DOI: 10.5061/dryad.j6q573nt7
title: Microbiome composition shapes temperature tolerance in a Hawaiian picture-winged <em>Drosophila</em>

description: Hawaiian picture-winged Drosophila are undergoing rapid biodiversity loss,
 with twelve species listed as endangered and others in decline. Microbiota
 are increasingly recognized as contributors to host adaptation and
 influence stress tolerance, reproduction, and survival. We investigated
 the role of microbial communities in two Drosophila basisetae populations
 from Hawaiian rainforests at 900m and 1200m elevation. Microbiome
 profiling of wild flies by high-throughput amplicon sequencing revealed
 distinct bacterial and fungal communities between sites. To test
 microbiome effects on host physiology, we conducted a fully factorial
 research design with microbiome inoculations in laboratory-reared flies
 acclimated at 18°C (control) or 24°C (stressful). Flies receiving
 low-elevation microbiota exhibited higher survival across temperatures,
 whereas those given high-elevation microbiota produced more eggs,
 indicating microbiome-mediated differences in survival and reproductive
 investment. Activity levels were higher when flies received microbiota
 from their native population, whereas critical thermal maximum and male
 accessory gland size revealed complex interactions among microbiome,
 temperature, and population. These population effects are consistent with
 whole-genome resequencing of wild-caught flies that identified outlier
 SNPs in genes associated with immunity, heat tolerance, and reproduction,
 despite signs of admixture and gene flow across populations. Overall,
 these results indicate that microbiome–host–environment interactions may
 modulate thermal tolerance and reproduction in population- and
 environment-specific ways and contribute to both phenotypic plasticity and
 evolutionary adaptation. These insights inform conservation strategies for
 Hawaiian Drosophila that incorporate microbial management to bolster
 adaptive potential and resilience in Hawaiian ecosystems.
description: Wild-caught flies from both locations were transported to the
 laboratory at UNLV and immediately placed in separate 1-gallon (∼3.8 l)
 breeding jars kept in a climate-controlled walk-in chamber and maintained
 at 18°C and 60–70% RH, the standard rearing temperature for picture-winged
 Drosophila, with a 12 h:12 h day: night cycle. For microbiome inoculation
 experiments, virgin female and male flies were first separated by sex
 within 7 days of eclosion and placed in adult food vials for 3 days at
 18°C and then subsequently placed in vials with an antimicrobial diet for
 7 days and housed in an incubator at 18°C.After the antimicrobial
 treatment, male and female D. basisetae from Tom’s Trail and Ola‘a
 populations were randomly placed (1 or 2 flies per vial) in sterilized
 glass vials containing a standard diet supplemented with 12.5 μl of a
 microbiome slurry from either Tom’s Trail or Ola‘a pipetted onto the
 surface of the food. The vials, along with the flies, were randomly placed
 in either an 18 or 24°C Percival incubator for 15 days; RH was 60–70% for
 both incubators.   To determine the acclimation
 survival during these 15 days, every 3–4 days (alternating between 3 and 4
 days), the flies were transferred to fresh sterile vials containing the
 same food and microbiome slurry. Every 2–3 days, the survival of the flies
 was recorded. To determine the basal movement at
 control temperature and critical thermal maximum (CTmax) of D. basisetae,
 flies that had been acclimated to either 18 or 24°C and the two microbiome
 inoculation treatments were next placed at 18°C for 3 days. After 3 days
 of acclimation to the control temperature, the flies were placed
 individually into the wells of a tissue culture plate (2 cm diameter),
 with each plate housing 12 flies.The Darwin chamber was programmed to
 initiate the temperature at 18°C for 15 min to allow the flies to
 acclimate for 5 min, then the basal movement of the flies was recorded at
 the control temperature for 10 min. The temperature in the chamber was
 gradually increased at a rate of 0.5°C per minute for 40 min and then
 slowed to a ramp of 0.25°C per minute for the next 60 min. The basal
 movement and CTmax for each fly were determined by analyzing video
 recordings and the temperature data with EthoVision XT software (Noldus)
 to identify the movement for 10 min. at the beginning of each trial, and
 when movement of each fly had ceased, indicating the CTmax for each
 individual fly. The flies that were stored in PBS in a
 4°C refrigerator after CTmax measurements were dissected within 5 days.
 The number of mature eggs was manually counted in dissected females under
 10× magnification using a Nikon SMZ25 dissecting microscope connected to a
 Windows computer with Nikon NIS Elements version 4.6 video software. The
 male accessory glands were also measured within 5 days after CTmax
 measurements. The males were dissected and the accessory glands were
 carefully positioned under the Nikon dissecting microscope and a picture
 was taken that included a micrometer for size standardization.
description: # Microbiome composition shapes temperature tolerance in a Hawaiian
 picture-winged *Drosophila* Dataset DOI:
 [10.5061/dryad.j6q573nt7](10.5061/dryad.j6q573nt7) ## Description of the
 data and file structure ### Files and variables #### File:
 Phenotyic_Data_D._basisetae_microbiome_and_temperature_tolerance_revised.csv **Description:**  ##### Variables * Block:  The data was collected over five time periods that are designated as Block. * FlyID:  The identification of the individual fly. * Fly Location:  The flies in the experiments were originally collected from two locations in the Hawaiian rainforest - olaa and toms * Sex:  F is female flies and M is male flies * Acclimation Temperature: There were two acclimation temperatures  18 Celsius and 24 Celsius. * Microbe Treatment:  There were two inoculation treatments of microbes (bacteria and fungi) from olaa and toms. * Survived\_ Acclimation:  The flies that survived the acclimation period are designated Y and the flies that did not survive the acclimation period are designated N. * ET CTmax C:   The Critical Thermal Maximum (CTmax) temperature was recorded using the EthoVision Software and recorded in degrees Celsius. * Basal minute:   The number of mm the flies moved during 10 minutes at 18 degrees Celsius prior to the temperature ramping procedure for the CTmax measurement. * Egg Count:  The female flies were dissected and the number of mature eggs were counted after taking a picture with a dissecting microscope. * Acc. Gland (mm):  The male flies were dissected and the size of the accessory gland was measure using Image J. * Missing data: n/a ## Access information Other publicly accessible locations of the data: * NA Data was derived from the following sources: * NA