Thermal tolerance and preference are both consistent with the clinal distribution of house fly proto-Y chromosomes

Selection pressures can vary within localized areas and across massive geographical scales. Temperature is one of the best studied ecologically variable abiotic factors that can affect selection pressures across multiple spatial scales. Organisms rely on physiological (thermal tolerance) and behavioral (thermal preference) mechanisms to thermoregulate in response to environmental temperature. In addition, spatial heterogeneity in temperatures can select for local adaptation in thermal tolerance, thermal preference, or both. However, the concordance between thermal tolerance and preference across genotypes and sexes within species and across populations is greatly understudied. The house fly, Musca domestica, is a well-suited system to examine how genotype and environment interact to affect thermal tolerance and preference. Across multiple continents, house fly males from higher latitudes tend to carry the male-determining gene on the Y chromosome, whereas those from lower latitudes usually have the male-determiner on the third chromosome. We tested whether these two male-determining chromosomes differentially affect thermal tolerance and preference as predicted by their geographical distributions. We identify effects of genotype and developmental temperature on male thermal tolerance and preference that are concordant with the natural distributions of the chromosomes, suggesting that temperature variation across the species range contributes to the maintenance of the polymorphism. In contrast, female thermal preference is bimodal and largely independent of congener male genotypes. These sexually dimorphic thermal preferences suggest that temperature-dependent mating dynamics within populations could further affect the distribution of the two chromosomes. Together, the differences in thermal tolerance and preference across sexes and male genotypes suggest that different selection pressures may affect the frequencies of the male-determining chromosomes across different spatial scales.

Methods
We performed our experiments using five nearly isogenic house fly strains, three with IIIM males and two with YM males. We reared each strain at 18°C, 22°C, and 29°C for two generations in order to evaluate how thermal acclimation affects thermal tolerance and thermal preference. Flies from each developmental temperature were assayed at equivalent physiological ages estimated by accumulated degree days. For our heat and cold tolerance assays, we used flies 22–50 total degree days after eclosion. For thermal preference assays, we used flies 96–115 total degree days after eclosion.

Thermal tolerance

We measured heat and cold tolerance in individual male and female flies. To measure heat tolerance, lightly anaesthetized individual flies were transferred to a 1.5 ml centrifuge tube that was sealed with fabric. We placed the 1.5 ml tube in a heat block set to 53°C. The time at which a fly fell to the bottom of the tube and could not make its way back to the top of the tube was considered the knockdown time. To measure cold tolerance, lightly anaesthetized flies were transferred to a fabric-sealed 20 ml glass vial individually, and the vials were placed in a 4°C refrigerator with a transparent door. Knockdown occured when a fly fell on its back to the bottom of the vial. We gently tapped the assay vial every 2–3 minutes to ensure flies were active. We also compared heat and cold tolerance between males raised at 22°C and 29°C, using the same approaches as described above.

Thermal preference

We measured thermal preference estimated from the position of individual flies along a 17–37°C thermal gradient. For each individual fly, we report mean thermal preference (Tpref) as the average position during a 10 minute assay window (measured once per minute). We also report thermal breadth, Tbreadth  as the coefficient of variation of individual-level Tpref during the assay window. Tbreadth provides an estimate of how individuals utilize thermal space within their environment