Discovering genes associated with dormancy in the monogonont rotifer Brachionus plicatilis

Protection against ROS and detoxification

Further searches for antioxidant enzymes identified 135 clones, which assembled into 11 putative transcripts coding for peroxiredoxins (E values between 10^-27 to 10^-77) and thioredoxin peroxidase activity (E value of 10^-57) (data not shown). Members of these families were found in all normalized libraries and Bpa-trpx-6 and Bpa-trpx-7 were additionally found in the sbs04 library associated with resting eggs (data not shown). Antioxidant activity is also associated with the enzyme phospholipid-hydroperoxide glutathione peroxidase, which protects membranes from oxidative stress by reducing the membrane hydroperoxides [40]. Twenty-nine clones were found to be associated with phospholipid-hydroperoxide glutathione peroxidase activity (GO:0047066) in the EST libraries and two putative transcripts were produced after contig assembly and were named gpx1 and gpx2. The transcript gpx1 was only found in the MS and sbs01 libraries, whilst gpx2 was found in all the normalized libraries. BLAST results for the two transcripts were quite different: gpx1 matched mammalian glutathione peroxidase 3 (E value = 10^-42) and gpx2 matched phosphlipid-hydroperoxide glutathione peroxidase of hydra and cattle tick (E value = 10^-20), and of mammals (E value 10^-18), although both confer antioxidant protection. The presence of two genes indicates a duplication of the gpx genes in the rotifer.

Dismutases catalyze the conversion of superoxide radicals into hydrogen peroxide, preventing their conversion into the more active hydroxyl radical [25]. Five putative transcripts were found to be associated with superoxide dismutase activity (GO:0004784). Two transcripts show homology with the Mn-SOD (E value = 10^-111) previously described by [19]. Three other transcripts were found to be similar to Cu/Zn-SOD. Transcripts were found across several different libraries and so could be designated as ubiquitous. However, the previously identified Mn-SOD of B. plicatilis was found to be over-expressed in rotifers with an extended life span resulting from caloric restriction [19]. Similarly in C. elegans, the DAF pathway (insulin, dauer associated) is also linked to caloric restriction and increased lifespan. Therefore these genes clearly have other roles in addition to putative functions associated with desiccation.

Maintaining the native folded conformation of proteins

Changes in environmental conditions (e.g. osmotic pressure, pH, temperature and desiccation), challenge protein structure and may cause the formation of cytotoxic protein aggregates and induce the production of "stress" proteins [41]. Therefore, desiccation tolerant resting eggs need to develop mechanisms for coping with denaturing and aggregation of proteins. The classical cellular response to this type of stress is the induction of "heat shock" or chaperone proteins [42–44] which facilitate the disaggregation of proteins and their refolding to native conformation, and/or the production of small heat shock proteins, which prevent initial protein aggregation [26].

Although members of the HSP70 family are regarded as the classical cellular stress response, the small heat shock proteins are being increasingly identified as having a pivotal role in survival in stressful conditions and metabolic arrest [23]. Encysted embryos of Artemia franciscana have been shown to contain substantial amounts of HSP26 [46, 47] along with a ferritin homologue [24], with both molecules acting as chaperones to prevent protein aggregation.

Regarding additional candidates for further investigation in resting-egg stage gene expression, a number of other heat shock proteins were identified (HSP60 and HSP80-100). Induction of the HSP60 protein was previously shown in B. plicatilis in response to various environmental pollutants [20, 48], and also in Plationus patulus in response to arsenic and heavy metal exposure [49] and therefore are potential "stress" proteins. Eleven putative transcripts with matches to HSP60 were found, as were putative transcripts with matches to other high molecular weight heat-shock proteins (HSP80-100). These candidates were found in all of the normalized libraries.

Late Embryogenesis Abundant (LEA) proteins

LEA proteins were originally identified in plant seeds during the late stages of embryonic development and are associated with desiccation tolerance throughout the life cycle of all major plant taxa [50]. They comprise a protein family with three major groups (Groups 1–3). They have also been found in non-plant species and to date almost all non-plant LEA proteins belong to Group 3 [51]. LEAs have been found in the nematode Aphelenchus avenae [52], bdelliod rotifers [53, 54] and desiccated A. franciscana cysts [55]. The exact function of LEA proteins is as yet, unknown, but their importance in desiccation and stress tolerance has been comprehensively demonstrated. For example, silencing of the lea gene in C. elegans dauer juveniles caused a significant reduction of worm survival during induction of desiccation and in osmotic and heat stresses [56]. LEA proteins were found to prevent protein aggregation in vitro [57]. Also, in vivo experiments using Aphelenchus avenae LEA proteins introduced into human cell lines demonstrated that these proteins played a role in anti-aggregation and protein stabilisation during desiccation procedures [58].

Three transcripts matching group 3 LEA proteins on BLAST sequence similarity analyses were identified (E values in the range of 1E^-11 – 5E^-25). These have been designated bpa-lea-1, bpa-lea-2 and bpa-lea-3 (Table 7). A rooted NJ tree was produced using translations of these transcripts with canonical plant LEA proteins from all three major groups [59] and the metazoan LEA proteins of C. elegans, A. franciscana, P. vanderplanki, and A. avenae (Figure 2). The putative rotifer genes were associated with the Group 3 protein family.

Contig	EST singnature	Contig size	Best BLAST match	E value	MS	RE	REH	FRE	sbs04	sbs02
bpa-lea-1	sb104P0049I13	25	Q6NMC2/LEA-like	5E-25	0	7	1	5	12	0
bpa-lea-2	sbs04P0011H05	35	Q9FKV7/LEA-like	1E-22	3	5	3	4	20	0
bpa-lea-3	sbs02P0007H12	3	Q9FKV7/LEA-like	1E-11	0	0	0	0	2	1
				Total	3	12	4	9	34	1

All match transcripts from Arabidopsis thaliana.

Trehalose metabolism

Trehalose is thought to play an important role in enhancing desiccation and stress tolerance [60]. For example, accumulation of trehalose has been shown in diapausing cysts of Artemia [61] and also the stress responses of nematodes [62, 63]. Trehalose is synthesized from glucose, catalyzed by the enzymes trehalose-6-phosphate synthase (tps) and trehalose phosphatase [64]. Trehalose can comprise ~17% of the dry mass in Artemia undergoing desiccation [65] and small amounts (0.35% of dry weight) have previously been found in B. plicatilis desiccated resting eggs [28]. Also a transcript [DDBJ: BJ979617] with high sequence similarity to the tps gene, encoding to trehalose phosphate synthase, was previously identified in an EST library of B. plicatilis [21].

Given the data and the nature of the way the libraries were produced it is not possible to determine the role of trehalose in resting-egg formation and survival solely using this data. In addition to the duplication of the trehalose-6-phosphate synthase gene in C. elegans, this species also shows a duplication of the trehalase gene, the enzyme which breaks down trehalose. In fact, there are four trehalase genes annotated in Ensembl [W05E10.4, F57B10.7, T05A12.2 and C23H3.7] [66]. BLAST searches of the rotifer data produced three singletons with matches to trehalase (data not shown). Although these were single reads and therefore sequence quality was variable, there were sufficient differences between the putative translations of these clones to indicate that they were potentially three different genes, demonstrating another situation analogous with the nematode. Although the C. elegans sequences are similar at the sequence level to other characterized trehalases (hence the annotation), they are designated as "unknown function", as RNAi studies produce no obvious phenotype. It has yet to be determined why there are four copies of this gene in C. elegans and what is the exact function of each paralogue. By extrapolation the same can be inferred for the three putative trehalases in the rotifer.

Aquaporins

Lipid and fatty acid metabolism

Expression experiments

Since all libraries were produced using either normalized or subtractive methods, real-time PCR experiments were conducted in order to assess the expression of selected genes in resting eggs and in resting-egg producing females (see [additional file 1] Table S1). The expression patterns of the selected genes were determined in resting eggs relative to amictic eggs, and in resting-egg producing females relative to amictic females (Fig. 3). It should be noted that in all cases the 95% confidence limits in the female samples were expanded compared to those of the egg samples. This may be attributed to the larger inherent variability between females, related to their age and size.

Genes upregulated in resting eggs include all the lea-like transcripts, a small heat shock protein and two of the genes involved in antioxidant activities: one of the glutathione S-transferases (Bpa-gst-8) and a superoxide dismutase (Mn-sod-2). Two gst-like transcripts were chosen for analysis: Bpa-gst-8, identified in the normalized libraries associated with resting eggs (RE, FRE) and also the subtracted library containing resting eggs, and gst-2 found in all the normalized libraries. As mentioned above, gst-8 is up-regulated in resting eggs and in resting-egg producing females. No significant change in the expression of gst-2 was found in resting eggs relative to amictic eggs but it was slightly up-regulated in resting-egg producing females. Therefore, the two gene family members clearly play different roles in cellular defense mechanisms.

The relative expression of tps-1 transcript was determined in order to evaluate the significance of trehalose synthesis in resting-egg production. The results do not show any significant change in the expression of the tps-1 like gene in resting eggs relative to amictic eggs or in resting-egg producing females relative to amictic females. Hence, the expression pattern of the tps-like transcript suggests that this gene may not be associated with resting-egg production, although it cannot be discounted that trehalose production is regulated at the translational level or enzyme activity rather than the transcriptional level.

Rotifer cultures and sample collection

Rotifers were hatched from resting eggs produced in the laboratory from rotifers collected at a seaside pond in Atlit, (40 km south of Haifa, Israel) in 1981. Some of the resting eggs were hatched in 2003 and resting eggs produced from them were stored in the laboratory. Resting eggs from 1981 and 2003 were stored in the dark at 4°C and hatched in 2005. Four groups of samples were collected for libraries; (I) mixed stage cultures containing both amictic and mictic females, (II) Resting eggs (RE), (III) resting eggs during hatching and (IV) mictic females with resting eggs

RNA extraction and library preparation

RNA was extracted with the TRIzol^® reagent (Invitrogen) following the manufacturer instructions. cDNA was synthesized using the SMART approach (SMART PCR cDNA synthesis kit, Clontech, U.S.A.) and subsequently normalized using duplex-specific nuclease (Trimmer kit, Evrogen, Russia) according to manufacturer's instructions and directionally cloned into pAL32 (Evrogen, Russia). Subtractive cDNAs were constructed by suppression subtractive hybridization (Evrogen, Russia) and cloned via the TOPO TA^® cloning system (Invitrogen, U.S.A.). Plasmids were transferred via electroporation into E. coli DH10B (Invitrogen, U.S.A.). Plasmids from the normalized libraries were 5' end sequenced using the pALforward primer (5'-CTCGGGAAGCGCGCCATT-3') and Big Dye Terminator chemistry. Clones of the subtractive libraries were sequenced from both ends using T7 and T3 primers.

Sequences were determined on ABI 3730XL capillary sequencers (Applied Biosystems, USA).

cDNA libraries construction and characterisation

Four normalized libraries and four subtractive libraries were constructed:

MS: Normalized library of a mixed population consisting of amictic females, mictic females and males. The library was generated from a combination of four cloned cultures and one non-cloned culture

RE: Normalized library of resting eggs. The resting eggs were obtained from a non-cloned culture.

REH: Normalized library of resting eggs in various stages of hatching. The library was constructed from resting eggs that were hatched for 20 or 30 hrs.

FRE: Normalized library of mictic females with resting eggs. The females were collected from a resting-egg producing clone (clone 1B₄).

sbs01: Subtractive library of a mixed stage population from a combination of cloned and non-cloned cultures (tester) vs. mixed stage population of a clone 1B₄ (driver)

sbs02: Subtractive library of a mixes stage population of clone 1B₄ (tester) vs. a mixed stage population of a combination of cloned and non-cloned cultures (driver).

sbs03: Subtractive library of a mixed stage population of clone 1B₄ (tester) vs. mictic females with resting eggs of clone 1B₄ (driver).

sbs04: Subtractive library of mictic females with resting eggs as tester vs. mixed stage population of clone 1B₄ (driver).

Sequence analysis and EST clustering

Sequence fasta files were processed using the script Trace2dbest [74], which incorporated the phred and crossmatch programmes [75, 76]. A minimum cut-off value of 100bp was applied after quality control processing for generating the submission file for EMBL (Accession numbers, FM897377–FM945301). Tgicl [77] was used for clustering the fasta files, incorporating quality scores, for each of the five libraries, as well as for all the libraries together. The clusters were database searched using Blastx [78] against the Uniprot/Swissprot and Uniprot/Trembl databases [79], with matches annotated for all scores with an expect score in excess of 1e-10. These annotations were then used to map Gene Ontology identifiers [80]. Sequence manipulation was carried out using the EMBOSS suite of programs [81]. Multiple sequence alignments of LEA proteins were performed using the ClustalW program. [82]. Phylogenetic trees were drawn with MEGA4 [83] using the Bacillus subtilis glucose inducible starvation protein B (Accession no: P26907) as an outgroup.

Real-Time PCR experiments

The relative abundance of bpa-lea-1, bpa-lea-2, bpa-lea-3, bpa-shsp-3, bpa-mnsod-2, bpa-cusod-1, bpa-gst-11, bpa-gst-4, bpa-tps-1 transcripts (see [additional file 1] Table S1 for the list of primers) were normalized to an elongation factor 1α housekeeping sequence using the equation: ratio = (E_target)^CP_target/(E _ef1a )^CP_ef1awhere E = 10^-1/slop, according to the method described by Pfaffl et al., [84]. The PCR mixture consisted of 1 μl of cDNA sample, 70 nM of each primer and 12.5 μl of SYBR Green master mix (ABgene, UK), in a final volume of 25 μl. Amplification were preformed with biological triplicate samples using a GenAmp 5700 thermocycler (PE Applied Biosystems, USA) according to the manufacturer's protocol.