Identification of anhydrobiosis-related genes from an expressed sequence tag database in the cryptobiotic midge Polypedilum vanderplanki (Diptera; Chironomidae)

Abstract

Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.

FIGURE 1.

Anhydrobiotic cycle of P. vanderplanki larvae. In laboratory conditions, the process of desiccation lasts 48 h, after active larvae have been removed from water. Water loss accelerates after 24 h of desiccation treatment, at which time trehalose accumulates in the body of larvae. After 48 h of desiccation, the larvae reach the anhydrobiotic state and can maintain this state for several months and even years. Once larvae are immersed in water, the process of rehydration takes place rapidly. The anhydrobiotic larvae quickly absorb water, and muscular contractions can be observed after a few minutes. They recover their original active state about 20 min to 1 h after the beginning of rehydration. In the present study, mRNAs used to construct the anhydrobiosis-related EST library were collected from active larvae and from individuals at 12 and 36 h after the beginning of desiccation treatment.

FIGURE 2.

EST clones classified by gene ontology. A and B, general proportions of ESTs (A) and clusters (B) for each ontology group in the whole database. Details of the percentages of ESTs and clusters for each ontology group in the three libraries (desiccation 0, 12, and 36 h) are shown in the tables on the right. Statistical significance (χ-square test) of the proportion changes in the three libraries is shown on the right side (ns, non significant; *, p < 0.05; **, p < 0.001; ***, p < 0.0001).

FIGURE 3.

General expression patterns. EST clusters were classified on the basis of their putative expression pattern, deduced from the changes in EST numbers for each cluster during desiccation. Nine expression patterns were defined for genes expressed constantly (pattern 0), up-regulated (patterns 1, 2, 3, and 4), or down-regulated (patterns 5, 6, 7, and 8). Further ranking of each expression pattern was established, based on the greatest amplitude of EST number variation, as compared with the value at 0 h (indicated by over 2 fold, over 5 fold, and over 10 fold).

FIGURE 4.

Relative expression patterns of selected genes. Expression levels were obtained by real-time quantitative PCR during the first 48 h of desiccation for globin 2 (A), hemoglobin CTT6 (B), thioredoxin 2 (C), desiccation-inducible protein 1 (D), LEA protein 1 (E), trehalose-6-phosphate synthase (F), aquaporin 1 (G), and trehalose transporter 1 (H). The corresponding accession numbers were respectively AB513664, AB513663, AB513662, AB513665, AB207255, AB490332, AB281619, and AB272983. Relative expression levels were calculated relative to elongation factor 1 expression (A–D) or ribosomal protein L32 expression (E–H) and were calibrated using the expression level at 0 h of desiccation treatment as 1.0. The numbers of ESTs at 0, 12, and 36 h of desiccation treatment in the corresponding clusters from the database are written in gray over each graph.

FIGURE 5.

Expression profiles of some selected groups of clusters, defined by functional keywords. x-axis, cluster numbers; y-axis, number of ESTs in each cluster; z-axis, expression pattern, represented by the number of ESTs at 0 (yellow bars), 12 (orange bars), and 36 h (red bars) after the beginning of the desiccation treatment. The total number of clusters in each group is indicated in parentheses. Statistical significance (χ-square test) of the proportion changes in the three libraries is shown on the right (***, p < 0.0001).