RNA sequencing data for polyphenic and monophenic Manduca sexta strains

How organisms evolve under extreme environmental changes is a critical question in the face of global climate change. Genetic accommodation is an evolutionary process by which natural selection acts on novel phenotypes generated through repeated encounters with extreme environments. In this study, polyphenic and monophenic strains of the black mutant tobacco hornworm, Manduca sexta, were evolved via genetic accommodation of heat stress-induced phenotypes, and the molecular differences between the two strains were explored. Transcriptomic analyses showed that epigenetic and hormonal differences underlie the differences between the two strains and their distinct responses to temperature. DNA methylation had diverged between the two strains potentially mediating genetic assimilation. Juvenile hormone (JH) signaling in the polyphenic strain was temperature-sensitive whereas in the monophenic strain, JH signaling remained low at all temperatures. Although 20-hydroxyecdysone titers were elevated under heat shock conditions in both strains, the strains did not differ in the titers. Tyrosine hydroxylase was also found to differ between the two strains at different temperatures, and its expression could be modulated by topical application of a JH analog. Finally, heat shock of unselected black mutants demonstrated that the expression of the JH-response gene, Krüppel-homolog 1 (Kr-h1), increased within the first 30 min of heat shock, suggesting that JH levels respond readily to thermal stress. Our study highlights the critical role that hormones and epigenetics play during genetic accommodation and potentially in the evolution of populations in the face of climate change.

Methods
Black mutant M. sexta used in this study derived from a single mutant that spontaneously arose in a colony of wildtype M. sexta in 1972. This mutant has low JH at the time of the sensitive period for cuticular melanization due to reduced JH production. The larvae were raised on artificial diet in an incubator under 16 hr light:8 hr dark conditions. Larvae were reared at 26.5°C until the end of the fourth instar when they began to undergo head capsule slippage. The larvae were then heat shocked at 40°C for 6 hrs in a portable incubator that was connected to a timer. The greenest fifth instar larvae were selected to create the polyphenic strain, and the darkest larvae were selected to establish the monophenic strain. Heat shocks were applied every generation for 16 generations. After this, the strains were maintained with occasional applications of heat shocks and selection for the appropriate coloration. Occasionally, larvae with the darkest coloration at 26.5°C were selected to maintain the polyphenism.

Three biological replicates were collected from monophenic and polyphenic larvae reared at 26.5°C, 32.5°C or heat shocked for 4 hrs at 40°C. Each biological replicate consisted of a pooled sample of seven brain/corpora allata complexes isolated from the larvae within approximately 4 hrs post head capsule slip. Samples were stored in Trizol and processed using standard RNA extraction procedures. The extracted RNA was sent to Genewiz for RNA-sequencing. Poly(A)+ selected mRNA-seq libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit from Illumina and sequenced (Illumina Hi-seq) with paired end 150-bp reads to a depth of ∼350 million paired reads per replicate.