A transposable element insertion is the switch between alternative life history strategies
TL;DR: The genetic basis of the ALHS switch in Colias crocea is mapped to a transposable element insertion downstream of the Colias homolog of BarH-1, a homeobox transcription factor, which arises via recruitment of a transcription factor previously known for its function in cell fate determination in pigment cells of the retina.
Abstract: Tradeoffs affect resource allocation during development and result in fitness consequences that drive the evolution of life history strategies. Yet despite their importance, we know little about the mechanisms underlying life history tradeoffs in wild populations. Many species of Colias butterflies exhibit an alternative life history strategy (ALHS) where females divert resources from wing pigment synthesis to reproductive and somatic development. Due to this reallocation, a wing color polymorphism is associated with the ALHS: individuals have either yellow/orange or white wings. Here we map the genetic basis of the ALHS switch in Colias crocea to a transposable element insertion downstream of the Colias homolog of BarH-1, a homeobox transcription factor. Using CRISPR/Cas9 gene editing, antibody staining, and electron microscopy we find morph-specific specific expression of BarH-1 suppresses the formation of pigment granules in wing scales. Lipid and transcriptome analyses reveal physiological differences associated with the ALHS. These findings characterize a novel mechanism for a female-limited ALHS and show that the switch arises via recruitment of a transcription factor previously known for its function in cell fate determination in pigment cells of the retina.
Summary (2 min read)
Bulk segregant analyses (BSA):
- The female informative cross data and mapping protocol described in Woronik and Wheat, 2017 46 was applied to the high quality reference genome to identify the contigs that made up the Alba chromosome.
- SAMTOOLS v1.2 48 was used to filter (view -f 3 -q 20), sort and index the bam files and generate mpileup files for the two pools and the orange mother.
- DNA was prepared as described above for 26 Alba and 28 orange female offspring resulting in two DNA pools.
- Library preparation (TruSeq PCR-free) and Illumina sequencing (150 bp paired-end reads with 350bp insert, HiSeqX), was performed at Science for Life Laboratory (Stockholm, Sweden).
- A site was considered an Alba SNP if 1) it was homozygous in the orange pool and 2) the allele frequency difference in the Alba pool compared to the orange was 0.45-0.55.
Antibody Generation and Staining:
- A Rabbit-anti-Bar antibody was generated against the full length sequence of the Vanessa cardui Bar homolog.
- Protein was generated by GenScript (Piscataway, NJ) and purified to >80% purity.
- Antibody staining was performed as described previously for Drosophila and butterfly tissues 54 .
- Staged pupal wings and retinas were dissected and fixed 48 hours post-pupation.
- Images were captured using standard confocal microscopy on a Leica SP5.
- The guide-RNA (gRNA) sequences were generated using the protocol described in Perry et al.
- Full gRNA constructs had the following configuration: an M13F region, a spacer sequence, a T7-promotor sequence, the Target specific sequence, a Cas9 binding sequence, and finally a P505 sequence.
- They were then mixed with Cas9-NLS protein (PNA Bio, Newbury Park, CA, USA) and diluted to a final concentration of 125-250 ng/µl.
- C. crocea females (n > 40) from Aiguamolls de l'Empordà, Spain were captured and kept in morph-specific flight cages in the lab at Stockholm University where they oviposited on alfalfa (Medicago sativa).
- To validate the mutation, Cas9 cut sites were PCR-amplified and a ~370bp region, centered on the intended cut site were sequenced using Illumina MiSeq 300bp paired-end sequencing.
- Sequences were amplified and ligated with Illumina adapter and indexes in a two-step process following the protocol certified by peer review) is the author/funder.
- Samples were coated in gold for 80 seconds using an Agar sputter coater and imaged under 5 kV acceleration voltage, high vacuum, and ETD detection using a scanning electron microscope (Quanta Feg 650, FEI, Hillsboro, Oregon, USA).
- The authors counted the number of pigment granules within each square and took the average, then conducted a two sample t-test in R.
- The total lipid content (nmol per abdomen) was calculated as a sum of pmol certified by peer review) is the author/funder.
Transcriptome assembly and differential expression analysis:
- Offspring from a wild caught Alba female from Catalonia, Spain were reared at Stockholm University.
- Pupae were dissected in PBS solution, and the abdomen and wings were flash frozen in liquid nitrogen and stored at -80 o C. RNA was extracted from the abdomen and wing tissues using Trizol.
- Raw data was cleaned and reads from all libraries were used in a de novo transcriptome assembly (Trinity version trinityrnaseq_r2013_08_14 with default parameters) 57 .
- SAMTOOLS v1.2 48 idxstats was then used to calculate the read counts per gene for each of the sorted bam files.
- BarH-1 is heterogeneously expressed in the scale building cells within this region.
Fig. 4. Physiological differences between female morphs of C. crocea.
- A) The mass corrected total neutral lipid content for female morphs in two temperature treatments.
- However there is an interaction between morph and temperature as the difference is only significant in the cold treatment.
- The X-axis is the log of the fold change (FC), positive log(FC) indicates the gene is upregulated in Alba individuals.
- C) Volcano plots to visualize gene expression differences between female morphs in pupal wing tissue.
- Certified by peer review) is the author/funder.
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