Mosquito rearing
Aedes albopictus used in the described experiments consisted of the National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (NIAID) strain from Gainesville, FL (MRA-804), contributed by Sandra A. Allan to BEI resources. Aedes aegypti used in these studies were from a colony strain collected from Corona, California. Eggs were hatched in 22 × 22 × 7.5 cm disposable plastic containers (Pactiv, Lake Forest, IL, USA) containing 500 mL aged liver powder solution (6 g liver powder/L) (Now Foods, Bloomingdale, IL). Larval pans and adult cages were housed in an incubator set to 28 ± 2.2 °C with a humidity of 85 ± 5% and a light (L):dark (D) cycle of 16:8 h. Eggs were hatched in a 1:1 solution of deionized water (DI) and fermented liver powder (0.6 g/L) (Now Foods, Bloomingdale, IL). After a 2-h egg hatch interval, first-instar larvae were separated into 21 cm × 21 cm × 7.5 cm disposable plastic rearing pans containing 500 mL of DI water at approximately 200 larvae per pan (Pactive, Lake Forest, IL). Larvae were provided a 60 g/L liver powder slurry for food ad libitum. Pupae were removed from larval rearing pans using a disposable plastic pipette and placed into 50 mL of DI water in a 140 mL plastic cups (Pactiv, Lake Forest, IL, USA). The plastic cup was then placed into a 24.5 × 24.5 × 24.5 cm BugDorm cage (MegaView Science Co., Taichung, Taiwan) where the pupae emerged into adults. Adults were provided with 10% sucrose solution ad libitum.
Mosquito survivorship and longevity
To examine for an effect of thermal stress on adult survivorship and longevity, mosquitoes were reared at either 22 °C (R22) and 28 °C (R28) and were exposed to either 22 °C (E22), 28 °C (E38), 32 °C (E32), and 38 °C (E38) as adults. In brief, eggs were hatched using the same methods described in the mosquito rearing section. After a 2-h egg hatch interval, first-instar larvae were separated into 21 cm × 21 cm × 7.5 cm disposable plastic rearing pans containing 500 mL of DI water at approximately 200 larvae per pan (Pactive, Lake Forest, IL). Larvae were provided a 60 g/L liver powder slurry for food ad libitum. Pupae were transferred in 50 mL of DI water in 140 mL plastic cups (Pactiv, Lake Forest, IL, USA) to 24.5 × 24.5 × 24.5 cm BugDorm cages (MegaView Science Co., Taichung, Taiwan).
In total, 75 approximately 48-h-old male and female mosquitoes were aspirated using a mechanical aspirator and anesthetized by placing the aspirator tip with mosquitoes in plastic bag with a chloroform soaked cotton ball for 15–20 s. The adults were then transferred to 1.8 L (11.4 height × 23.7 cm diameter) plastic buckets modified to serve as mosquito cages (Airlite Plastics Co., Omaha NE). The buckets were modified into cages by adding a tubular stockenett (Richardson Products Inc., Frankfort, IL, USA) to provide access to the cage and a portion of the top of the lids removed and covered with no-see-um netting (Skeeta, Inc., Bradenton, FL, USA). Each cage type consisted of three to four replicates. The cages with mosquitoes were placed in an incubator set to the initial rearing temperature for 24 h. After the 24 h, the cages were moved to the respective exposure temperature. The cages were monitored daily for dead adults.
Mosquito respiration as a measure of thermal stress
For respiration experiments, Ae. aegypti and Ae. albopictus were reared at either R22 or R28 as described in the previous section. Eggs were hatched using the same methods described in the mosquito rearing section. After a 2-h egg hatch interval, first-instar larvae were separated into 21 cm × 21 cm × 7.5 cm disposable plastic rearing pans containing 500 mL of DI water at approximately 200 larvae per pan (Pactive, Lake Forest, IL). Larvae were provided a 60 g/L liver powder slurry for food ad libitum. Pupae were transferred in 50 mL of DI water in 140 mL plastic cups (Pactiv, Lake Forest, IL, USA) to 24.5 × 24.5 × 24.5 cm BugDorm cages (MegaView Science Co., Taichung, Taiwan). Adults were provided with 10% sucrose solution ad libitum.
Mosquito respiration rates were determined by a Li-Cor gas analyzer (LI-6800–89) with an attached insect chamber (Chamber volume 49.9 cm3) (LICOR Corporate Offices—US, Lincoln, NE). Approximately 24 h post emergence, 50 males or females were aspirated from their respective cage and placed into the Li-Cor respiration chamber. The Li-Cor gas analyzer pump speed was set to auto, the flow rate was set to 400 μmol/s, the press valve was set to 0.0 kPa, H2O was set to on, the relative humidity was set to 85%, CO2 injector was set to on, the soda lime was set to scrub auto, and carbon dioxide concentration in the reference cell (CO2_r) was set to a setpoint of 400 μmol−1. The adult mosquitoes were allowed to acclimate to the exposure temperature for 20 min before data collection began. A measurement of CO2 production was taken every 1 min for 120 min.
Heat shock gene expression
Real-time quantitative polymerase chain reaction (qPCR) was performed to examine the expression of heat shock genes: AeaHsp26, AeaHsp83, and AeaHsc70. Primers for these genes are found in supplemental Supplementary Table S1 [20]. Ae. aegypti and Ae. albopictus female whole mosquitoes from each rearing and exposure temperature combination at 2 and 24 h postexposure were flash frozen using liquid nitrogen. RNA was extracted using Qiagen RNeasy kit following the manufacturer’s instructions. Following the RNA extraction, complementary DNA (cDNA) was made using a Lunascript cDNA synthesis kit (New England Biolabs, Ipswich, MA) following the manufacturer’s instructions. All amplifications were performed as two technical replicates using Platinum 10X SYBR Green qPCR SuperMix-UDG (ThermoFisher) on a Bio-Rad CFX96 qPCR system (Bio-Rad, Hercules, CA USA). Thermocycler amplification cycling conditions consisted of an initial denaturing step of 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 54–60 °C for 1 min, and 72 °C for 30 s, and an elongation step at 72 °C for 8 min.
Wing collection and measurement
Ae. albopictus and Ae. aegypti were reared at R22 and R28. Eggs were hatched using the same methods described in the mosquito rearing section. After a 2-h egg hatch interval, approximately 200 first-instar larvae of each species were placed into a separate 21 cm × 21 cm × 7.5 cm disposable plastic rearing pan containing 500 mL of DI water (Pactive, Lake Forest, IL). Larvae were provided a 60 g/L liver powder slurry for food ad libitum. Pupae were transferred in 50 mL of DI water in 140 mL plastic cups (Pactiv, Lake Forest, IL, USA) to 24.5 × 24.5 × 24.5 cm BugDorm cages (MegaView Science Co., Taichung, Taiwan). Once the mosquitoes reached adulthood, approximately 10–15 mosquitoes from both sexes (one for each sex and each rearing temperature) were aspirated and placed into a 2.5 mL Eppendorf tube containing mineral oil to prevent desiccation and stored at −20 °C. Wings were removed, placed on a slide, and imaged using an Leica S9 stereomicroscope and attached camera (Leica microsystems, Wetzlar, Germany). Wings were then measured from the base of the wing to the axial vein using the images and ImageJ software (vs. 1.54 m).
Statistical analyses
Survivorship curves and log-rank analyses were performed using JMP Pro 17 (JMP Statistical Discovery LLC, Cary, NC). Comparisons of CO2 production from respiration trials was performed in R using a generalized estimation equation (GEE) model. Kruskal–Wallis tests followed by pairwise Wilcoxon tests were used to determine differences in the HSP gene expression and wing lengths using JMP Pro 17 (JMP Statistical Discovery LLC, Cary, NC).