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.

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.