Stage-specific divergence in larval thermal tolerance among populations of the Culex pipiens Assemblage

Climate change is shifting the distribution of insect vectors of disease, with consequences for human pathogen exposure. Predicting these range shifts requires understanding vectors' physiological limits. In North America, West Nile virus is transmitted primarily by two closely related and interbreeding Culex species-Cx. pipiens in the north and Cx. quinquefasciatus in the south-which, together with Cx. pipiens f. molestus comprise the North American Cx. pipiens Assemblage. Although the assemblage is widely distributed, little is known about how temperature shapes its species' ranges, particularly in the larval stage, where selection by temperature extremes is likely stronger than in adults. We compared larval thermal tolerance of first and fourth instars under high- and low-temperature extremes among five assemblage populations from the eastern United States. Southern Cx. quinquefasciatus populations were more tolerant of high temperatures than northern Cx. pipiens populations, whereas northern populations showed greater freezing tolerance in later stages. We also quantified larval macronutrient composition by colorimetric assay to characterize population-level differences in fitness-relevant energy stores. Populations differed in composition, but these differences did not align with thermal-tolerance patterns. Finally, we extended the first-instar high-temperature assay to two mid-latitude populations from within and near the hybrid zone in Maryland. Their thermal tolerance was intermediate to northern and southern populations', and LT50 was marginally correlated with collection-site climate after correcting for multiple comparisons. These findings provide empirical data on physiological variation within the Cx. pipiens Assemblage and inform future investigation of the genetic basis of thermal tolerance and range shifts.