Adult mosquitoes were collected from households in 3 cities over a four-day period, once a month. Each city had between 15 and 40 trapping sites (Fig. 1b, 1c, 1d, Supp. Table 1). The study cities occupy a latitudinal transect at the northern edge of the geographic range of Ae. aegypti (Fig. 1a). At the southern end of this transect is Hermosillo, Sonora, Mexico (29.0989° N, 110.9542° W); A city where the Ae. aegypti population has maintained local, seasonal transmission of the dengue viruses. At the center of the transect is Nogales, Sonora, Mexico (31.1907° N, 110.5645° W) which saw its first cases of local transmission in 2014, during the study period; At the northern end of this transect is Tucson, Arizona, USA, (32.2217° N, 110.9264° W) which has no documented cases of locally-acquired dengue fever before or during the study period. This transect of the Sonoran Desert occupies 394.2 km and a range in elevation from 210 m (Hermosillo, Mexico) above sea level to 1,199 m (Nogales, Mexico). Collections were limited to the 3 months of the monsoon season, July, August, and September (sometimes in October), due to the significant seasonal increase in mosquito abundance following summer monsoon precipitation events (Supp. Table 2).
Mosquito sampling and testing for bias
Biogents BG-Sentinel traps were baited with octanol and lactic acid lures and were either connected to a battery or to a household electric supply. BG Sentinel traps have been found to be about as efficient as human landing rate or backpack aspirators and more efficient than oviposition traps for evaluating abundance in the field [28–30]. BG Sentinel traps have a slight bias for host-seeking Ae. aegypti females and the location of the trap is a potential source of bias against nulliparous females . Also, adult mosquito abundance is not affected by the use of insecticides indoors . In essence, known trapping biases would result in over-sampling of our target group of blood-fed females, which is preferable for public health surveillance.
Adults collected from traps were aspirated into containers and taken to the laboratory for analysis. Dead adult females were counted and included in abundance data but not included in any parity or age analyses. Live females were stored in a -80º C freezer until processing (N= 3,920 measures of individual size and N = 4,739 individuals analyzed for parity status). Since traps were only checked once a day during the collection periods, some mortality occurred in the field-collected females that could have caused a bias in the body size of the surviving females. To test if there was a size bias due to differential survival in the trap, dead females from Tucson (N = 60) were measured and compared to live females (N = 78) from the same subset of sites and months.
Mosquito age and parity assessments
Ovaries were dissected to determine parity. Visual inspection of trachea in the ovaries allowed us to determine whether a female had completed a reproductive cycle, or not [33,34]. Tracheae that are tightly coiled are considered nulliparous, having never completed a reproductive cycle. Individuals with extended tracheae are considered parous, since once the tracheae extend to transport oxygen to developing eggs they will not recoil. Individuals determined to have completed a reproductive cycle, and/or had a visible, undigested blood meal, and/or eggs were all considered as parous. Parity serves as a physiological marker of age and for observing changes over time in biting persistence and the human/mosquito contact rate, for a given location [34–36].
Classification of individuals into age groups was done with a genomic age-grading technique using real-time PCR assays of an age-dependent gene, SCP-1 . Females tested for age could be classified categorically as being either 0-5, 6-14, or >15 days old. A continuous measure of age was also adapted from the abundance values of SCP (Ernst, unpublished data), and was used for testing the regression models.
Wing measurements and weather data
Wings were removed from field-collected females and affixed onto glass microscope slides with a drop of water. Samples were secured onto the slides with a glass cover slip fixed with tape on the sides. Length was measured from the proximal to the distal end for each wing, as described in .
Seven-day averages of temperature, diurnal temperature range, average daily maximum and minimum temperature, and percent relative humidity were estimated using city-specific, historical weather data from the National Oceanic and Atmospheric Association (NOAA) and using site-specific (sites within cities) averages from remote climate loggers (HOBO Pro v2, Onset). Weather averages from NOAA and the HOBOs were each tested against wing length to determine which data source was a better fit.
Using our sample-specific age data, we also tested a new technique for estimating when a particular female developed. This technique involves back-casting by different periods of time starting from the date a sample was captured, based on the results of the age-dependent gene expression analyses (Supp. Table 3). Estimating the developmental period of individual mosquitoes in order to study the impact of environmental factors on adult longevity is a novel approach, considering previous studies typically assign the same estimated development period to all mosquitoes sampled .
A number of dates are missing HOBO data: Nogales 2013 August, Age group 1 was generated from 6 days (missing one day of weather data). No HOBO data exists for Hermosillo 2015 or 2013 August and Nogales 2013 August for age groups 2 and 3, or July 2013 for any city.
All data was analyzed on R 1.0.143  and JMP . ANOVA and linear regression were used to test the impact of the explanatory variables temperature during development, wing length, and relative humidity in the 1 wk prior to capture and temperature in the 1 wk prior to capture on the response variable, age.
To test direct and indirect effects of explanatory factors on wing length and age at death, we used a combination of factor analysis and regression analysis known as multivariate path analysis or structural equation modeling. R was used to do the path analyses on average temperature during development, wing length, relative humidity in the one week prior to capture and temperature in the one week prior to capture, and age. The strength of the models tested were evaluated by comparing their AIC values which take into account indirect effects and impose a penalty for each additional variable used. Using AIC enables prioritization of simplicity in model selection.