Discovering genes associated with dormancy in the monogonont rotifer Brachionus plicatilis

Abstract

Background: Microscopic monogonont rotifers, including the euryhaline species Brachionus plicatilis, are typically found in water bodies where environmental factors restrict population growth to short periods lasting days or months. The survival of the population is ensured via the production of resting eggs that show a remarkable tolerance to unfavorable conditions and remain viable for decades. The aim of this study was to generate Expressed Sequence Tags (ESTs) for molecular characterisation of processes associated with the formation of resting eggs, their survival during dormancy and hatching.

Results: Four normalized and four subtractive libraries were constructed to provide a resource for rotifer transcriptomics associated with resting-egg formation, storage and hatching. A total of 47,926 sequences were assembled into 18,000 putative transcripts and analyzed using both Blast and GO annotation. About 28-55% (depending on the library) of the clones produced significant matches against the Swissprot and Trembl databases. Genes known to be associated with desiccation tolerance during dormancy in other organisms were identified in the EST libraries. These included genes associated with antioxidant activity, low molecular weight heat shock proteins and Late Embryonic Abundant (LEA) proteins. Real-time PCR confirmed that LEA transcripts, small heat-shock proteins and some antioxidant genes were upregulated in resting eggs, therefore suggesting that desiccation tolerance is a characteristic feature of resting eggs even though they do not necessarily fully desiccate during dormancy. The role of trehalose in resting-egg formation and survival remains unclear since there was no significant difference between resting-egg producing females and amictic females in the expression of the tps-1 gene. In view of the absence of vitellogenin transcripts, matches to lipoprotein lipase proteins suggest that, similar to the situation in dipterans, these proteins may serve as the yolk proteins in rotifers.

Conclusion: The 47,926 ESTs expand significantly the current sequence resource of B. plicatilis. It describes, for the first time, genes putatively associated with resting eggs and will serve as a database for future global expression experiments, particularly for the further identification of dormancy related genes.

Figure 1

The life cycle of Brachionus plicatilis showing asexual and sexual reproduction and formation of resting eggs. In the asexual life cycle, diploid amictic females produce parthenogenetic diploid amictic eggs. A mixis signal initiates the occurrence of a sexual cycle, whereby, diploid mictic females produce haploid eggs via meiosis. The haploid eggs develop into either haploid males or, if fertilized, they form diploid dormant (or diapausing) resting eggs. The internal insemination of diploid mictic females carrying haploid eggs, is possible for only a few hours after birth. Mictic females are shaded in grey and include mictic females producing male eggs or mictic females that form diploid resting eggs. All females are diploid while males are haploid.

Figure 2

Rooted NJ tree of lea-like deduced proteins, LEA proteins of other invertebrates and canonical plant LEA proteins from the three major groups. The out-group used was of glucose starvation inducible protein of Bacillus subtilis (Accession No. 26907; defined as LEA protein by [51]). The canonical plant LEA proteins were chosen after [59]. The LEA proteins of invertebrates are highlighted in yellow.

Figure 3

Expression pattern of selected genes in resting eggs (RE) vs. amictic eggs (AE) and resting-egg producing females (FRE) vs. amictic females (FA). Genes that were tested include: the Late embryonic abundant protein (lea-1, lea-2, lea-3), small heat shock proteins (shsp-3), manganase supreroxide dismutase (mn-sod-2), copper or zinc superoxide dismutase (cu/zn-sod-1), glutathione S-trasferase (gst-2, gst-8) and trehalose phosphate synthase (tps-1).