DOI: 10.5061/dryad.1vhhmgr4t
title: RNA sequencing data for polyphenic and monophenic Manduca sexta strains

description: 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.
description: <em>Black</em> mutant <em>M. sexta</em>
 used in this study derived from a single mutant that spontaneously arose
 in a colony of wildtype <em>M. sexta</em> 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. 
description: # RNA sequencing data for polyphenic and monophenic Manduca sexta strains
 [https://doi.org/10.5061/dryad.1vhhmgr4t](https://doi.org/10.5061/dryad.1vhhmgr4t) ## Description of the data and file structure In this study, the expression of differentially expressed genes were probed in the corpora allata/brain complex. 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.  ### Files and variables #### File: 26M1Y4\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 26.5°C. Replicate 1 of 3; read 1 #### File: 26M1Y4\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 26.5°C. Replicate 1 of 3; read 2 #### File: 26M2Y5\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 26.5°C. Replicate 2 of 3; read 1 #### File: 26M2Y5\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 26.5°C. Replicate 2 of 3; read 2 #### File: 26M3Y6\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 26.5°C. Replicate 3 of 3; read 1 #### File: 26M3Y6\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 26.5°C. Replicate 3 of 3; read 2 #### File: 26P1Y1\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 26.5°C. Replicate 1 of 3; read 1 #### File: 26P1Y1\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 26.5°C. Replicate 1 of 3; read 2 #### File: 26P2Y2\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 26.5°C. Replicate 2 of 3; read 1 #### File: 26P2Y2\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 26.5°C. Replicate 2 of 3; read 2 #### File: 26P3Y3\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 26.5°C. Replicate 3 of 3; read 1 #### File: 26P3Y3\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 26.5°C. Replicate 3 of 3; read 2 #### File: 32M1Y10\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta *reared at 32.5°C. Replicate 1 of 3; read 1 #### File: 32M1Y10\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 32.5°C. Replicate 1 of 3; read 2 #### File: 32M2Y11\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 32.5°C. Replicate 2 of 3; read 1 #### File: 32M2Y11\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 32.5°C. Replicate 2 of 3; read 2 #### File: 32M3Y12\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 32.5°C. Replicate 3 of 3; read 1 #### File: 32M3Y12\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* reared at 32.5°C. Replicate 3 of 3; read 2 #### File: 32P1Y7\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 32.5°C. Replicate 1 of 3; read 1 #### File: 32P1Y7\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 32.5°C. Replicate 1 of 3; read 2 #### File: 32P2Y8\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 32.5°C. Replicate 2 of 3; read 1 #### File: 32P2Y8\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 32.5°C. Replicate 2 of 3; read 2 #### File: 32P3Y9\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 32.5°C. Replicate 3 of 3; read 1 #### File: 32P3Y9\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* reared at 32.5°C. Replicate 3 of 3; read 2 **File: 40M1Y16_R1_001.fastq.gz** **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* that was heat shocked. Replicate 1 of 3; read 1 #### File: 40M1Y16\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* that was heat shocked. Replicate 1 of 3; read 2 #### File: 40M2Y17\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* that was heat shocked. Replicate 2 of 3; read 1 #### File: 40M2Y17\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* that was heat shocked. Replicate 2 of 3; read 2 #### File: 40M3Y18\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* that was heat shocked. Replicate 3 of 3; read 1 #### File: 40M3Y18\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the monophenic strain of *Manduca sexta* that was heat shocked. Replicate 3 of 3; read 2 #### File: 40P1Y13\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* that was heat shocked. Replicate 1 of 3; read 1 #### File: 40P1Y13\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* that was heat shocked. Replicate 1 of 3; read 2 #### File: 40P2Y14\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* that was heat shocked. Replicate 2 of 3; read 1 #### File: 40P2Y14\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* that was heat shocked. Replicate 2 of 3; read 2 #### File: 40P3Y15\_R1\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* that was heat shocked. Replicate 3 of 3; read 1 #### File: 40P3Y15\_R2\_001.fastq.gz **Description:** Raw RNA-seq sequencing data from a pooled sample of 7 brain-corpora allata complexes isolated from the polyphenic strain of *Manduca sexta* that was heat shocked. Replicate 3 of 3; read 2 ## Code/software Any simple Text editor can open these FASTA files. The reference genome for this species can be found at [https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_014839805.1/](https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_014839805.1/) (Genome assembly JHU_Msex_v1.0).