Gene Duplication and Gene Expression Changes Play a Role in the Evolution of Candidate Pollen Feeding Genes inHeliconiusButterflies

Abstract

Heliconius possess a unique ability among butterflies to feed on pollen. Pollen feeding significantly extends their lifespan, and is thought to have been important to the diversification of the genus. We used RNA sequencing to examine feeding-related gene expression in the mouthparts of four species of Heliconius and one nonpollen feeding species, Eueides isabella. We hypothesized that genes involved in morphology and protein metabolism might be upregulated in Heliconius because they have longer proboscides than Eueides, and because pollen contains more protein than nectar. Using de novo transcriptome assemblies, we tested these hypotheses by comparing gene expression in mouthparts against antennae and legs. We first looked for genes upregulated in mouthparts across all five species and discovered several hundred genes, many of which had functional annotations involving metabolism of proteins (cocoonase), lipids, and carbohydrates. We then looked specifically within Heliconius where we found eleven common upregulated genes with roles in morphology (CPR cuticle proteins), behavior (takeout-like), and metabolism (luciferase-like). Closer examination of these candidates revealed that cocoonase underwent several duplications along the lineage leading to heliconiine butterflies, including two Heliconius-specific duplications. Luciferase-like genes also underwent duplication within lepidopterans, and upregulation in Heliconius mouthparts. Reverse-transcription PCR confirmed that three cocoonases, a peptidase, and one luciferase-like gene are expressed in the proboscis with little to no expression in labial palps and salivary glands. Our results suggest pollen feeding, like other dietary specializations, was likely facilitated by adaptive expansions of preexisting genes—and that the butterfly proboscis is involved in digestive enzyme production.

Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon

“…emphasizing that these 70 cocoonase genes were found within moth and butterfly families, which indicates that the cocoonase is Lepidoptera-specific (Fig 1). In addition, multiple copies of cocoonase were identified in the butterflies, consistent with recent research on Heliconius butterflies, which uncovered the role of duplicated cocoonase genes in pollen feeding [37][38][39]. While, there was single copy in most moths except Plutella xylostella, Trichoplusia ni, Plodia The phylogenetic tree was constructed using the Maximum Likelihood method, based on the Poisson correction model with 1,000 bootstrap replicates, and bootstrap support values more than 50% were shown in the tree.…”

Drift and Directional Selection Are the Evolutionary Forces Driving Gene Expression Divergence in Eye and Brain Tissue ofHeliconiusButterflies

“…De novo transcriptome assemblies for H. charithonia , H. sara , and H. doris were downloaded from Dryad with data identifier doi: 10.5061/dryad.ds21fv5 (Catalán et al 2018). Transcriptomes from H. erato and H. melpomene were downloaded from Dryad with data identifier doi: 10.5061/dryad.8d724 (Smith et al 2016). RNA-sequencing (RNA-seq) data for all the species were downloaded from ArrayExpress: E-MTAB-6810 (Catalán et al 2018).…”

Pollen feeding inHeliconiusbutterflies: the singular evolution of an adaptive suite

“…Like all butterflies, Heliconius lack cocoons, and it has been suggested that cocoonase homologues may have been coopted for use in digesting pollen proteins [62,63], potentially weakening the pollen wall. Cocoonase underwent several duplications along the lineage leading to Heliconius and their sister genus, Eueides, with an additional duplication specific to Heliconius and a further duplication in H. melpomene [63]. However, a functional role for cocoonase in pollen feeding has not yet been directly demonstrated.…”