, were higher in the Þrst instars of Ae. albopictus than of Ae. aegypti, especially at 10 C (Table 1). However, in the fourth instars, the LT 50 of Ae. albopictus was 2.31 and 4.20 h shorter than that of Ae. aegypti at 2.5 and 5 C but was h longer at 10 C. Cold Adaptation Selection Experiments. Exposure times at 5 C temperature had a signiþcant effect on the survival of Ae. aegypti (F 5, ; P ), but the generation did not (WilksÕ 0.88; F 3, ; P ). However, the exposure times and generation had signiþcant nonadditive effects (WilksÕ 0.13; F 15, ; P ) on the survival of the immatures. To better understand this result, we compared the Þrst generation with the fourth generation. A cold adaptation of Ae. aegypti was identiþed after longer exposures but not after short exposures. The survivorship of the F 3 generation was signiþcantly higher than that of the F 0 generation at 36 h (t 5.04, df 5, P 0.005), 48 h (t 3.1, df 5, P 0.05), and 60h(t 5.85, df 5, P 0.005) but the opposite after shorter exposures (Fig. 3A). For Ae. albopictus, both exposure times (F 5, ; P ) at 5 C temperature and the generation (WilksÕ 0.48; F 3, ; P ) had Fig. 3. (A) Mean survivorships of the Þrst generation (solid circles) and the fourth generation (solid triangles) of Ae. aegypti through selection at 5 C. (B) Mean survivorship of the Þrst generation (solid circles) and the fourth selected (solid triangles) generation of Ae. albopictus through selection at 5 C. Vertical bars represent standard errors.

5 March 2007 CHANG ET AL.: SURVIVAL OF Ae. aegypti AND Ae. albopictus 209 Fig. 4. Larval mortality in Ae. aegypti (open circles) and Ae. albopictus (solid circles) in the Þeld studies in northern Taiwan. The solid bold line represents the daily average temperature, and the other two lines represent the maximum and minimum daily averages. Temperatures at 13.4 and 10 C are the thresholds of larval development of Ae. aegypti in Taiwan and the global limit in the distribution of this species, respectively (Chen and Huang 1988, Mitchell 1995). signiþcant effects on survivorship. In addition, the exposure times and generation had signiþcant nonadditive effects (WilksÕ 0.28; F 15, ; P 0.001) on survival of the immatures. When further comparing the Þrst generation with the fourth generation, cold adaptation was found at all exposure times. However, only middle exposure times of 72 h (t 5.01, df 5, P 0.005) and 96 h (t 2.69, df 5, P 0.05) demonstrated a signiþcant difference between the Þrst and fourth generations (Fig. 3B). Field Experiments. Ae. aegypti had a signiþcantly higher mortality rate than Ae. albopictus during the exposure to cold fronts in the winter of 2004 (Fig. 4). More Ae. aegypti immatures died than Ae. albopictus immatures in the Þrst batch ( , df 1, P ), second batch ( , df 1, P ), and third batch ( , df 1, P ). However, in the fourth batch there was no signiþcant difference between these two species ( , df 1, P 0.05). Both species had the longest developmental time from the Þrst instar to adulthood in January, and this period lasted 31 d. During this experiment (Þrst, second, and third batch), the two mosquito species experienced three to four cold fronts, and the mean water temperature ranged between 14 and 15 C. There was no cold front during the last batch, and the average water temperature was 20 C (Table 2). The lowest temperatures detected were 8.35, 8.75, and 6.16 C in January, February, and March. Discussion Our study demonstrated that, in most conditions, Ae. aegypti were more susceptible to low temperature than Ae. albopictus both in the laboratory and in the Þeld. A short exposure time to low temperature did not affect the acclimatization of Ae. aegypti immatures, but a longer exposure increased survivorship in later generations exposed to low temperatures. For Ae. albopictus, exposure to low temperatures increased the acclimatization of subsequent generations of this species. Overall, low temperatures in northern and central Taiwan, observed in cold fronts, had a negative impact on the range of Ae. Aegypti, but this factor alone is not sufþcient to prevent Ae. aegypti from occupying the rest of Taiwan. Our results showed that Ae. albopictus had a higher cold tolerance than Ae. aegypti, similar to the Þndings of Tsuda and Takagi (2001). They determined that Ae. albopictus larvae had a greater ability to survive at temperatures below 10 C in Nagasaki, Japan, in which circumstances Ae. aegypti larvae all died. The Þeld mortality of Ae. aegypti in this study was as high as Table 2. Effects of outdoor exposures on the development and mortality of the immature stages of Ae. aegypti and Ae. albopictus from January to April 2005 in Taipei, Taiwan Species Batch Front no. Mean temp SD ( o C) n Mortality (%) a Ae. aegypti Ae. albopictus b a Mortality was calculated by the no. of dead larvae 100/no. of larvae tested in each batch. b This mean temperature was calculated from 1 to 15 April. Data from 16 to 19 April were lost.

6 210 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no % (Fig. 4). Hawley et al. (1989) showed that eggs of Ae. aegypti and tropical Ae. albopictus experienced complete or near-complete mortality, whereas temperate strains of Ae. albopictus could survive the winter when overwintering in Indiana. Therefore, in the temperate zone, the temperature in winter is an important determinant of the survivorship of both species and limits the distribution of Ae. aegypti. In Taiwan, containers occupied by both species were found more frequently outdoors than indoors (Hwang 1991, Teng et al. 1996). Low temperatures in the winter, occurring especially during cold fronts, played a more important role in the survival and development of these two species. Hence, low temperatures are likely to limit the expansion of Ae. aegypti due to low survivorship of larvae, and a longer developmental time. The frequent travel of people and goods inside Taiwan by train, automobile, and truck may carry Ae. aegypti eggs, larvae, or adults into areas where this species is absent. Aedes albopictus was introduced into the United States of America through the importation of used tires (Hawley et al. 1987) and lucky bamboo (Madon et al. 2002). The spread of Ae. aegypti also may occur in a similar way because both species are commonly found in these artiþcial containers (Juliano 1998). Ae. aegypti eggs may survive transport, and some of their larvae may develop the ability to tolerate lower temperatures by acclimatization. Research on the impact of relative humidity on egg mortality of these two species under low temperatures between 5 and 20 C should be performed in the future to elucidate this point. Acknowledgments We appreciate the statistical assistance of Shwn-Bin Horng, Jen-Pei Liu, and Jr-Rung Lin. We also thank Simon J. Yu for valuable comments on the manuscript. 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