Global warming trends, human-assisted transport, and urbanization have allowed poleward expansion of many tropical vector species, but the specific mechanisms responsible for thermal mediation of range changes and ecological success of invaders remain poorly understood.  Aedes aegypti (Diptera: Culicidae) is a tropical mosquito currently expanding into many higher-latitude regions including the urban desert region of Maricopa County, Arizona. Here, adult populations virtually disappear in winter and spring, and then increase exponentially through summer and fall, indicating that winter conditions remain a barrier to development of A. aegypti. To determine whether cold limits the winter development of A. aegypti larvae in Maricopa County, we surveyed for larval abundance, and tested their capacity to develop in ambient and warmed conditions. Aedes aegypti larvae were not observed in artificial aquatic habitats in winter and spring but were abundant in summer and fall, suggesting winter suppression of adults, larvae or both. Water temperatures in winter months fluctuated strongly; larvae were usually cold-paralyzed at night but active during the day. Despite daytime temperatures that allowed activity, larvae reared under ambient winter conditions were unable to develop to adulthood, perhaps due to repetitive cold damage. However, warming average temperature by 1.7°C allowed many larvae to successfully develop to adults. Because daytime highs in winter will often allow adult flight, it is possible that relatively minor additional winter warming may allow A. aegypti populations to develop and reproduce year-round in Maricopa County.

From September of 2017, till August of 2019, water temperatures and A. aegypti larval presence was recorded in nine 19 liter buckets placed in the backyard of Jon Harrison’s home in Tempe, Arizona (33.339, -111.924), as it was known to experience high abundances of A. aegypti. Buckets were 5 – 10 m apart, and so should not be considered ecologically independent. Onset HOBO Pendant® UA-002-08 data loggers (Bourne, Massachusetts) were used to record temperature levels, and larval presence was observed every 1-10 days depending on season (frequently in the summer, less so in winter). If mosquito larvae were observed, they were collected from the bucket with a net and their species identity confirmed with a dissection scope. The data set labeled Figure 2 data provides the water temperatures in one representative bucket from 2017-2019 as shown in Fig. 2 of the manuscript.

Larval rearing for mesocosm experiments

The parents of larvae used in the mesocosm overwintering experiments were reared from Maricopa County, AZ, origin eggs collected by Maricopa County Vector Control from September to November of 2019. These eggs were placed in a 500 ml beaker, submerged, and hatched in a solution of 0.25 g/L baker’s yeast (Byttebier et al. 2014). As the 1^st instar larvae emerged, they were fed TetraMin fish flakes every 1-2 days, making sure that an excess amount of food was visible in the container. The rearing density for the larvae was maintained at fewer than 500 animals per liter of water. As pupae began to appear, the beaker of larvae was placed in a 95-liter polymer-screened cage to contain the expected adults. Cotton balls saturated with 10% sucrose solution were made available for the adults as they began to emerge; these were taken away two days prior to blood feeding. One week after emerging, the adults were blood-fed using mice (IACUC protocol: 18-1662R). After a three-day gestation period, the females were supplied with moist seed-germinating paper to encourage oviposition. Once the females had finished ovipositing, the eggs were kept moist for an additional 48 hours before being dried, and placed in open zip lock sandwich bags which were stored at 100% humidity and 24°C. High humidity in the egg storage containers was achieved by storing damp paper towels along with the opened egg bags within a larger 3.8L bag. These eggs were kept for less than one month before the hatching procedure was repeated to produce the larvae for the experiment. In the lab, across all life stages, the mosquitoes were exposed to a 12:12 L/D photoperiod at 24°C.

After hatching, the 2^nd instar larvae were moved to their outdoor experimental mesocosms. The larvae were randomly distributed with 20 larvae supplied per each of three ambient mesocosms (Amb1, Amb2, Amb3) and six to warmed mesocosms (W1 – W6), which were warmed by varying amounts (W1 = least warmed, W6 = most warmed). The goal was to achieve a range of warming from very small warming (1-2°C in the least-warmed mesocosm (W1), to near-summer conditions in the most-warmed mesocosm (W6). Each mesocosm was a 150 ml clear plastic container, filled with 125 mL dechlorinated tap water. TetraMin fish flakes were supplied to each mesocosm, with more added every three days or when food was completely consumed. Although the mesocosms were open, we observed no mosquitoes flying in the field, and none were captured in local water buckets, and all A.a. in the mesocosms were of uniform stage, so we believe that this experiment was not affected by oviposition from wild mosquitoes.

Manipulation of thermal conditions for larval outdoor rearing

All mesocosms were placed on a table one meter above the ground and protected from rain, wind, and sunlight by a roof. The mesocosms were placed within individual lidless pine boxes (10x10x14 cm, 0.95 cm thick walls), and so were exposed to normal fluctuations in air temperature.  Each warmed mesocosm was placed on 40mm² thermoelectric plates with 40mm² aluminum heatsinks attached using thermally conductive adhesive on each side. The warming orientation of the thermoelectric plate was positioned upwards, towards the mesocosms, to ensure adequate energy transfer from the heating units to the water. Each thermoelectric device was powered by two KORAD KD3005D 30V, 5A power supplies (Shenzhen, China). The thermoelectric plates were wired in parallel. Variable warming was produced by changing the supplied voltage. Temperatures were measured in the cups using HOBO Pendant® UA-002-08 data loggers submerged in the center of each cup. We did not measure temperature gradients within the mesocosms, but believe that they are likely to be small except possibly in the mesocosms that were maximally-warmed, as the mesocosms were small and mostly not strongly warmed above air temperature. Temperatures were logged each hour in each warmed mesocosm, and in one ambient treatment mesocosm. The data file labeled Figure 3 data provides the wate temperatures at hourly intervals during the experiment for one mesocosm at ambient temperature, mesocosm W1 (the least warmed mesocosm) and mesocosm W6 (the most warmed mesocosm) as shown in Fig. 3 of the manuscript.