Gene expression analysis
Eastern (n=10; 5 males, 5 females) and western (n=10; 5 males, 5 females) monarchs were randomly selected following the flight trials described above. They were subsequently flown on the flight mill for an additional two minutes and then immediately frozen in liquid nitrogen. Tri Reagent (Sigma) was used to extract RNA from the thorax of frozen samples. cDNA was synthesized from 600ng of total RNA using High Capacity cDNA Reverse Transcription kit (Thermofisher) according to manufacturer instructions.
Gene expression of six candidate genes was quantified relative to two housekeeping genes: 18S and 28S (Panet al. , 2015). The six candidate genes included two dynein genes (DPOGS201379, DPOGS211203) and a myosin gene associated with motor activity (DPOGS200868) that were related to monarch migration phenotypes in the genomic analysis by Zhan et al. (Zhan et al. , 2014). We also measured expression of a neurotransmitter gated ion channel (GABA receptor) gene (DPOGS202675) that is involved in the invertebrate neuromuscular system (Lummis, 1990; Schuske, Beg & Jorgensen, 2004), and a putative protein (DPOGS211604) whose homolog was found to be expressed in the wing disc of the silkworm (Mitaet al. , 2003). Finally, we quantified expression of a myosin heavy chain gene associated with non-muscular motor activity (DPOGS215054) that controls flight in fruit flies (Wells, Edwards & Bernstein, 1996). For each monarch, we carried out three replicate PCR reactions for 18S, 28S and each of the six candidate genes. Expression of candidate genes relative to 18S and 28S was calculated as: