DOI: 10.5061/dryad.z8w9ghxgc
title: Data for: Phenotypic adaptation to temperature in the mosquito vector, Aedes aegypti

description: Most models exploring the effects of climate change on mosquito-borne
 disease ignore thermal adaptation. However, if local adaptation leads to
 changes in mosquito thermal responses, ‘one size fits all’ models could
 fail to capture current variation between populations and future adaptive
 responses to changes in temperature. Here we assess phenotypic adaptation
 to temperature in Aedes aegypti, the primary vector of dengue, Zika, and
 chikungunya viruses. First, to explore whether there is any difference in
 existing thermal response of mosquitoes between populations we used a
 thermal knockdown assay to examine five populations of Ae. aegypti
 collected from climatically diverse locations in Mexico, together with a
 longstanding laboratory strain. We identified significant phenotypic
 variation in thermal tolerance between populations. Next, to explore
 whether such variation can be generated by differences in temperature we
 conducted an experimental passage study by establishing six replicate
 lines from a single field-derived population of Ae. aegypti from Mexico,
 maintaining half at 27°C and the other half at 31°C. After 10 generations
 we found a significant difference in mosquito performance, with the lines
 maintained under elevated temperatures showing greater thermal tolerance.
 Moreover, these differences in thermal tolerance translated to shifts in
 the thermal performance curves for multiple life history traits, leading
 to differences in overall fitness. Together, these novel findings provide
 compelling evidence that Ae. aegypti populations can and do differ in
 thermal response, suggesting that simplified thermal performance models
 might be insufficient for predicting the effects of climate on
 vector-borne disease transmission. 
description: <strong>Mosquito
 Collection</strong>
 <em>Aedes
 aegypti</em><em>
 </em>mosquitoes were collected from the field using ovitraps in five
 different locations in Mexico (Cabo San Lucas, Acapulco, Monterrey, Ciudad
 Juárez, and Jojutla) and compared to a standard laboratory population
 (Rockefeller strain) maintained at Penn State University. The field
 locations were chosen to capture a gradient of the landscape and climate,
 driven primarily by variation in altitude. Populations were founded with
 sufficient viable eggs collected from multiple ovitraps
 across the cities to yield at least 100 adult mosquitoes in the F1
 laboratory generation. Mosquitoes were
 reared for a generation (F2) in standard laboratory conditions (27°C, 80%
 humidity, 12:12hr photoperiod, 0.60 mg of bovine liver powder per 600
 larvae and ad libitum access to 10% sugar solution for the adults) prior
 to experimentation to remove the influence of any maternal effects. Two
 populations, Jojutla and Juárez, were reared for an additional generation
 (F3) to ensure a large enough population for subsequent
 experiments.  <strong>Knockdown assays to estimate thermal
 tolerance</strong> We followed
 methods developed recently by Ware-Gilmore et al. to examine the thermal
 tolerance of adult <em>Ae. aegypti</em> mosquitoes. These
 methods were adapted from numerous studies in
 <em>Drosophila</em> and were shown to be sufficiently
 sensitive to demonstrate the effects of infection with either dengue virus
 or the bacterial endosymbiont, <em>Wolbachia</em>, on thermal
 sensitivity. In brief,
 three-to-four-day-old female mosquitoes were placed into individual sealed
 40mL glass vials. The vials were submerged into a tank filled
 with water at a regulated temperature of 41°C. Individuals were allowed
 two minutes to acclimate, after which they were monitored and the time to
 knockdown (immobility or death) was recorded. We monitored mosquitoes until all were knocked
 down. In the common garden experiment, we conducted six replicate runs of
 10 mosquitoes giving a total of 60 mosquitoes per population. The passage
 experiment had 18 total replicates (6 per independent passaged line) of 10
 mosquitoes for each temperature treatment.
 <strong>Experimental
 passage </strong> We used the F1 population of <em>Ae. aegypti
 </em>mosquitoes collected from Monterrey, Mexico, to establish six
 replicate lines, half of which were maintained at a standard insectary
 temperature of 27°C (80% humidity, 12:12hr photoperiod), which also
 approximates the overall mean temperature in Monterrey summer months, and
 the other half were maintained at an elevated temperature of 31°C (80%
 humidity, 12:12hr photoperiod) (SI Appendix, Fig. S1 and S2, Table S1).
 The elevated temperature of 31°C represents an increase of 4°C as might be
 expected under future climate warming. However, neither temperature
 simulates realistic environmental variation in the natural home
 environment and so both treatments were under some level of artificial
 selection to lab conditions. Each replicate line was initiated with 600 first
 instar larvae. Larvae were added into 5.7 L containers containing 3 L of
 deionized water and 0.60mg of bovine liver powder (MP Biomedicals) and
 placed in controlled temperature incubators (3 replicate containers at
 27°C and 3 containers at
 31°C). Every other day we
 added 0.60 mg of bovine liver powder to each container until larvae began
 to pupate when we scaled the food to the number of remaining larvae. We
 removed pupae and placed them in a small cup (30 mL) with water from their
 original environment to allow for eclosion. Cups containing pupae were
 added to a large cage with ad libitum access to 10% sugar solution made
 with dextrose anhydrous and deionized water to sustain the adults as they
 emerged. We counted total
 pupae per container, along with the number of pupae that eclosed
 successfully. When the adult mosquitoes were of reproductive age (3-5 days
 after eclosion), any dead adult mosquitoes were counted. These measures
 were used to get an accurate count of surviving adult mosquitoes in each
 cage.  To ensure balanced selection between lines and account for
 potential effects of genetic bottlenecks or drift that could result from
 different population sizes, adult mosquitoes were culled (3-5 days after
 eclosion) before blood feeding so that each line had the same number of
 mosquitoes. The lines were culled in pairs, for example replicate one at
 27°C was paired with
 replicate one in 31°C, for
 all ten generations to ensure independence between replicates. Mosquitoes
 were culled with a 50:50 sex
 ratio to maintain possible
 differences in selection on sex. Once the adult cages were established, mosquitoes
 were fed a human blood meal every four days for 16 days using a standard
 membrane feeder. Eggs were collected every day and maintained on dry
 filter paper to prevent hatching. At the end of the 16-day egg laying
 period, the eggs were transferred to larval containers to initiate
 hatching, thus maintaining a uniform age structure. Mosquitoes were reared
 through to adult as described. We followed this protocol for ten
 generations (SI Appendix, Fig. S5).
 At the end of the experimental
 period, we measured thermal tolerance for the six lines using the
 knockdown methods described above. In addition, to extend
 beyond this proxy variable and fully explore the effects of the passage
 treatments, egg-to-adult survival, mosquito development rate, mean adult
 survival, and fecundity were measured in mosquitoes reared in
 environmentally controlled incubators at 13°C, 17°C, 21°C, 25°C, 27°C,
 29°C, 31°C, 33°C, 35°C, 37°C, each ± 0.2°C and 80%± 10% relative humidity.
 Eggs from the 6 passaged lines were hatched at 27°C.  After 24 hours, 200
 first instar larvae were put into 1.89 L containers with 1 L of deionized
 water and 0.20 mg of larvae bovine liver powder (MP Biomedicals) and
 placed in the respective incubator. We fed larvae 0.20 mg of liver powder
 every other day until pupation. Once larvae began to pupate, we scaled
 their food to the number of remaining larvae. We removed and counted
 living and dead pupae the day of pupation and placed them in a small cup
 (30 mL) with water from their original environment to allow for eclosion.
 Cups containing pupae were added to a small cage (17.5 cm3) with ad
 libitum access to 10% sugar solution made with dextrose anhydrous and
 deionized water. We then counted the number of adults that eclosed every
 day. After 95% of females emerged, we blood fed females who were then 3-5
 days old.  We used blood from de-identified human donors (BioIVT, Corp.)
 so IRB approval and human subjects’ approval was not needed. Immediately
 after blood-feeding, we counted the total number of blood-fed females and
 placed up to 10 individual females into separate containers (50 mL
 polypropylene centrifuge tubes) lined with filter paper that contained 7
 mL deionized water to measure individual fecundity. We recorded the day
 that females in individual containers first laid eggs and let them lay
 eggs for three total days, after which we removed them from their
 containers. We extracted the water from the containers to let the filter
 paper dry in their respective incubators and then we counted the eggs. We
 counted and determined the sex of the number of adults that died every
 day. We censored this experiment 4 weeks after the first egg lay at each
 temperature (SI Appendix, Fig. S5).
description: # Associated data for "Phenotypic adaptation to temperature in the
 mosquito vector, *Aedes aegypti*"
 [https://doi.org/10.5061/dryad.z8w9ghxgc](https://doi.org/10.5061/dryad.z8w9ghxgc) We used explored mosquito adaptation to temperature in the vector species *Aedes aegypti* through thermal knockdown of field mosquitoes and life history experiments of experimentally adapted mosquitoes. We used survival analysis and a Bayesian approach to analyze these data. ## Description of the data and file structure There are three types of files found in this set, excel/csv files containing raw data, R code and text files (containing models for Bayesian models). There are three sets of data containing field mosquito knockdown data, experimental passage mosquito knockdown data, and life history data for experimental passage mosquitoes. Both knockdown data sets correspond to R code labeled knockdown models. Selection experiment data correspond to models using a Bayesian approach. Models for mosquito development rate, egg-to-adult survival, and juvenile mortality rate models along with the fitness model are all contained in one R code. Each of these traits requires a .bug/.txt file which contains the model. Separately, we have the code for the hierarchical eggs per female per gonotrophic cycle code and the adult survival analysis code (and corresponding models). This dataset includes three separate files: "Mexico Knockdown Data.csv": \- Includes data from a thermal knockdown experiment using 5 populations of *Aedes aegytpi* from Mexico (Cabo San Lucas, Acapulco, Monterrey, Ciudad Juárez, and Jojutla) along with one laboratory adapted line of mosquitoes. This CSV file includes the replicate assay (6 total, 10 individuals in each), the population, the status (1=knocked down, 0= resisted knockdown), time to knockdown (amount of time until an individual was knocked down at 41°C), and Seconds (time to knockdown converted to seconds). This dataset corresponds to the R file "Dennington\_GCB\_ Knockdown models". "Selection Experiment Knockdown Data.csv": \- Includes data from a thermal knockdown experiment using six lines of mosquitoes selected over 10 generations\, three at a control temperature of 27°C and three lines at an increased thermal pressure of 31°C\. We compare these results to the population of mosquitoes from Monterrey\, Mexico before selection\. This CSV file includes the replicate assay \(6 total\, 10 individuals in each\)\, the population \(three selected at 27°C and three at 31°C\)\, the status \(1=knocked down\, 0= resisted knockdown\)\, time to knockdown \(amount of time until an individual was knocked down at 41°C\)\, Seconds \(time to knockdown converted to seconds\) and Pressure \(the selection pressure over 10 generations\- N=no selection\, L=Selected at 27°C and H=Selected at 31°C\)\. This dataset corresponds to the R file "Dennington\_GCB\_ Knockdown models"\. "Dennington\_SelectionExp\_LarvalDevelopmentSurvival.csv": Includes data from life history measurements including columns Population (the replicate lines at either 27°C or 31°C), Temp (Temperature at which the groups were reared), Rep (replicate measurements of each selected line), Pupae (number of pupae formed), Adults (number of adults eclosed), Larv\_Surv (proportion of larvae that survived to adult from starting 200 larvae), Pupal death (number of pupae that formed but did not eclose), Time to pupa (days to pupation), Time to adult (days to adult), dev rate p (pupation rate= 1/ time to pupa), dev rate a (mosquito development rate= 1/time to adult), Pressure (the selection pressure over 10 generations- L=Selected at 27°C and H=Selected at 31°C), number attempted (total number of starting mosquitoes), and juvenileMU (juvenile mortality rate- approximation using the -(natural log of Larval survival)/ Time to adult). This dataset corresponds to the R file "Dennington\_GCB\_TPC Models. R". "Dennington\_ SelectionExp\_Fecundity.csv": Includes data from life history measures for fecundity including Population (the replicate lines at either 27°C or 31°C), Temp (Temperature at which the groups were reared), Rep (replicate measurements of each selected line), number (Individual within each experiemtal group), eggs (number of eggs for the first gonotrophic cycle), larvae (number of larvae that hatched), hatch\_rate (number of larvae/number of eggs), date\_fed (date of the first blood feed), date\_first\_lay (date of the first time eggs were layed), and cycle\_length (amount of time in days between first blood feed to first egg lay). This dataset corresponds to the R file "Dennington\_Fecundity Model.R". "Dennington\_SelectionExp\_AdultSurvivalSummary.csv": Includes data from life history measures for adult survival in a summary form. Columns are Population (the replicate lines at either 27°C or 31°C), Temp (Temperature at which the groups were reared), Rep (replicate measurements of each selected line), M (number of dead males), F (number of dead females), status (acknowledging that these individuals died), Day (day that we censored the experiment), Total (sum of dead individuals), Pressure (the selection pressure over 10 generations- L=Selected at 27°C and H=Selected at 31°C), NumberAdultsEclosed (number of individuals that made it to adulthood), and ProportionDead (total number that died over the total number that eclosed). This dataset corresponds to the R file "Adult Survival Anlysis (by LRJ).R". "Dennington\_SelectionExp\_AdultSurvivalRaw.csv": Includes data from life history measures for adult survival in a raw data form (counting how many individuals died on each day). Columns are Population (the replicate lines at either 27°C or 31°C), Temp (Temperature at which the groups were reared), Rep (replicate measurements of each selected line), Date (the date that the measurement was taken), Day (the day of the experiment, with day 1 being the first day), Dead\_M (number of dead males, Dead\_F (number of dead Females). This dataset corresponds to the R file "Adult Survival Anlysis (by LRJ).R". <br> <br> <br> <br> The files including the code to produce all models are: "Dennington\_GCB\_ Knockdown models.R": Includes analysis for thermal knockdown data using Kaplan-Meier survival curves with ggsurvplot (including both models in the main document and the supplementary documents). "Dennington\_Fecundity Model.R": Analysis for eggs per female per first gonotrophic cycle model, with an interaction using Bayesian inference. This code uses "Dennington\_SelectionExp\_LarvalDevelopmentSurvival.csv" and "JAGS\_Modelexample.bug" and "JAGS\_quad\_interaction.bug" "Dennington\_GCB\_Code to get summary data for TPCs.R" Example code to produce raw data plots (Figure 3). This code uses "Dennington\_SelectionExp\_Fecundity.csv". "Dennington\_GCB\_TPC Models.R": Code for for mosquito development rate, egg-to-adult survival, and juvenile mortality rate models along with fitness model. The fitness model integrates juvenile mortality rate, mosquito development rate, fecundity and adult survival models. This code requires "JAGS\_model\_briere.bug". "Adult Survival Anlysis (by LRJ).R": Code for adult survival analysis as used in the fitness model. This code uses the data set "Dennington\_SelectionExp\_AdultSurvivalRaw.csv" and "Dennington\_SelectionExp\_AdultSurvivalSummary.csv". This code requires "surv\_gengamma\_temp.txt". <br> ## Sharing/Access information All data and code were produced for the purpose of this manuscript. Figure to produce the map in the supplemental material (SI figure 1) was adapted from published code by Valle-Jones D (2022). *mxmaps: Create Maps of Mexico*. https://www.diegovalle.net/mxmaps/, [https://github.com/diegovalle/mxmaps](https://github.com/diegovalle/mxmaps)). ## Code/Software This code was run on R/Rstudio Version 2023.06.1.