Overview of the study system and transcriptome characterization. (A) Phylogenetic relationship between the genera Eucorethra, Mochlonyx and Chaoborus, adapted from Borkent and Borkent (2008) and Ogawa (2007), with a description of tracheal morphology and the three tissues of interest indicated. (B) De novo transcriptomes were generated from RNA-sequencing of three tissues from each species (from left to right: Eucorethra underwoodi, Mochlonyx cinctipes and Chaoborus trivittatus). Transcripts with full open-reading frames (ORFs) were selected for further analysis. Following clustering by sequence similarity (combining transcripts with >95% identity), reciprocal best hits were identified between each transcriptome (the number of clustered transcripts with reciprocal best hits between species pairs is indicated in red). (C) Venn diagram describing the number of Drosophila melanogaster candidate genes involved in epithelial function that were the target of best hit queries from Eucorethra, Mochlonyx and Chaoborus.
ABSTRACT
Aquatic larvae belonging to the genus Chaoborus are the only truly pelagic insects, possessing the ability to regulate their buoyancy and position in the water column by using two pairs of closed, air-filled sacs derived from the longitudinal trunks of their tracheal system. Previous work revealed that the volume of these air sacs is controlled using a unique mechanochemical system, where alternating bands of resilin and tracheal cuticle produce a composite material which expands and contracts with changing pH. While a simple epithelium enveloping each air sac is known to control the pH of the resilin bands, the molecular mechanisms underpinning its ability to regulate pH, and thus the air sac's volume, are unknown. To reveal these mechanisms, we compared the transcriptome of air sacs from Chaoborus trivitattus larvae with that of tracheal tissue from the aquatic larvae of two other genera within the Chaoboridae: Eucorethra underwoodi, whose larvae possess an unmodified tracheal system which they use to breathe air using a posterior respiratory siphon, and Mochlonyx cinctipes, whose larvae possess a tracheal system with dilated anterior and posterior sac-like regions, but which remains open through a posterior siphon. We found that C. trivitattus air-sac epithelia show strong expression of ion channels related to pH regulation relative to E. underwoodi tracheal tissue, including orthologs of NHA1, Nhe2, Ae2 and pHCl-2, as well the aquaporin Drip. This suggests that the tracheal epithelium of the ancestral chaoborid possesses all functionality required to control pH and deal with the flux of water associated with the swelling and contraction of resilin as part of the ancestral liquid-clearing function of the tracheal epithelium, enabling the evolution of a novel buoyancy control mechanism.