Comparative genome sequencing reveals genomic signature of extreme desiccation tolerance in the anhydrobiotic midge

Abstract

Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki.

Figure 1. Desiccation tolerance and phylogeny of two chironomid species.

(a) Adult male of the sleeping chironomid, P. vanderplanki (left), and anhydrobiotic cycle of the larvae (right). During the dry season, larvae desiccate slowly to reach an ametabolic, quiescent state, termed anhydrobiosis. On rehydration, dried larvae rapidly recover normal activity. (b) Adult male of the congeneric chironomid, P. nubifer (left). P. nubifer larvae can survive mild desiccation for 24 h like other chironomids, but they cannot enter anhydrobiosis and are killed by severe dehydration (right). Scale bar, 2 mm. (c) Phylogenetic tree inferred from the amino-acid sequence of cytochrome oxidase I (COI) showing the relationship between P. vanderplanki, P. nubifer and other Diptera. The scale shows the evolutionary distance between species in million years (MYA).

Figure 2. Putative mechanism for the evolution of ARId in the P. vanderplanki genome.

ARIds are genomic regions containing clusters of duplicated genes that are transcriptionally active during anhydrobiosis. (a) A gene of foreign origin (for example, LEA protein) is incorporated into P. vanderplanki genome by HGT and undergoes extensive duplications and shuffling. (b) A pre-existing P. vanderplanki gene originally not involved in anhydrobiosis and originating from another region of P. vanderplanki genome was inserted to a new locus by intragenomic duplication (IGD) and undergoes extensive duplications and shuffling to acquire or improve a specific function for desiccation tolerance. All the genes in the ARIds from both a,b become highly upregulated during anhydrobiosis (red arrows).

Figure 3. Amino-acid motif distribution in PvLEA proteins and a bacterial hypothetical protein.

The distribution was determined by MEME motif analysis (version 4.9.1). The closest non-eukaryotic genes resembling LEA proteins for P. vanderplanki and prokaryotes were identified by a cross-Blast search and used for analysis. The height of the motif block is proportional to –log(P value), truncated at the height for a motif with a P value of 1e−10. MEME parameter settings were as follows: any number of repetitions for the distribution of motif occurrences; 11 for minimum and maximum motif width; and 2 for maximum number of motifs to find.

Figure 4. Evolutionary relationships of the classical and novel desiccation-responsive TRX and PIMT proteins.

(a) Phylogenetic tree of TRX proteins showing the clusters of classical insect TRX-1 (green), TRX-2 (grey), TRX-3 (blue) and the cluster of desiccation-responsive TRX, specific to P. vanderplanki (pink). (b) Phylogenetic tree of PIMT proteins showing the cluster of the classical PIMT-1 conserved among Diptera (green) and the cluster of desiccation-responsive PIMT proteins, which are specific to P. vanderplanki (pink). The evolutionary history was inferred using the neighbor-joining method and the evolutionary distances were computed using the maximum likelihood estimation (units: amino-acid substitutions per site). Pv, P. vanderplanki; Pn, P. nubifer; Aa, Aedes aegypti; Ag, Anopheles gambiae; Am, Apis mellifera; Cq, Culex quinquefasciatus; At, Arabidopsis thaliana; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus; Nv, Nasonia vitripennis; XI, Xenopus laevis.