Most models exploring the effects of climate change on mosquito-borne disease ignore thermal adaptation. However, if local adaptation leads to changes in mosquito thermal responses, ‘one size fits all’ models could fail to capture current variation between populations and future adaptive responses to changes in temperature. Here we assess phenotypic adaptation to temperature in Aedes aegypti, the primary vector of dengue, Zika, and chikungunya viruses. First, to explore whether there is any difference in existing thermal response of mosquitoes between populations we used a thermal knockdown assay to examine five populations of Ae. aegypti collected from climatically diverse locations in Mexico, together with a longstanding laboratory strain. We identified significant phenotypic variation in thermal tolerance between populations. Next, to explore whether such variation can be generated by differences in temperature we conducted an experimental passage study by establishing six replicate lines from a single field-derived population of Ae. aegypti from Mexico, maintaining half at 27°C and the other half at 31°C. After 10 generations we found a significant difference in mosquito performance, with the lines maintained under elevated temperatures showing greater thermal tolerance. Moreover, these differences in thermal tolerance translated to shifts in the thermal performance curves for multiple life history traits, leading to differences in overall fitness. Together, these novel findings provide compelling evidence that Ae. aegypti populations can and do differ in thermal response, suggesting that simplified thermal performance models might be insufficient for predicting the effects of climate on vector-borne disease transmission. 

Mosquito Collection

Aedes aegypti mosquitoes were collected from the field using ovitraps in five different locations in Mexico (Cabo San Lucas, Acapulco, Monterrey, Ciudad Juárez, and Jojutla) and compared to a standard laboratory population (Rockefeller strain) maintained at Penn State University. The field locations were chosen to capture a gradient of the landscape and climate, driven primarily by variation in altitude. Populations were founded with sufficient viable eggs collected from multiple ovitraps across the cities to yield at least 100 adult mosquitoes in the F1 laboratory generation. Mosquitoes were reared for a generation (F2) in standard laboratory conditions (27°C, 80% humidity, 12:12hr photoperiod, 0.60 mg of bovine liver powder per 600 larvae and ad libitum access to 10% sugar solution for the adults) prior to experimentation to remove the influence of any maternal effects. Two populations, Jojutla and Juárez, were reared for an additional generation (F3) to ensure a large enough population for subsequent experiments. 

Knockdown assays to estimate thermal tolerance

We followed methods developed recently by Ware-Gilmore et al. to examine the thermal tolerance of adult Ae. aegypti mosquitoes. These methods were adapted from numerous studies in Drosophila and were shown to be sufficiently sensitive to demonstrate the effects of infection with either dengue virus or the bacterial endosymbiont, Wolbachia, on thermal sensitivity. In brief, three-to-four-day-old female mosquitoes were placed into individual sealed 40mL glass vials. The vials were submerged into a tank filled with water at a regulated temperature of 41°C. Individuals were allowed two minutes to acclimate, after which they were monitored and the time to knockdown (immobility or death) was recorded. We monitored mosquitoes until all were knocked down. In the common garden experiment, we conducted six replicate runs of 10 mosquitoes giving a total of 60 mosquitoes per population. The passage experiment had 18 total replicates (6 per independent passaged line) of 10 mosquitoes for each temperature treatment.

Experimental passage

We used the F1 population of Ae. aegypti mosquitoes collected from Monterrey, Mexico, to establish six replicate lines, half of which were maintained at a standard insectary temperature of 27°C (80% humidity, 12:12hr photoperiod), which also approximates the overall mean temperature in Monterrey summer months, and the other half were maintained at an elevated temperature of 31°C (80% humidity, 12:12hr photoperiod) (SI Appendix, Fig. S1 and S2, Table S1). The elevated temperature of 31°C represents an increase of 4°C as might be expected under future climate warming. However, neither temperature simulates realistic environmental variation in the natural home environment and so both treatments were under some level of artificial selection to lab conditions.

Each replicate line was initiated with 600 first instar larvae. Larvae were added into 5.7 L containers containing 3 L of deionized water and 0.60mg of bovine liver powder (MP Biomedicals) and placed in controlled temperature incubators (3 replicate containers at 27°C and 3 containers at 31°C). Every other day we added 0.60 mg of bovine liver powder to each container until larvae began to pupate when we scaled the food to the number of remaining larvae. We removed pupae and placed them in a small cup (30 mL) with water from their original environment to allow for eclosion. Cups containing pupae were added to a large cage with ad libitum access to 10% sugar solution made with dextrose anhydrous and deionized water to sustain the adults as they emerged. We counted total pupae per container, along with the number of pupae that eclosed successfully. When the adult mosquitoes were of reproductive age (3-5 days after eclosion), any dead adult mosquitoes were counted. These measures were used to get an accurate count of surviving adult mosquitoes in each cage.  To ensure balanced selection between lines and account for potential effects of genetic bottlenecks or drift that could result from different population sizes, adult mosquitoes were culled (3-5 days after eclosion) before blood feeding so that each line had the same number of mosquitoes. The lines were culled in pairs, for example replicate one at 27°C was paired with replicate one in 31°C, for all ten generations to ensure independence between replicates. Mosquitoes were culled with a 50:50 sex ratio to maintain possible differences in selection on sex. Once the adult cages were established, mosquitoes were fed a human blood meal every four days for 16 days using a standard membrane feeder. Eggs were collected every day and maintained on dry filter paper to prevent hatching. At the end of the 16-day egg laying period, the eggs were transferred to larval containers to initiate hatching, thus maintaining a uniform age structure. Mosquitoes were reared through to adult as described. We followed this protocol for ten generations (SI Appendix, Fig. S5).

At the end of the experimental period, we measured thermal tolerance for the six lines using the knockdown methods described above. In addition, to extend beyond this proxy variable and fully explore the effects of the passage treatments, egg-to-adult survival, mosquito development rate, mean adult survival, and fecundity were measured in mosquitoes reared in environmentally controlled incubators at 13°C, 17°C, 21°C, 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, 37°C, each ± 0.2°C and 80%± 10% relative humidity. Eggs from the 6 passaged lines were hatched at 27°C.  After 24 hours, 200 first instar larvae were put into 1.89 L containers with 1 L of deionized water and 0.20 mg of larvae bovine liver powder (MP Biomedicals) and placed in the respective incubator. We fed larvae 0.20 mg of liver powder every other day until pupation. Once larvae began to pupate, we scaled their food to the number of remaining larvae. We removed and counted living and dead pupae the day of pupation and placed them in a small cup (30 mL) with water from their original environment to allow for eclosion. Cups containing pupae were added to a small cage (17.5 cm3) with ad libitum access to 10% sugar solution made with dextrose anhydrous and deionized water. We then counted the number of adults that eclosed every day. After 95% of females emerged, we blood fed females who were then 3-5 days old.  We used blood from de-identified human donors (BioIVT, Corp.) so IRB approval and human subjects’ approval was not needed. Immediately after blood-feeding, we counted the total number of blood-fed females and placed up to 10 individual females into separate containers (50 mL polypropylene centrifuge tubes) lined with filter paper that contained 7 mL deionized water to measure individual fecundity. We recorded the day that females in individual containers first laid eggs and let them lay eggs for three total days, after which we removed them from their containers. We extracted the water from the containers to let the filter paper dry in their respective incubators and then we counted the eggs. We counted and determined the sex of the number of adults that died every day. We censored this experiment 4 weeks after the first egg lay at each temperature (SI Appendix, Fig. S5).