Comparative Transcriptomics Reveal The Molecular Basis Of Firefly Adaptation To Freshwater Environments

Fireflies generally refer to Coleoptera fireflies,more than 2000 species have been recorded in the world,among which aquatic fireflies are rare,only 9 species have been recorded.The larvae of Aquatica leii are aquatic,and mainly inhabit on the substrates of shallow rivers,streams and ditches;the larvae of Lichnuris praetexta are terrestrial and widely distributed.L.praetexta is one of the most common fireflies in the genus.Although the behavior and morphology of fireflies in different habitats have been studied and recognized,little is known about the molecular basis of fireflies' adaptation to the water environment.This dissertation mainly discusses the molecular basis of the adaptation of the firefly to the aquatic environment from the changes of gene sequence and expression level,the intraspecific and interspecific comparison of aquatic and terrestrial fireflies.In this paper,the mitochondrial transcriptome of aquatic firefly(A.leii)and terrestrial firefly(L.praetexta)were sequenced by high-throughput sequencing technology.PRANK was used to align the sequence of orthologous genes to construct a nucleotide and amino acid data set,with Dendroctonus pokerosae and Tribolium castaneum as outgroups for phylogenetic analysis;Through the selection pressure test,identify the rapidly evolving and positive selection gene sets in the aquatic firefly;Through the analysis of differential gene expression between and within different types of fireflies,identify the specific differentially expressed genes of aquatic fireflies;The functional analysis of these genes was carried out by enrichment analysis to reveal the molecular basis of the firefly adapting to the water environment.In this paper,the transcriptome of aquatic fireflies A.leii was assembled de novo and then combined with the transcriptome sequences of the genomes of one other aquatic fireflies(Luciola aquatilis)and two terrestrial beetles(T.castaneum and D.pokerosae)to reconstruct the maximum likelihood and Bayesian tree agreement among the five species.Based on 2,675 single-copy orthologous genes among five species,the evolution rate of each branch was evaluated.The analysis results show that the average evolution speed of aquatic fireflies is slower than that of terrestrial fireflies,indicating a genomewide evolutionary constraint in the aquatic fireflies.This paper also identified 341 rapidly evolving genes and 116 positive selection genes and specific differentially expressed genes in aquatic firefly.The main functions of these genes involve energy metabolism,mitochondrial function,intracellular protein transport,cytoplasm,hypoxia response,and mitochondrial ATP production.Among them,76 genes are both rapidly evolving and subject to positive selection,mainly involved in the production of ATP,energy metabolism and hypoxic response.In addition,7,271 differentially expressed genes(DEGs)were identified between A.leii larvae and adults,and 8,309 DEGs were identified between L.praetexta larvae and adults.Through the comparison of DEGs between species(A.leii and L.praetexta),it was found that 1,445 genes were specifically differentially expressed in aquatic fireflies.These genes are mainly related to metabolic efficiency(such as ATP production,hypoxia and immune response)and certain aquatic firefly aquatic adaptation morphology(such as chitin epidermis,respiratory gills and compound eye morphology,etc.)related genes.These results indicate that sequence and expression-level changes in genes associated with both metabolic efficiency and morphological attributes related to the freshwater lifestyle contributed to freshwater adaptation in fireflies.This study provides new insights into the molecular mechanisms of aquatic adaptation in insects.