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, depending on the year (Fig. 1, 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.Thistransect of the Sonoran Desert occupies 394.2 km and a range in elevation from 210 m (Hermosillo, Mexico) above sea levelto 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 and subsequent dengue transmission (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[47–49]. 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 locations within each city for quantification and then transported to the central laboratory in Tucson 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. Dead females from Nogales and Hermosillo were discarded and unable to be analyzed for this purpose.
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[52,53]. 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[53–55]. In this study we characterized females with a visible bloodmeal as parous, therefore it is more accurate to consider parity in this context as a measure of the percent of blood-fed females in a population.
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. Host seeking in Ae. Aegypti females begins at 36-48 hours post-emergenceand the average extrinsic incubation period of the dengue virus ranges from 6.5-15 days. This means that the youngest age group cannot participate in disease transmission, the 6-14 day old group can but is unlikely to include possible vectors, and the oldest age group will consist of likely vectors. These age groups were used to get a general estimate of when the developmental period occurred. In addition, a continuous measure of age was adapted from the abundance values of SCP-1 (Schmidt, unpublished data) using data from, and was used in the regression models. The relationship between transcript abundance of SCP and chronological age was not significantly different between fed and unfed mosquitoes. The R code used to generate this continuous age variable can be found in the Supplementary Materials.
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 along the major axis, from the proximal to the distal end for each wing, as described in. In a previous publication by this study’s authors, wing length of the major axis was found to be more tightly associated with age at death than wing area or length of the minor axis.
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. Although female mosquitoes collected at the same time, of the same age group, are still likely to have variation in development time, it is not currently possible to estimate development time in field-collected mosquitoes. For this reason, we chose to use seven-day averages for estimating average developmental temperature. We previously found an average developmental period of about 8 days for Ae. aegypti derived from eggs collected from three locations in Tucson, Arizona.
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 August 2013,Nogales in August of 2013 for age groups 2 and 3, or July 2013 for any city. This lack of data due to missing or defective HOBOs reduced the total N in our path analyses from 3,920 to 1,125.
All data was analyzed on R 1.0.143and JMP. ANOVA and linear regression were used to test the impact of the explanatory variables temperature during development, wing length, female abundance, 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 (SEM). R was used for the SEM using the variables average temperature during development, wing length, female abundance, relative humidity in the one week prior to capture and temperature in the one week prior to capture, and age. The R packages used for the SEM were car, QuantPsyc, ggm, semPlot, lavaan, nlme, and devtools.
The strength of the models tested were first evaluated by comparing their AIC values which consider indirect effects and impose a penalty for each additional variable used. Using AIC enables prioritization of simplicity in model selection. Models with the lowest AIC scores were then evaluated by their adjusted R2 to determine goodness-of-fit.