DOI: 10.5061/dryad.pvmcvdnv9
title: Data from: Prior heat waves improve survival of field but not domesticated populations of tobacco hornworm exposed to repeated bacterial infections

description: In insects and other invertebrates, prior pathogen exposures can improve
 immune responses and survival to subsequent infections through immune
 priming. Alternatively, stress and metabolic costs of multiple infections
 can impair host immunity and survival. The impact of high temperature
 extremes on host-pathogen interactions are not well-understood despite the
 increasing occurrence of heat waves caused by climate change. The response
 of insects to heat waves and pathogens depends on recent evolutionary
 history with selective pressures. Domestication of insect pests has
 occurred in lab colonies of model species, reducing selective pressures
 for immune and heat stress responses. Lab strains are often used in
 immunological or heat stress experiments to represent wild field strains,
 but the efficacy of this approach is seldom evaluated. Using the tobacco
 hornworm (Manduca sexta), we tested the impact of a heat wave during
 initial pathogen exposure on survival of a secondary infection with
 Bacillus thuringiensis bacteria. We used a domesticated lab population and
 a naturally-occurring field population of M. sexta to evaluate impacts of
 recent domestication on immune and thermal responses. A heat wave during
 initial infection significantly increased survival of the secondary B.
 thuringiensis infection in the field, but not the lab population of M.
 sexta. In the field population, survival of the repeated infection was
 temperature-dependent: exposure to an initial infection event reduced
 survival of the secondary infection at the control temperature regime,
 consistent with a stress effect. However, a heat wave during the initial
 infection event increased survival of the secondary infection, consistent
 with immune priming effects. The results of this study demonstrate that a)
 insect response to thermal stress and pathogens can depend on recent
 domestication, and b) responses of hosts to repeat pathogen exposures can
 be temperature-dependent, suggesting  that cross-talk between the
 heat stress and immune memory pathways may have important consequences for
 host-pathogen outcomes under heat wave events.
description: Data were collected from a lab experiment conducted in 2023. Data
 files (.csv files) contain raw data and can be processed with the
 associated R script to produce all statistical analyses and figures used
 in the manuscript. For detailed information on methods, see
 manuscript.
description: # Data from: Prior heat waves improve survival of field but not
 domesticated populations of tobacco hornworm exposed to repeated bacterial
 infections
 [https://doi.org/10.5061/dryad.pvmcvdnv9](https://doi.org/10.5061/dryad.pvmcvdnv9) Primary dataset (finFieldimmunepriming_HS_datasheet_2023data.csv) contains data collected from the immune priming experiment run in lab and field *M. sexta* with initial low dose exposure and subsequent high dose of Bacillus thuringiensis, testing the impact of heat wave during the initial exposure on survival following secondary infection. Data collected summer 2023. ## Description of the data and file structure **FE_primecleanedcode.Rmd** is the code used to conduct analyses and generate figures (in R). It uses both .csv files included. The primary datasheet (**finFieldimmunepriming_HS_datasheet_2023data.csv**)** **is used for all analyses and figures used in the manuscript. The second datasheet (**bledLarvae_from_prime.csv**) contains data used in the supplementary material. **finFieldimmunepriming_HS_datasheet_2023data.csv** is the primary data file for this paper. It contains growth and development data, survival data, and data for the timing of experimental injections and experimental heat wave exposure. Below is a readme for the columns in the data sheet: | column | unit | description | | :--------------------- | :-------------- | :------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | sample.ID | numeric | unique sample identification number used to distinguish samples throughout the experiment | | block | categorical | experimental block the samples were run under. Not applicable for this experiment, as all samples were run in the same block | | expt\_part | categorical | experiment part the samples belonged to (similar to block). Not applicable for this experiment, as all samples were from the same experiment part (i.e. used the same dosages of infections) | | trt.group | categorical | treatment group (factorial) the sample was assigned to. combination of prime, temperature, and challenge. | | origin | categorical | population the sample originated from. Lab or field | | prime | categorical | initial infection treatment the sample received. Bt=low dose Bacillus thuringiensis injection in 10uL volume; n=sham injection of sterile PBS in 10uL volume; na=unmanipulated control (no injection). | | temp | categorical | temperature treatment regime the insect was assigned starting at 1st day of fourth instar. Lasted 3 days. HW=heat wave of 32 degrees C +/-10; noHW=25 degrees C+/-10 | | challenge | categorical | immune challenge during secondary infection treatment the sample received. Bt=Bacillus thuringiensis high dose infection in 25uL volume; N=sham injection with sterile PBS in 25uL volume; NA=unmanipulated control (no injection) | | date.PMD | date | date of premature death (premature is defined as death prior to the 5th instar). If NA, data are not applicable (i.e., the sample did not die before 5th instar). | | stage.PMD | categorical | life stage at premature death. indicates day of initial infection ('prime' treatment), day after, etc. | | date.died | date | date of death after 5th instar. if NA, data are not applicable (i.e., the sample either died prematurely or survived to wandering) | | col.stage | categorical | collection stage for field M. sexta. Typically they were collected in the field as eggs, but sometimes 1st or 2nd instar larvae. All lab M. sexta were collected as eggs. | | species | categorical | species collected in the field. Manduca quinquemaculata is identical to M. sexta until 3rd instar, so were sometimes found in the experiment accidentally. We ran the experiment with them included, and kept note of the species here. In the Rmd file, we remove them from analyses as we did not have enough to be statistically significant | | date.hatch | date | date the insect hatched. NA means not applicable (if the insect was collected after hatching, or exact hatch date was not observed) | | date.4th | date | date of molt to 4th instar | | mass.4th | numeric (mg) | mass on the day of molt to 4th instar. NA is not available (mass was missed for this sample on this day) | | date.5th | date | date of molt to 5th instar. NA is not applicable (insect did not survive to 5th instar) | | mass.5th | numeric (mg) | mass on the day of molt to 5th instar.  NA is not available if there is a date in date.5th (mass was missed for this sample on this day). If no date in date.5th, data are not applicable (sample did not survive to 5th instar. | | date.6th | date | date of molt to 6th instar. NA is not applicable (insect did not survive to 6th instar or wandered in the 5th instar) | | mass.6th | numeric (mg) | Mass on the day of molt to 6th instar. NA is not available if there is a date in date.6th (mass was missed for this sample on this day). If no date in date.6th, data are not applicable (sample did not survive to 6th instar, or wandered prior to this point). | | mass.7th | numeric (mg) | Mass on the day of molt to 7th instar. NA is not available if there is a date in date.6th (mass was missed for this sample on this day). If no date in date.7th, data are not applicable (sample did not survive to 7th instar, or wandered prior to this point). | | date.7th | date | date of molt to 7th instar. NA is not applicable (insect did not survive to 7th instar or wandered in the 5th or 6th instar) | | date.wander | date | date of observation of wandering. Defined as observing visible dorsal vein and characteristic wandering behavior (insect is highly mobile walking circles around its enclosure). | | mass.wander | numeric (mg) | mass upon observation of wandering. NA is not applicable if no date in date.wander (insect did not survive to wandering), or not available if there is a date in date.wander (data were not collected for mass of this sample) | | notes\_dev | notes | notes on development | | date.temp.in | date | date the sample was entered into the treatment temperature in environmental chambers. Always same day of molt to 4th instar. NA in this column means data were not applicable (sample died prior to entering the temperature treatment). | | chamber.time.temp.in | time | time of day the sample was entered into the treatment temperature in environmental chambers.  NA in this column means data were not applicable (sample died prior to entering the temperature treatment). | | time.temp.in | time | time of day outside the chambers that the sample was entered into the treatment temperature in environmental chambers. Chambers were set to mimick an earlier time of day to facilitate logistical experimental work during a more agreeable time of day.  NA in this column means data were not applicable (sample died prior to entering the temperature treatment). | | date.prime | date | date of initial infection | | chamber.time.prime | time | time of day the sample was initially infected.  NA in this column means data were not applicable (sample died prior to the initial infection treatment). | | time.prime | time | time of day outside the chambers that the sample received the initial infection. Chambers were set to mimick an earlier time of day to facilitate logistical experimental work during a more agreeable time of day.  NA in this column means data were not applicable (sample died prior to this treatment). | | primedose | dose (CFU/10uL) | dose of initial infection included in the 10uL syringe. Refers to the actual number of CFUs the insect received. NA in this column means not applicable (insect died prior to this point) | | prime\_inj\_vol | numeric (uL) | microliters (uL) of injection received. Always 10uL for Bt or sham injections, and 0 for unmanipulated controls | | time.temp.out | time | time of day outside the chambers that the sample was removed from the temperature treatment and placed at 25C+-10 baseline temperature regime. Chambers were set to mimick an earlier time of day to facilitate logistical experimental work during a more agreeable time of day.  NA in this column means data were not applicable (sample died prior to this timepoint). | | chamber.time.temp.out | time | time of day the sample was removed from the treatment temperature in environmental chambers.  NA in this column means data were not applicable (sample died prior to this timepoint). | | date.temp.out | date | date the sample was removed from the temperature treatment and returned to 25C+-10 baseline thermal regime. the temperature treatment lasted 3 days. | | mass.challenge | numeric (mg) | mass of the sample at the time of the secondary infection immune challenge. NA in this column means not applicable if there is a date in date.challenge, and not available if there is no date in date.challenge (ie the insect died prior to this timepoint) | | date.challenge | date | date of secondary infection immune challenge. NA in this column means not applicable (insect died prior to this time point) | | chamber.time.challenge | time | time of secondary infection treatment. NA in this column means not applicable (insect died prior to this time point) | | time.challenge | time | time of day outside the environmental chamber that the secondary infection treatment took place. Chambers were set to mimick an earlier time of day to facilitate logistical experimental work during a more agreeable time of day. NA in this column means not applicable (insect died prior to this time point) | | challenge\_goalCFU | dose (CFU/25uL) | dose of secondary infection included in the 25uL syringe. Refers to the actual number of CFUs the insect received. NA in this column means not applicable (insect died prior to this point) | | challenge\_vol | numeric (uL) | microliters (uL) of injection received. Always 25uL for Bt or sham injections, and 0 for unmanipulated controls | | notes | notes | notes about the experiment and the way it was conducted for this sample if it deviated in any way from the standardized experimental protocol | **bledLarvae_from_prime.csv** is a datasheet for larvae that were bled to test for presence of bacterial colonies (in supplement). there's a section of the code that uses it to visualize, but is not necessary for the manuscript. Below is a readme for the columns in the data sheet. | **column** | **units** | **description** | | :---------------------------------------------- | :---------- | :---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Population | categorical | population of Manduca sexta the sample originated from. lab or field | | Initial infection | categorical | initial infection the sample received. Bt=Bacillus thuringiensis low dose; Sham=sham injection with sterile PBS | | Temperature | categorical | temperature treatment regime the insect was assigned starting at 1st day of fourth instar. Lasted 3 days. HW=heat wave of 32 degrees C +/-10; noHW=25 degrees C+/-10 | | No. larvae setup | count | number of cultures set up from larval blood | | No. with colonies 1 day post initial infection  | count | number of culture plates with bacterial cultures present the day after infection took place. Blood was taken 1 day after the infection and allowed to incubate for 24 hours before reading plates.  | | No. with colonies day of molt to 5th | count | number of culture plates with bacterial cultures present on the day of molt to 5th instar. Blood was taken the day of molt to 5th instar and allowed to incubate for 24 hours before reading plates.  | | No. with colonies day after molt to 5th | count | number of culture plates with bacterial cultures present on the day after molt to 5th instar. Blood was taken the day after molt to 5th instar and allowed to incubate for 24 hours before reading plates.  | ## Code/Software The code provided (.Rmd file) uses the two datasheets listed above. All data cleaning, analysis, and figure generation are in the code. All coding done in R (version of software and libraries reported in main manuscript).