Post-eclosion temperature effects on insect cuticular hydrocarbon profiles

The insect cuticle is the interface between internal homeostasis and the often harsh external environment. Cuticular hydrocarbons (CHCs) are key constituents of this hard cuticle and are associated with a variety of functions including stress response and communication. CHC production and deposition on the insect cuticle vary among natural populations and are affected by developmental temperature; however, little is known about CHC plasticity in response to the environment experienced following eclosion, during which time the insect cuticle undergoes several crucial changes. We targeted this crucial phase and studied post-eclosion temperature effects on CHC profiles in two natural populations of Drosophila melanogaster. A forty-eight hour post-eclosion exposure to three different temperatures (18, 25, & 30 °C) significantly affected CHCs in both ancestral African and more recently derived North American populations of D. melanogaster. A clear shift from shorter to longer CHCs chain-length was observed with increasing temperature, and the effects of post-eclosion temperature varied across populations and between sexes. The quantitative differences in CHCs were associated with variation in desiccation tolerance among populations. Surprisingly, we did not detect any significant differences in water loss rate between African and North American populations. Overall, our results demonstrate strong genetic and plasticity effects in CHC profiles in response to environmental temperatures experienced at the adult stage as well as associations with desiccation tolerance, which is crucial in understanding holometabolan responses to stress.

Methods
Body Mass Data: Body weight data was collected in the groups of five flies. The wet weight and dry weight data are given in miligrams. To obtain dry weight flies were dried at 50 degree centrigrade overnight. The wet mass, dry mass, and water content values were used to generate residuals for ANOVA analysis of desiccation tolerance. 

Cuticular Hydrocarbon Data: Hydrocarbons were extracted in hexane before running through gas chromatography column. Data file contains raw data (retention time). Peak areas of the GC chromatograms corresponding to individual CHCs were converted into relative proportions of the sum of CHC peak areas. The relative data was arcsine transformed and the overall quantitative variability among samples and treatments visualized using principal component analysis (PCA) performed in Statistica 8.0.

Desiccation Assay: Flies in the groups of five were used for desiccation assay. To measure desiccation tolerance desiccating environemnt was created in the tube (using Silica gel). Data contains hourly dead flies number. We used LT100 data. We ran a mixed model ANOVA where population cages were nested within geographic source population.

Respirometry Data (Water Loss Rate): Water-loss rate (WLR) was measured using flow-through respirometry. Flies in the groups were placed in a glass chamber where dry air was allowed to get in and outlet was connected with a humidity sensor. Water-loss rate was calculated from water vapor released by flies into the air stream. We ran a mixed model ANOVA where lines were nested within geographic source population.