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.

BackgroundSeveral organisms display dormancy and developmental arrest at embryonic stages. Long-term survival in the dormant form is usually associated with desiccation, orthodox plant seeds and Artemia cysts being well documented examples. Several aquatic invertebrates display dormancy during embryonic development and survive for tens or even hundreds of years in a hydrated form, raising the question of whether survival in the non-desiccated form of embryonic development depends on pathways similar to those occurring in desiccation tolerant forms.Methodology/Principal FindingsTo address this question, Illumina short read sequencing was used to generate transcription profiles from the resting and amictic eggs of an aquatic invertebrate, the rotifer, Brachionus plicatilis. These two types of egg have very different life histories, with the dormant or diapausing resting eggs, the result of the sexual cycle and amictic eggs, the non-dormant products of the asexual cycle. Significant transcriptional differences were found between the two types of egg, with amictic eggs rich in genes involved in the morphological development into a juvenile rotifer. In contrast, representatives of classical “stress” proteins: a small heat shock protein, ferritin and Late Embryogenesis Abundant (LEA) proteins were identified in resting eggs. More importantly however, was the identification of transcripts for messenger ribonucleoprotein particles which stabilise RNA. These inhibit translation and provide a valuable source of useful RNAs which can be rapidly activated on the exit from dormancy. Apoptotic genes were also present. Although apoptosis is inconsistent with maintenance of prolonged dormancy, an altered apoptotic pathway has been proposed for Artemia, and this may be the case with the rotifer.ConclusionsThese data represent the first transcriptional profiling of molecular processes associated with dormancy in a non-desiccated form and indicate important similarities in the molecular pathways activated in resting eggs compared with desiccated dormant forms, specifically plant seeds and Artemia.

Numerous aquatic invertebrates remain dormant for decades in a hydrated state as encysted embryos. In search for functional pathways associated with this form of dormancy, we used label-free quantitative proteomics to compare the proteomes of hydrated encysted dormant embryos (resting eggs; RE) with nondormant embryos (amictic eggs; AM) of the rotifer A total of 2631 proteins were identified in rotifer eggs. About 62% proteins showed higher abundance in AM relative to RE (Fold Change>3; = 0.05). Proteins belonging to numerous putative functional pathways showed dramatic changes during dormancy. Most striking were changes in the mitochondria indicating an impeded metabolism. A comparison between the abundance of proteins and their corresponding transcript levels, revealed higher concordance for RE than for AM. Surprisingly, numerous highly abundant dormancy related proteins show corresponding high mRNA levels in metabolically inactive RE. As these mRNAs and proteins degrade at the time of exit from dormancy they may serve as a source of nucleotides and amino acids during the exit from dormancy. Because proteome analyses point to a similarity in functional pathways of hydrated RE and desiccated life forms, REs were dried. Similar hatching and reproductive rates were found for wet and dried REs, suggesting analogous pathways for long-term survival in wet or dry forms. Analysis by KEGG pathways revealed a few general strategies for dormancy, proposing an explanation for the low transcriptional similarity among dormancies across species, despite the resemblance in physiological phenotypes.

Rotifer resting eggs retain their viability for several decades in a non-desiccated form and are of interest in discerning the processes associated with dormancy, since in most organisms this phenomenon is linked with desiccation. The expression pattern of candidate genes with biological functions associated with dormancy in several other organisms was examined in rotifers. High-throughput transcriptome profiling revealed three patterns of gene expression in resting eggs: (1) relatively highly expressed genes coding for LEA proteins and putative paralogs of the small heat shock protein family (shsp); (2) genes coding for ferritin (ferr), glutathione-6-transferase (gts) and HSP70, where some of the putative gene paralogs of these families show relatively high expression levels and other putative paralogs show relatively low expression levels in resting eggs; and (3) genes with relatively low expression levels in resting eggs, for trehalose-6-phosphate synthase (tps), fatty-acid binding proteins (fab) and of lipoprotein lipase (lpl) and the aquaporins gene family (aqp). Changes in the expression pattern of some members of putative gene families occurred during the obligatory dormant period of resting eggs. A transition was observed from an expression pattern of diapausing embryos to an expression pattern of amictic females, during hatching. Differences were also found in the expression pattern in the different types of females, especially in those carrying resting eggs, and in males compared with females. These results suggest putative functional significance to genes associated with dormancy in non-desiccated resting eggs. It could also be proposed that their occurrence in resting eggs is developmentally programmed to facilitate survival in case of desiccation.

Two genes encoding for late embryogenesis abundant proteins (LEAs) are expressed in encysted diapausing embryos (or resting eggs) of rotifers (Brachionus plicatilis O.F. Müller) and females forming them. The two genes (bpa-leaa and bpa-leab) share approximately 50% of their nucleotides sequence, and bpa-leaa is more than twofold longer than bpa-leab. The deduced amino acid sequences show high abundance of alanine, glycine, lysine, and glutamic acid; a hydropathy index of lower than one; and a relatively high (81-82%) predicted probability of forming alpha-helices in their secondary structure, all of which are characteristic features of LEAs. The predicted molecular masses of bpa-LEAA ( approximately 67 kDa) and bpa-LEAB ( approximately 27 kDa) are similar to the molecular mass determined by Western-blot analyses, suggesting a low probability of posttranslational modifications. In silico analysis reveals that the two LEAs resemble group 3 LEAs based on the repeats for 11mer motifs, although they also display several putative amino acids typical of the 20mer motif of group 1 LEAs. The rotifer LEAs do not contain a predicted target sequence and are more likely localized in the cytosol. LEAs were expressed in resting eggs and females producing them, but not in other female forms or males. LEA transcripts and proteins are degraded during hatching, suggesting that LEAs are developmentally programmed during resting egg formation and hatching. LEAs probably equip the resting eggs to withstand desiccation if that occurs during dormancy. The present study expands our knowledge about the biological pathways associated with formation of rotifer resting eggs and also demonstrates the occurrence of LEAs in dormant, nondesiccated, encysted animal embryos.