1. Hatching Response of Aedes aegypti Eggs Following Exposure To Low Air Temperatures Emily Barry with Dr. Matthew Aliota University of Wisconsin – Madison Department of Pathobiological Sciences Abstract With global temperatures on the rise, the geographic range of certain mosquito species is predicted to expand. As ranges expand, the threat of mosquito-transmitted pathogens, which can infect humans, also increases. For example, Chikungunya virus and Dengue virus both are transmitted by Aedes aegypti, a mosquito species anticipated to have its geographic range expand. Ae. aegypti are currently located throughout the Americas and other tropic regions and are found in over a quarter of the United States, primarily in the southeast, although also in the east and southwest. Currently, the geographic range of this mosquito species is limited, in part, by low temperature and cool range margins. However, characterization of the effect of temperature shifts on the egg stage of Ae. aegypti are needed to more accurately define the risk of this mosquito species invading new areas. Here, we determined the hatching response of both field and colonized Ae. aegypti egg batches stored for different time periods (3d, 7d, 14d, 21d) at 4°±1°C with 21% relative humidity or 26.5°±1°C with 75±10% relative humidity. For both the field and colonized strains, the hatch rate continuously decreased after exposure at 4°C, with no hatching after 7 days exposed. Hatch rates at 26.5°C remained relatively constant, with a small decrease in rates as exposure time increased. Interestingly, temperature that eggs are exposed to impacted larval development. Larval development decreased as exposure time at 4°C extended, with deaths of larvae, pupae, and adults observed. In sum, Ae. aegypti eggs and larvae were able to hatch and develop after exposure to temperatures below their defined low temperature limit. These data suggested that Ae. aegypti has the potential to invade more temperate areas as eggs are able to withstand the cold season and hatch again when favorable conditions return. Experimental Procedure Aedes aegypti, black-eyed Liverpool (LVP) strain have been highly colonized at the University of Wisconsin. Ae. aegypti (COL) field eggs were collected in Medellin, Colombia in August 2013, and reared at the University of Wisconsin for 11 generations. Egg strips were separated into containers, and labeled with according temperatures and durations of exposure. Strips were then placed into their specified temperature chamber, either a 26.5°±1°C with 75±10% relative humidity chamber on a 16:8 light:dark cycle, or at 4°±1°C with 21% relative humidity chamber with constant dark. After the designated time periods of 3d, 7d, 14d, or 21d were completed, egg strips were removed from chambers and hatched in water for four days to determine hatch rate. Once hatched, equal numbers of larvae were placed into pans and development was observed and recorded. Each pan received the same amount of food. Ae. aegypti eggAe. aegypti larvae Ae. aegypti pupaeAe. aegypti adult Figure 1: Representation of the life stages of an Ae. aegypti mosquito. The mosquito develops in water and then emerges into an adult. Results Figure 2: Graphs representing the hatching rate of each exposure time. Each line representing a specific strain and temperature exposure. Total Mortality LVP 4°CLVP 26.5°CCOL 4°CCOL 26.5°C 3 Days 5631561056225620 7 Days 237 6 9 10 14 Days 2210002331000 21 Days 001010221170 # observed# dead# observed# dead# observed# dead# observed# dead Table 1: Mortality of mosquitoes was observed. Mortality values were combined from larvae, pupae, and adults. Some exposure periods were observed longer than others. Figure 3: Map of the actual and potential geographic distribution of Ae. aegypti (WHO). As temperatures rise, mosquito populations increase, and the geographic range may expand. Discussion and Conclusion From analysis of data it is seen that as time progresses for temperature exposure, the hatch rate decreases and mortality increases, especially for eggs exposed to 4°C. From observing the graphs of Figure 2, it is seen that at some time between 7 and 14 days, mosquito eggs exposed to cold temperatures will likely not survive and if they do, only a small percentage survives. Between 7 and 14 days of exposure to the temperatures, it is seen that hatching rates with 4°C exposure lower by nearly 80%. This means that mosquito eggs may remain dormant for a time between 7 and 14 days and have successful hatch rates before facing small or nonexistent rates. For the exposures to 26.5°C, the eggs may remain dormant for a time between 14 and 21 days before the hatch rate decreases. As seen in Table 1, mortality was highest among the Liverpool strain at 4°C. This is likely due to the high colonization of the LVP strain. Since the strain has been raised in a constant 26.5°C chamber, their likelihood of survival after exposure to a drastic temperature change is small. The opposite goes into affect for COL. As this is a field strain, the eggs are more likely to be adapted to lower temperatures due to their likely previous exposure to colder temperatures in the environment. It is also seen in Table 1 that mortality is highest among both LVP 4°C and COL 4°C due to their exposure to cold temperatures. If global temperatures continue to rise as predicted, Ae. aegypti will spread geographically. Looking at Figure 3, it is seen that Ae. aegypti are already as far north as the Chicago area and Maine. If the temperatures become warmer, it may be seen that Ae. aegypti could be farther spread due to their ability to withstand temperatures of 4°C for over 7 days before facing large mortality rates. There are numerous factors that effect the hatch rate, such as water temperature and humidity (Eisen, L. et al. 2014), air temperature exposure and time length is a major factor. Due to Ae. aegypti being the carriers of specific mosquito-transmitted pathogens, such as Dengue virus, Chikungunya virus, and yellow fever virus, which can infect humans, their threat increases. If there are greater mosquito populations in a greater area, humans face a larger danger of becoming infected with dangerous diseases. Although these diseases are not a common risk, rising mosquito populations will make the threat more widespread. In conclusion, temperature and the duration of the exposure affects the hatch rate and survival of mosquitoes. Although not clearly negative until after 7 days, mosquitoes are able to sustain their population at 4°C affecting the geographic spread of Ae. aegypti and their transmittable diseases. Acknowledgments Selected References I would like to thank Professor Matthew Aliota for agreeing to take me on for the summer, and teaching me invaluable information. I also give him my greatest gratitude for being incredibly patient and supportive of me throughout the entire process. I would also like to thank the MMSD, specifically Lisa Wachtel and Carmen Lombard for allowing me to participate in such a spectacular program. E. M. McCray Jr., H. F. Schoof, (Dec. 1972). “The Effects of Low Temperature on Eggs of Aedes aegyti” Mosquto News 1972. Brady, Golding, Pigott, Kraemer, Messina, Reiner Jr, Scott, Smith, Gething, Hay, (2014). “Global temperature constraints on Aedes aegypti and Aedes albopictus persistence and competence for dengue virus transmission”, Parasite & Vectors. Eisen, L. et al. (May 2014). “The Impact of Temperature on the Bionomics of Aedes aegypti, with Special Reference to the Cool Geographic Range Margins”. Journal of Medical Entomology. Ciota, A. et al. (January 2014). “The Effect of Temperature on Life History Traits of Culex Mosquitoes.” Journal of Medical Entomology. Figure 4: Mean longevity was calculated from data. Note that sufficient data was not collected for 14 or 21 days.