High altitude favours long chained cuticular hydrocarbons in Drosophila

Cuticular hydrocarbons (CHCs) are key components of the insect cuticle and contribute to the wide geographical distribution of this taxon. Many studies have investigated sex and population differences in CHC profiles, with these investigations mostly focusing on latitudinal CHC variation, whereas CHC variation across altitudinal transects is less well-studied. Here, we tested whether CHC profiles vary along an altitudinal gradient in the cosmopolitan vinegar fly Drosophila melanogaster. We collected three populations of D. melanogaster from the Western Himalayas at altitudes ranging from 760 to 2592 m above sea level and studied their CHC profiles for standing and plastic differences. We found quantitative differences in 25 CHCs across populations, and at higher elevations, males and females expressed higher amounts of particular long-chained hydrocarbons. We also found an overall shift in CHCs in all three populations when flies were exposed to desiccating conditions. Overall, our findings suggest that there is an altitudinal cline in CHCs; however, this does not mirror the well-established latitudinal clines in fly hydrocarbons.

Methods
Drosophila melanogaster populations were collected from three altitudinal locations in the Western Himalayas (between 760-2592 metres above sea level (a.s.l); Rohru (31.2°N, 77.81°E, 2592.84 m), Chamba (32°N, 76°E, 996 m), and Mandi (31°N, 76.9°E, 760 m). Hereafter, HL (highland), ML (midland), and LL (lowland) correspond to Rohru, Chamba, and Mandi, respectively. Fly collection was undertaken in and around apple orchards during the winter months (Oct and Nov 2019) using both mouth aspirators and bait-trap methods. Field-collected flies were transported to the laboratory, sorted by sex under a stereo-zoom microscope (Leica S9i) using a mild CO2 stream, and placed under controlled conditions: 23 ± 0.5°C, 12:12 light: dark cycle. The females were placed individually in fresh food vials and allowed to oviposit until larval activity was visible. In total, 120 founder iso-female lines (from three sites; HL, 41 lines; ML, 53 lines; and LL, 26 lines) were established and maintained under controlled conditions (see above 23± 0.5 °C). Successful lines were scanned for species identification based on taxonomic keys (e.g. sex combs arrangement, pigmentation, and genitalia structures). Of the 120 iso-female lines, 58 were identified as D. melanogaster (37 lines: HL; 15 lines: ML; 6 lines: LL). Three separate population cages (one for each altitude) were created by pooling iso-female lines from the respective collection sites (LL, ML, and HL). Each population cage was then maintained for five, non-overlapping, generations (ca. 1000-1200 flies/cage). All stocks (iso-female lines and population cages) were maintained on ad libitum corn-meal agar food medium under controlled conditions (23±0.5 °C) and a 12:12h light: dark cycle). These conditions were maintained throughout the experiment unless stated otherwise.

Experimental Design

Eggs were collected from the three population cages (LL, ML, and HL) by placing two food plates (per cage) containing active yeast for 3-4 hours. Food chunks with ca. 30 eggs were handpicked from these plates and transferred into separate vials containing fresh food medium (n=20 vials per population cage). The vials were then incubated at 23°C until adults eclosed. A total of 600 virgin flies were sorted within 8 hours of eclosion from each population and kept isolated for 3 days. On the fourth day, these flies were split across two experimental treatments: control (no desiccation) and desiccation (8 hours desiccation). Five replicates (R1-R5) were created for each sex with each replicate consisting of 20 adults (see S1).