Genomics and transcriptomics of the molting gland (Y-organ) in the blackback land crab, Gecarcinus lateralis

Abstract

Molting is required for growth and development in crustaceans. In the blackback land crab Gecarcinus lateralis, molting is stimulated by ecdysteroids, hormones produced in the Y-organ (YO). Throughout the molting cycle, the YO demonstrates phenotypic plasticity. The phenotypic plasticity is correlated with the stages of the molt cycle, during which YO ecdysteroid production varies. During intermolt, the longest stage of the molt cycle, the circulating ecdysteroid titers are low and molting is suppressed. In preparation for molting, the YO increases ecdysteroid production during premolt. Circulating ecdysteroids continue to rise, dropping right before the ecdysis and remaining low in the subsequent postmolt period. During the molt cycle, the YO's sensitivity to inhibitory cues also varies, which contributes to ecdysteroid fluctuations. To better understand how changes in gene expression modulate the YO's phenotypic plasticity, a YO transcriptome from five molt stages was generated. Using over 5.6 million reads from Illumina, 229,278 contigs were assembled to comprise the reference transcriptome. By comparing expression levels of the transcripts between the molt stages, 13,189 unique differentially expressed contigs were identified in G. lateralis. Based on differential expression, insect hormone biosynthesis and oxidative phosphorylation pathways were enriched, validating the YO transcriptome identity. Using GO enrichment, MAP kinase was identified as a possible candidate gene for regulating YO ecdysteroid synthesis. To complement and validate the transcriptome, claw muscle genomic DNA was sequenced and assembled using 2.6 million reads. 375,152 scaffolds ≥ 500 bp were built, with an N50 of 1,841 bp. Using k-mer frequencies, the genome size was estimated to be 3.07 Gb, similar to mammalian vertebrates. The median gene size of G. lateralis was approximated to be 6,300 bp; the disparity between the median estimate and the N50 prohibited further computational analysis. Genome scaffolds were sufficient in length for manual comparison. Alignment of the transcriptome and genome sequences of the Rheb gene showed 100% nucleotide alignment in the open reading frame, and extended the sequence by 7.7 fold, including the identification of four introns. The sequence comparison validated both genome and transcriptome assemblies and extended the gene sequence. Next-generation sequencing provided us with a global perspective of molecular variations within the YO throughout the molt cycle. We hypothesize variations in gene expression regulate YO phenotypic plasticity by varying ecdysteroid production. YO transitions throughout molting are essential for regulation. YO activation and commitment, both corresponding to increased ecdysteroids, are required to induce ecdysis. YO repression, during which circulating ecdysteroid titers are low, is needed to prevent precocious molting. Identifying changes in gene expression and key regulatory elements correlating with variations in YO phenotype will increase our understanding of molt cycle regulation, which is critical for crustacean development, growth, and repair.