Skip to main content
Download PDF
  • Research article
  • Open access
  • Published:

Comparative transcriptomics in Syllidae (Annelida) indicates that posterior regeneration and regular growth are comparable, while anterior regeneration is a distinct process

BMC Genomics volume 20, Article number: 855 (2019) Cite this article

Abstract

Background

Annelids exhibit remarkable postembryonic developmental abilities. Most annelids grow during their whole life by adding segments through the action of a segment addition zone (SAZ) located in front of the pygidium. In addition, they show an outstanding ability to regenerate their bodies. Experimental evidence and field observations show that many annelids are able to regenerate their posterior bodies, while anterior regeneration is often limited or absent. Syllidae, for instance, usually show high abilities of posterior regeneration, although anterior regeneration varies across species. Some syllids are able to partially restore the anterior end, while others regenerate all lost anterior body after bisection. Here, we used comparative transcriptomics to detect changes in the gene expression profiles during anterior regeneration, posterior regeneration and regular growth of two syllid species: Sphaerosyllis hystrix and Syllis gracilis; which exhibit limited and complete anterior regeneration, respectively.

Results

We detected a high number of genes with differential expression: 4771 genes in S. hystrix (limited anterior regeneration) and 1997 genes in S. gracilis (complete anterior regeneration). For both species, the comparative transcriptomic analysis showed that gene expression during posterior regeneration and regular growth was very similar, whereas anterior regeneration was characterized by up-regulation of several genes. Among the up-regulated genes, we identified putative homologs of regeneration-related genes associated to cellular proliferation, nervous system development, establishment of body axis, and stem-cellness; such as rup and JNK (in S. hystrix); and glutamine synthetase, elav, slit, Hox genes, β-catenin and PL10 (in S. gracilis).

Conclusions

Posterior regeneration and regular growth show no significant differences in gene expression in the herein investigated syllids. However, anterior regeneration is associated with a clear change in terms of gene expression in both species. Our comparative transcriptomic analysis was able to detect differential expression of some regeneration-related genes, suggesting that syllids share some features of the regenerative mechanisms already known for other annelids and invertebrates.

Background

Growth and regeneration are remarkable developmental abilities of annelids. Most annelids grow during their entire life by addition of segments from a segment addition zone (SAZ) located between the pygidium and the last segment [1,2,3,4,5,6,7,8]. Moreover, nearly all annelid species are able to completely restore the posterior body, while only some groups are able to regenerate the anterior body [6,7,8,9,10]. Whereas several studies describing the process of (anterior and posterior) regeneration are available, the molecular background of this ability remains largely unknown in annelids [6,7,8, 10].

Studies including molecular data during regeneration in annelids have been published for the clitellates Enchytraeus japonensis Nakamura, 1993 [11,12,13,14,15] and Pristina leidyi Smith, 1896 [1, 16,17,18,19,20]; and the non-clitellates Alitta virens Sars, 1835 [21,22,23,24], Capitella teleta Blake, Grassle and Eckelbarger, 2009 [25,26,27,28,29], and Platynereis dumerilli (Audouin and Milne Edwards, 1833) [3, 30,31,32,33,34,35]. All those species regenerate the posterior body, but only E. japonensis and P. leidyi exhibit anterior regeneration [1, 11,12,13,14,15, 17,18,19,20]. Studies on anterior regeneration in non-clitellates have been limited to morphological approaches so far (e.g. [36,37,38,39,40,41,42,43,44]). Interestingly, some genes that are expressed in the SAZ during regular growth/development have been detected in different stages of posterior regeneration in annelids, for example, Hox genes [21,22,23, 27, 45], β-catenin [17], and genes of the germline multipotency program such as piwi, vasa, nanos, and PL10 [27, 46,47,48].

Within Annelida, Syllidae are known to completely regenerate their tails [8, 49]. However, when dealing with anterior regeneration, many species can only regrow the prostomium and few segments, e.g. Eusyllis blomstrandi Malmgren, 1867 [49,50,51]; while others additionally regenerate all missing segments and also a characteristic differentiation of the digestive tube called proventricle (e.g. Syllis gracilis Grube, 1840 [37, 52,53,54,55]). Interestingly, the molecular background of regeneration in syllids has not been explored.

We used RNA-seq to generate gene expression profiles of the anterior and posterior regeneration processes, as well as the regular posterior growth of two species of syllids: Sphaerosyllis hystrix Claparède, 1863 [56] (Exogoninae), and Syllis gracilis (Syllinae). Our aim was to analyse the changes in gene expression during the first stages of posttraumatic anterior regeneration (AR) and posterior regeneration (PR) by comparing them with the non-regenerating condition (NR) (i.e. intact individuals in regular posterior growth), and between themselves (AR and PR). Additionally, selected genes previously shown to be (highly) expressed during regeneration in other annelids and other invertebrates have been investigated. Finally, we also documented the morphological changes during anterior and posterior regeneration in both species, and identified regeneration-related genes that could be of interest for future studies in syllid regeneration.

Results

Illumina NGS and assembly

We used a comparative transcriptomic approach in order to compare gene expression in three conditions: anterior regeneration (AR), posterior regeneration (PR), and non-regenerating (NR), i.e. intact individuals in regular posterior growth (see Figs. 1, 2 and 3 for experimental design and morphological data). mRNA samples of S. hystrix and S. gracilis were sequenced for each condition using an Illumina sequencing platform. Considering all three conditions, we generated a total of 79.5 GB raw reads for S. hystrix and 74.3 GB for S. gracilis (Table 1). After trimming the reads, 84.0 and 88.3% of reads remained for S. hystrix and S. gracilis, respectively (Table 1). Those cleaned reads were assembled, generating 315,224 contigs for S. hystrix (average length = 733.43, N50 = 1158) and 526,860 contigs for S. gracilis (average length = 626.48, N50 = 858). According to BUSCO [57], both transcriptomes were highly complete 97,8% (S. hystrix) and 98,6% (S. gracilis), despite showing a high level of redundancy with 73.8 and 80.6%, respectively (Table 1). We found 179,841 predicted proteins in the transcriptome of S. hystrix and 309,576 predicted proteins in the one of S. gracilis (Table 1). The raw reads were uploaded at the NCBI Sequence Read Archive (SRA). Assemblies and transdecoder predicted proteins are available under https://github.com/rannypribeiro/Regeneration_transcriptomics.

Fig. 1
figure 1

Regeneration timeline of the specimens sequenced for transcriptomic data. Bisection was performed in the midbody site and the amputees were fixed for sequencing in the first stages of regeneration: stage 1 (healing), stage 2 (early blastema development), stage 3 (late blastema development), and stage 4 (patterning/cap regeneration). Anterior regeneration sequencing cover stages 1–3; posterior regeneration covers all the stages. Time-scale of experimentation: 12 days for Sphaerosyllis hystrix and 8 days for Syllis gracilis (see Methods)

Full size image
Fig. 2
figure 2

Light microscopy pictures of the regenerating Sphaerosyllis hystrix. a, b, c, g, h, i anterior regeneration. d, e, f, j, k, l posterior regeneration. Amputation was performed in the midbody region and the regenerating animals were observed for 14 days post amputation (dpa). Immediately after body bisection, the wound is closed by invagination through muscle contraction. Anterior regeneration starts by wound healing (1–3 dpa) and the formation of a small blastema (a). The anterior blastema is formed after 4–6 dpa and no differentiated organ is regenerated until 12 dpa (b, c, g). An incomplete prostomium (head) appeared after 13 dpa, bearing eyes (h), and a pair of minute antennae in 14 dpa (i). Posterior regeneration proceeds more quickly: healing occurred in 2 dpa, the blastema developed from 2 to 4 dpa, and a pygidium with a pair of cirri was first seen after 9 dpa (d, e, f). From 10 to 14 dpa, amputees had regrown new pygidia and a maximum of four posterior segments (j–l). All pictures are in dorsal view. Scale bar 0.2 mm. White dashed lines show amputation level. Black dashed lines show the regenerated eyes. Abs: an, antenna; ey, eye

Full size image
Fig. 3
figure 3

Light microscopy pictures of the regenerating Syllis gracilis. a, b, c, g, h, i anterior regeneration. d, e, f, j, k, l posterior regeneration. Anterior and posterior regeneration of S. gracilis were observed during 8 dpa. The wound is completely healed after 2 dpa and a blastema develops during the following days in both anterior and posterior regeneration. After 8dpa, the blastema was still elongating during anterior regeneration (a–c, g–i). Regarding posterior regeneration, the blastema differentiated between 4 and 7 dpa; after 8 dpa a pygidium bearing three short cirri was restored (d–f, j–l). All pictures are in dorsal view. Scale bar 0.2 mm. White dashed lines show amputation region

Full size image
Table 1 Statistical summary of raw data, transcriptome assembly, and functional annotation of Sphaerosyllis hystrix and Syllis gracilis
Full size table

Functional annotation of transcripts and gene ontology

Around 35.7% (S. hystrix) and 31.3% (S. gracilis) of the assembled transcripts were annotated. The annotation results showed hits with human and mouse genes mostly, and less than 1% with known annelid genes (Additional file 1). Within Annelida, most transcripts were annotated with Lumbricus sequences: 38% (S. hystrix) and 28% (S. gracilis) (Additional file 1). Gene ontology (GO) categories were assigned to 28.5 and 24.5% of the transcripts of S. hystrix and S. gracilis, respectively. Our results showed that both species have a similar distribution of genes associated to the categories of cellular component, molecular function and biological process (Additional file 1).

Comparison of gene expression profiles

In order to identify differentially expressed (DE) genes, we compared the transcriptomic profiles of anterior regeneration and posterior regeneration (AxP), anterior regeneration and non-regenerating condition (AxN), and posterior regeneration and non-regenerating (PxN) of both studied species.

Sphaerosyllis hystrix

Considering the overall results, we detected 4771 DE genes in S. hystrix (FDR < 0.001) (Fig. 4a; Additional file 2: Tables S1–S4). Analysing the comparisons separately, 108 genes were found to be differentially expressed in AxP, and 4768 genes in AxN. No DE genes were found in PxN. Four thousand six hundred sixty-three of the DE genes were exclusively found in AxN; 105 genes were present in both AxN and AxP; and only 3 genes were exclusive of AxP. Most of the DE genes were up-regulated in AR (4699) rather than in PR (161) or in NR (58) (Fig. 4a). AR up-regulated genes had similar expression levels in both PR and NR (see Additional files 2: Table S1). Gene Ontology analysis showed that 76% of the DE genes were annotated. The most prominent GO terms in AxP and AxN belong to the cellular component category (e.g. secretory granule, zymogen granule membrane, motile cilium, apical lamina of hyaline layer, ribosomal and mitochondrial parts) (Fig. 5a, b; Additional file 2: Tables S5 and S6).

Fig. 4
figure 4

Heatmaps of differentially expressed genes during regeneration (FDR < 0.001). a Sphaerosyllis hystrix results. b Syllis gracilis results. Note that some of the genes can be up-regulated in more than one condition. Values in centred log2(fpkm+ 1). AR: anterior regeneration, PR: posterior regeneration, NR: non-regenerating. See Additional file 2: Table S1 and Additional file 3: Table S7 for detailed results

Full size image
Fig. 5
figure 5

Results of gene ontology annotation of DE genes. Only the ten most significant enriched GO terms are plotted. a AxP comparison and b AxN comparison for Sphaerosyllis hystrix. c AxP comparison and d AxN comparison for Syllis gracilis. CAT: category; BP: biological process, CC: cellular component, MF: molecular function. Z-score is useful to know if the expression of genes belonging to a certain GO term is more likely to be decreasing (negative) or increasing (positive) and it is calculated as the number of up-regulated genes minus the number of down-regulated genes divided by the square root of the gene count [58]. Up-regulated genes have logFC> 0, and down-regulated genes have logFC< 0. Inner boxes size is based on the p-value and represents the significance of the enrichment of each GO term. Output data of the GOplot analyses is available in Additional file 2: Tables S5 and S6, and Additional file 3: Tables S11 and S12

Full size image

Syllis gracilis

The overall results of the differential expression analysis showed 1997 DE genes among the three experimental conditions of S. gracilis (FDR < 0.001) (Fig. 4b; Additional file 3: Tables S7–S10). Of those genes, 1863 and 1428 were found in AxN and AxP, respectively. Similarly, to the results obtained for S. hystrix, no DE genes were found in PxN (FDR < 0.001). Of the DE genes, 529 were exclusive of AxN; 1334 were present simultaneously in AxN and AxP; and only 134 were exclusively detected in AxP. One thousand nine hundred forty genes were up-regulated in AR rather than in PR (33) or in NR (42) (Fig. 4b). In terms of gene ontology, 86% of genes with differential expression were annotated and the most prominent GO terms in AxP and AxN belong to the cellular component category (e.g., ribosome, intracellular ribonucleoprotein complex, ribosomal unit, macromolecular complex annotated) (Figs. 5c, d; Additional file 3: Tables S11 and S12).

Identification of candidate regeneration genes

In order to identify putative regeneration-related genes in these species, BLAST searches were performed against our transcriptomes using publicly available sequences of those genes that have been previously shown to be (highly) expressed during regeneration in other annelids (Table 2; Additional file 4) [1, 2, 12, 13, 17, 21, 23, 27, 32, 35, 45, 46, 48, 59,60,61,62,63].

Table 2 Results of BLAST searches for candidate regeneration genes
Full size table

A total of 71 regeneration-related candidates were found in the literature. From those, 57 were identified in the transcriptome of S. hystrix and 54 in the transcriptome of S. gracilis. Multiple gene isoforms were identified after BLAST searches in S. hystrix (e.g. for paics and slit) and S. gracilis (e.g. even-skipped, FGFR, gcs1a, glutamine synthetase, hedgehog, JNK, Msx, piwi1, Sfrp1/2/5 and Wnt) (Additional file 4), indicating that there might be multiple unique homologs of some of those genes in these species. Of the resulting homologs, paics in S.hystrix; and β-catenin, cycB3, glutamine synthetase, paics, and PL10 in S. gracilis were detected to have differential expression, being all of them up-regulated in AR (FDR < 0.001). If we consider the significance threshold to be FDR < 0.01, the number of candidate regeneration genes with differential expression increases to 14, including JNK and rup2, in S. hystrix; and brat, elav, FGFR, gcs1a, slit, Hox7, Lox2 in S. gracilis (Table 2; Additional file 4). Interestingly, all the Hox genes reported to be involved in the regeneration and development of other annelids [2, 3, 23, 46, 64] were found in the transcriptome of S. hystrix but none of them presented differential expression in any of the pairwise comparisons. In the case of S. gracilis, all Hox genes were found in the assembly, except Hox2 and Hox3. Interestingly, Hox7 and Lox2 were among differentially expressed genes in the comparisons AxP and AxN, being up-regulated in AR (FDR > 0.01) (Table 2, Additional file 4).

Morphological results of regeneration

The herein studied species exhibited a complete posterior regeneration, but anterior regeneration developed to different degrees. Sphaerosyllis hystrix regenerated an incomplete prostomium after 14 dpa and, even in advanced stages (around 50 dpa), they did not restore new segments. Thus, like in many other syllids [51, 65], the anterior regeneration of S. hystrix seems to be limited. Regarding Syllis gracilis, our own field observations and previous studies provide solid evidence that they are able to restore a complete anterior body with up to 18 segments and all digestive structures [37, 55, 66]. Moreover, specimens of S. gracilis from the same area showing advanced anterior regeneration have also been documented in detail by Parapar et al. [55]. Syllis gracilis was expected to regenerate the prostomium after 8 dpa, based on previous studies [37, 66]. However, we noticed only a blastema elongation during anterior regeneration after 8 dpa. This observed difference might be a result of the reduced temperature in our study (14 °C) compared to the one used by Boilly and Thibaut [37] (18 °C), as lower temperatures seem to delay the whole regeneration process in syllids [51].

Discussion

Posterior regeneration resembles regular posterior growth

In this study, we investigate regenerative processes of two species of syllids Sphaerosyllis hystrix (Exogoninae) and Syllis gracilis (Syllinae). Using comparative transcriptomics, we analyse three conditions: anterior regeneration, posterior regeneration, and regular growth. In both investigated species, our analyses revealed no differentially expressed (DE) genes between posterior regeneration (PR) and regular growth (NR); whereas the anterior regeneration (AR) significantly differed from those other conditions by having a high number of up-regulated genes. The absence of DE genes in the PxN comparisons of both species indicates that genes in PR and NR have similar expression levels. This result suggests that the genetic mechanisms behind the posterior regeneration and regular growth are similar in syllids with lifelong growth.

Previous studies provided similar results indicating that several genes expressed in the SAZ are also expressed in the blastema during posterior regeneration in annelids [3, 5, 21,22,23, 29, 46]. These two regions contain undifferentiated cells (blastema) and pluripotent cells (teloblasts in the SAZ), which require the activity of certain genes linked to stem-cellness, differentiation, reestablishment of antero-posterior and dorso-ventral axes, and elongation of the nervous system, among other processes [1, 3, 4, 7, 27, 46, 59]. Those processes are present during regeneration, growth, and homeostasis in planarians and acoels, and have been shown to be regulated by similar genetic pathways, e.g. Wnt and FGFRL signalling, TOR (target of rapamycin) control, and germline multipotency program activity [67,68,69,70,71]. Body growth and regeneration, therefore, are somehow similar programs in animals with high regenerative capacity.

Gene up-regulation in the anterior regeneration

The high number of up-regulated genes in AR may be due to the combination of two different factors: First, the presence of two proliferative zones acting at the same time (the SAZ and the blastema of anterior regeneration (see Fig. 1). Second, as suggested by a previous study in flatworms [72], some DE genes in AR might be involved in the reestablishment of anterior identity and the regeneration of anterior-specific structures, such as the brain. The presence of two proliferative zones in AR implies the existence of a higher number of cells simultaneously expressing certain genes involved in regeneration and growth. Therefore, the overall number of reads of transcripts related to these processes might be higher in AR than in PR or NR. The GO annotation showed that most of the DE genes were assigned to the cellular component category, thus suggesting functions related to cellular proliferation.

However, many of the up-regulated genes in AR of both species could not be identified and, hence, their functions remain unknown. This is probably due to the scarcity of annelid genomic data in the databases used for annotation. In addition, the generated assemblies had high duplication levels, which resulted in artificially large transcriptomes. These high duplication levels were probably a consequence of pooling different individuals for each sequencing library, which can introduce allelic variation, splicing differences, and assembly artefacts [73]. Nevertheless, our transcriptomic analyses relied on highly complete assemblies based on BUSCO criteria and the comparison with other annelid assemblies [57, 74, 75]; and, since we performed the differential expression analyses at the level of Trinity ‘genes’ (which sums up the expression values of all isoforms of a ‘gene’), this redundancy does not affect our results.

Regeneration-related genes

Using BLAST searches, we were able to identify regeneration-related candidates among the DE genes of our analysis. Those candidates have been associated to regenerative processes such as wound healing, blastema formation, stem cell regulation, cell proliferation, segmentation, and morphogenesis by several studies in annelids [1, 2, 12, 13, 17, 21, 23, 27, 32, 35, 45, 46, 48, 59,60,61,62,63]. Among the regeneration-related genes explored in this study, we found 12 DE genes in S. gracilis and 3 DE genes in S. hystrix; all of them were up-regulated in AR (FDR > 0.01, see Table 2).

Some of the DE genes are associated to cell proliferation and nervous system elongation, processes that support the two-proliferation-zones hypothesis suggested above; they are paics, JNK, PL10, slit, elav, glutamine synthetase (gs), and rup [29, 46, 76, 77]. Of these candidate genes, only paics (phosphoribosylaminoimidazole) was differentially expressed in both species (Table 2). paics is required for de novo biosynthesis of purines during cellular proliferation, and it has been reported to be highly expressed during regeneration in the clitellate Enchytraeus japonensis [12]. Similarly, JNK (up-regulated homolog in AR of S. hystrix) translates signals into apoptotic cell death and controls cell proliferation and differentiation to coordinate regeneration in planarians [78, 79]. Also, a homolog of PL10 was differentially expressed in S. gracilis results, up-regulated in AR (AxN comparison). PL10, like vasa, piwi, and nanos, is one of the germline multipotency program genes [80]. These genes are linked to somatic differentiation and stem-cellness, and can be considered conserved markers of the SAZ in annelids [14, 26, 34, 48, 62, 74, 80, 81].

The genes gs, elav and slit play an important role in nervous system regeneration and growth in annelids [12, 13, 46]. Homologs of those genes were found to be up-regulated in AR (AxP and AxN comparisons of S. gracilis). The enzyme glutamine synthetase (encoded by gs) plays a role in cell metabolism, ammonia detoxification, glutamate transmitter degradation in the nervous system, and was found to be expressed in early stages of regeneration in Enchytraeus japonensis [12, 13, 82]. The genes slit and elav, on the other hand, encode signalling and a RNA-binding proteins, respectively [83, 84]. They are expressed, for example, in the ventral midline cells (slit) and differentiating neurons (elav) during posterior regeneration in Platynereis dumerilii [46], and are evolutionary conserved across animal evolution [83, 84].

Interestingly, in both transcriptomes we identified homologs of Ej-rup 1–5 (E. japonensis regeneration up-regulated genes 1–5), regeneration-related genes previously reported for the clitellate E. japonensis (Table 2) [12]. However, only in S. hystrix one of them (Shy-rup2) was up-regulated in AR (AxN comparison). The function of this gene is not clear, but Ejrup2 was detected in epidermal cells of the blastema during anterior regeneration and might be a regeneration-specific gene [12].

Supporting the hypothesis of AR gene up-regulation being related to the reestablishment of anterior identity and structures, we detected some DE genes probably related with the specification of the antero-posterior axis, e.g. Hox genes and β-catenin [21,22,23, 64, 69, 85, 86]. In this study, Sgr-Hox7 and Sgr-Lox2 were up-regulated in AR (AxP and AxN comparisons; FDR > 0.01), which means that they are expressed in similar levels during tail regeneration and regular posterior growth, but are required during anterior regeneration of S. gracilis. In studies on the annelids P. dumerilii and A. virens (which cannot regenerate anteriorly), the expression of Hox7 and Lox2 was detected during larval development, growth and posterior regeneration [21,22,23, 64]. In addition, we detected up-regulation of a homolog of β-catenin in AR of S. gracilis (AxP, AxN comparisons; FDR < 0.001). In annelids, for example, β-catenin expression has been found in the blastema of P. leidyi during anterior and posterior regeneration, and in fission zones during asexual reproduction [17]. Additionally, Demilly et al. [59] suggested that the Wnt/β-catenin pathway is involved in neural cell proliferation/differentiation in P. dumerilii. In planarians, Wnt/β-catenin signalling is known to be required for the establishment of the antero-posterior axis during regeneration, promoting homeostasis and proper brain regeneration [67, 69, 85].

Conclusions

We studied the regenerative abilities of two syllid species. Both species can completely regenerate the posterior body after one to 2 weeks post amputation. However, only Syllis gracilis is able to regenerate the entire anterior body and, in contrast, Sphaerosyllis hystrix has a limited anterior regeneration. By using RNA-seq, we found that, for both species, individuals in posterior regeneration and intact individuals have comparable gene expression profiles. On the other hand, anterior regeneration shows a significant up-regulation of DE genes, including some candidate regeneration genes related to cellular proliferation (paics and JNK), nervous system development (gs, elav, slit), stem-cellness (PL10), and reestablishment of antero-posterior axis (Hox genes and β-catenin). Those results lead to two main conclusions, first that posterior regeneration is similar to the postembryonic process of growth in annelids, while anterior regeneration is markedly different from both; and second, that syllids regenerate using common genetic pathways (regeneration-related genes) already described for other annelids and other groups of invertebrates, supporting the importance of comparative studies to illuminate the evolution of regeneration in Metazoa.

Methods

Sampling

Animals were collected in intertidal rocky shores from Ferrol, Galicia, Spain (43°27′17.0″N; 8°18′39.8″W) during third quarter moon in April 2017. Specimens of Syllis gracilis and Sphaerosyllis hystrix were sorted for regeneration experiments. Syllis gracilis is a species complex with eight recognized lineages, from which a specimen from Galicia, Spain has been shown to belong to ‘lineage 8′ [87]. We confirmed that our specimens also belong to this lineage through phylogenetic analysis using sequences from the transcriptome assembly and those provided by a previous study [87] (see Additional files 5 and 6). For each molecular marker (COI, 16S, 18S, 28S), alignments were performed using MAFFT version 7 [88] (G-INS-I iterative method), and the datasets produced were concatenated using FASconCAT-G version 1.02 [89]. Then, a maximum likelihood analysis was conducted using RAxML, with 1000 bootstrap pseudoreplicates, and a partition scheme allowing for optimization of the three genes separately [90, 91].

Experimental procedures

Intact non-reproducing adults were selected for regeneration experiments. The animals were anesthetized in a 3.5% MgCl2 solution dissolved in seawater. Bisection was performed in the midbody of 48 individuals of S. hystrix (after chaetigers 13–18), and of 30 individuals of Syllis gracilis (after chaetigers 25–48) (Fig. 1). The animals were kept in one-litre aquariums with flowing filtered natural seawater at 14 °C for up to 14 dpa. Since there were only few specimens of S. gracilis, and some of them died during experimentation, the regeneration process could only be followed until 8 dpa. Anterior and posterior amputees were separated in different aquaria and kept in starvation during the experiment. Two amputees of each condition were fixed in 4% PFA every day for morphological observations (up to 14 dpa in S. hystrix and 8 dpa in S. gracilis, see Fig. 1). Optical microscopy images of fixed animals were taken to document morphological changes using an Olympus CX31 microscopy and a BQ Aquaris V. For transcriptome sequencing, amputees were fixed in RNA later (Ambion, Darmstadt, Germany) to represent four stages of regeneration: stage 1 (healing response/cicatrisation); stage 2 (early blastema development); stage 3 (late blastema development); stage 4 (patterning/cap regeneration), only observed during posterior regeneration (see Fig. 1). Two amputees of S. hystrix were fixed per stage: 1 dpa (stage 1), 5 dpa (stage 2), 9 dpa (stage 3) and 12 dpa (stage 3/ stage 4), summing up a total of 16 amputees; i.e. 8 for anterior regeneration (AR) and 8 for posterior regeneration (PR). Additionally, five intact individuals of S. hystrix were fixed as non-regenerating condition (NR). For S. gracilis, one amputee of each regenerative condition (AR, PR) was fixed in 1 dpa (stage 1), 3 dpa (stage 2), 6 dpa (stage 3) and 8 dpa (stage 3/stage 4), and two whole animals were fixed for NR (Fig. 1). All experimental procedures were conducted in April to May 2017 at the Marine Biological Station of A Graña (Ferrol, Galicia, Spain).

Illumina sequencing and de novo assembly

Three libraries were prepared for each of the two species: anterior regeneration (AR), posterior regeneration (PR), and non-regenerating adults (NR), i.e. intact individuals in regular posterior growth. RNA extraction was conducted by pooling together all individuals belonging to the same condition and species, i.e. amputees in different stages were pooled for each regenerative condition (AR, PR) and intact specimens were pooled together to prepare the non-regenerating condition (NR). Considering that the sequences represent samples of pooled individuals, there were no biological replicates from which to estimate inter-individual variability in gene expression. Although replicates provide a robust statistical support in differential expression analyses [92, 93], pooling samples for transcriptomic sequencing can be a useful strategy to establish a good framework of DE genes from small animals and neglected organisms [94]. RNA was isolated using NZYTech’s Total RNA isolation kit and the pure RNA was eluted in a final volume of 30 μL. Quality and quantity of RNA were checked in an Agilent 2100 Bioanalyzer using Agilent RNA 6000 kit. To prepare the libraries, we used Illumina’s TruSeq Stranded mRNA Library Prep Kit following manufacturer’s instructions. Fragment size distribution and concentration were checked in the Agilent Bioanalyser. Qubit dsDNA BR Assay kit (Thermo Fisher Scientific) was used to quantify the libraries, which were then sequenced in an Illumina HiSeq 4000 PE100 lane. All procedures of RNA isolation, library construction, and sequencing were performed by AllGenetics & Biology SL (A Coruña, Spain).

We used FastQC v0.11 (http://bioinformatics.babraham.ac.uk/projects/fastqc/) to assess sequence quality, and Trimmomatic v0.33 [95] to trim the raw sequences based on quality results with options HEADCROP:10 LEADING:20 SLIDINGWINDOW:5:20 MINLEN:70. De novo transcriptome assembly was perform using Trinity v2.3.2 [96, 97] and transcripts with ≥200 bases were kept. Assembly statistics were obtained using the TrinityStats tool of Trinity, and BUSCO [57] was used to estimate transcriptome completeness.

Functional annotation and gene ontology

Transcripts were annotated following the Trinotate pipeline (https://github.com/Trinotate/Trinotate.github.io/wiki). For that, TransDecoder v3.0.1 (https://transdecoder.github.io/) was used to predict protein sequences from the assembled transcripts. Then, both the assembled transcripts and the predicted proteins were used for functional annotation, which was performed using BLAST v2.5.0 [98], HMMER v3.1b2 (http://hmmer.org), signalp v4.1 [99], RNAmmer v1.2 [100], and tmHMM v2.0c [101] to find known sequences and domains. Gene ontology (GO) terms analysis was used to classify the functions of the predicted genes. Trinotate v3.0.1 (http://trinotate.github.io) and WEGO Web Server [102] were used to summarise the results of gene ontology (GO).

Differential expression analyses

Using the Differential Expression module of Trinity v.2.3.2.

(https://github.com/trinityrnaseq/trinityrnaseq/wiki/Trinity-Differential-Expression), we performed pairwise comparisons at the Trinity ‘gene’ level between each condition of our experiment: anterior regeneration against posterior regeneration (AxP), anterior regeneration against non-regenerating condition (AxN), and posterior regeneration against non-regenerating (PxN). We ran RSEM [103] to estimate per-condition transcript abundance, and edgeR [104, 105] to perform the differential expression analyses. At this point, we tried several values for the dispersion parameter of edgeR, and concluded that 0.5 was the best fitting value for our data.

After that, we used the analyze_diff_expr.pl script of Trinity to create differential expression subsets for each pairwise comparison, and included the examine_GO_enrichment flag that, by combining the results of differential expression and the functional annotation, can inform which Gene Ontology categories are enriched or depleted in each experimental condition for each pairwise comparison. We used GOplot to illustrate the results of gene ontology (GO) enrichment analyses [58]. The outputs files of the differential expression analyses and the functional enrichment analyses are available under https://github.com/rannypribeiro/Regeneration_transcriptomics. Overall results are summarised in Additional files 2 and 3.

Identification of candidate regeneration genes

BLASTn and BLASTp searches were used to detect homologs of genes related to animal regeneration that had been previously reported in the literature. The top hits in the BLAST results were analysed via a reciprocal BLASTn against the nr/nt database (NCBI) to verify the putative identity of candidate regeneration genes in the transcriptomes of S. hystrix and S. gracilis.

Availability of data and materials

The raw reads are available at the NCBI Sequence Read Archive (SRA) for Sphaerosyllis hystrix (BioProject ID PRJNA517681, SRX5314036–38) and Syllis gracilis (BioProject ID PRJNA517687, SRX5314271–73). Assemblies, transdecoder predicted proteins, and differential gene expression results generated in this study can be accessed in https://github.com/rannypribeiro/Regeneration_transcriptomics.

Abbreviations

AR:

Anterior regeneration, as experimental condition

AxN:

Anterior regeneration versus non-regenerating

AxP:

Anterior regeneration versus posterior regeneration

BP:

Biological process

BUSCO:

Benchmarking Universal Single-Copy Orthologs

CC:

Cellular component

DE genes:

Differentially expressed genes

dpa:

Day(s) post amputation

Ej-rup:

Enchytraueus japonensis regeneration up-regulated gene

FGFR:

Fibroblast growth factor receptor

Gcs1a:

Glucosidase 1

GO:

Gene ontology

gs:

Glutamine synthetase

GSK3β:

Glycogen synthase kinase-3 β

JNK:

c-Jun N-terminal kinase

MF:

Molecular function

NR:

Non-regenerating, as experimental condition

paics:

Multifunctional protein ADE2

PR:

Posterior regeneration, as experimental condition

PxN:

Posterior regeneration versus non-regenerating

References

  1. Bely AE, Wray GA. Evolution of regeneration and fission in annelids: insights from engrailed- and orthodenticle-class gene expression. Development. 2001;128:2781–91.

    CAS  PubMed  Google Scholar 

  2. Pfeifer K, Dorresteijn AWC, Fröbius AC. Activation of Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii. Dev Genes Evol. 2012;222:165–79.

    Article  CAS  PubMed  Google Scholar 

  3. Gazave E, Béhague J, Laplane L, Guillou A, Préau L, Demilly A, et al. Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev Biol. 2013;382:246–67.

    Article  CAS  PubMed  Google Scholar 

  4. Balavoine G. Segment formation in annelids: patterns, processes and evolution. Int J Dev Biol. 2014;58:469–83.

    Article  PubMed  Google Scholar 

  5. Bely AE, Zattara EE, Sikes JM. Regeneration in spiralians: evolutionary patterns and developmental processes. Int J Dev Biol. 2014;58:623–34.

    Article  PubMed  Google Scholar 

  6. Zattara EE, Bely AE. Phylogenetic distribution of regeneration and asexual reproduction in Annelida: regeneration is ancestral and fission evolves in regenerative clades. Invertebr Biol. 2016;135:400–14.

    Article  Google Scholar 

  7. Özpolat BD, Bely AE. Developmental and molecular biology of annelid regeneration: a comparative review of recent studies. Curr Opin Genet Dev. 2016;40:144–53.

    Article  PubMed  CAS  Google Scholar 

  8. Ribeiro RP, Bleidorn C, Aguado MT. Regeneration mechanisms in Syllidae (Annelida). Regeneration. 2018;5:26–42.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Bely AE. Early events in annelid regeneration: a cellular perspective. Integr Comp Biol. 2014;54:688–99.

    Article  PubMed  Google Scholar 

  10. Boilly B, Boilly-Marer Y, Bely AE. Regulation of dorso-ventral polarity by the nerve cord during annelid regeneration: a review of experimental evidence. Regeneration. 2017;4:54–68.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Nakamura Y. A new fragmenting enchytraeid species, Enchytraeus japonensis from a cropped Kuroboku soil in Fukushima, northern Japan (enchytraeids in Japan 5). Edaphologia. 1993;50:37–9.

    Google Scholar 

  12. Myohara M, Niva CC, Lee JM. Molecular approach to annelid regeneration: cDNA subtraction cloning reveals various novel genes that are upregulated during the large-scale regeneration of the oligochaete, Enchytraeus japonensis. Dev Dyn. 2006;235:2051–70.

    Article  CAS  PubMed  Google Scholar 

  13. Niva CC, Lee JM, Myohara M. Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis. Dev Genes Evol. 2008;218:39–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tadokoro R, Sugio M, Kutsuna J, Tochinai S, Takahashi Y. Early segregation of germ and somatic lineages during gonadal regeneration in the annelid Enchytraeus japonensis. Curr Biol. 2006;16:1012–7.

    Article  CAS  PubMed  Google Scholar 

  15. Takeo M, Yoshida-Noro C, Tochinai S. Morphallactic regeneration as revealed by region-specific gene expression in the digestive tract of Enchytraeus japonensis (Oligochaeta, Annelida). Dev Dyn. 2008;237:1284–94.

    Article  CAS  PubMed  Google Scholar 

  16. Smith F. Notes on species of north American Oligochaeta. II. Illinois Nat Hist Surv Bull. 1896;4:396–413.

    Google Scholar 

  17. Nyberg KG, Conte MA, Kostyun JL, Forde A, Bely AE. Transcriptome characterization via 454 pyrosequencing of the annelid Pristina leidyi, an emerging model for studying the evolution of regeneration. BMC Genomics. 2012;13:287.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zattara EE, Bely AE. Evolution of a novel developmental trajectory: fission is distinct from regeneration in the annelid Pristina leidyi. Evol Dev. 2011;13:80–95.

    Article  PubMed  Google Scholar 

  19. Zattara EE, Bely AE. Investment choices in post-embryonic development: quantifying interactions among growth, regeneration, and asexual reproduction in the annelid Pristina leidyi. J Exp Zool B Mol Dev Evol. 2013;320:471–88.

    Article  PubMed  Google Scholar 

  20. Zattara EE, Turlington KW, Bely AE. Long-term time-lapse live imaging reveals extensive cell migration during annelid regeneration. BMC Dev Biol. 2016;16:6.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Kulakova M, Bakalenko N, Novikova E, Cook CE, Eliseeva E, Steinmetz PRH, et al. Hox gene expression in larval development of the polychaetes Nereis virens and Platynereis dumerilii (Annelida, Lophotrochozoa). Dev Genes Evol. 2007;217:39–54.

    Article  CAS  PubMed  Google Scholar 

  22. Bakalenko NI, Novikova EL, Nesterenko AY, Kulakova MA. Hox gene expression during postlarval development of the polychaete Alitta virens. Evodevo. 2013;4:13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Novikova EL, Bakalenko NI, Nesterenko AY, Kulakova MA. Expression of Hox genes during regeneration of nereid polychaete Alitta virens (Annelida, Lophotrochozoa). Evodevo. 2013;4:21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sars M. Beskrivelser og Iagttagelser over nogle moerkelige eller nye i Havet ved den Bergenske Kyst levende Dyr af Polypernes, Acalephernes, Radiaternes, Annelidernes og Molluskernes classer, med en kort Oversigt over de hidtil af Forfatteren sammesteds fundne Ar. Thorstein Hallagers Forlag hos Chr. Dahl, R.S: Bergen; 1835.

    Google Scholar 

  25. Blake JA, Grassle JP, Eckelbarger KJ. Capitella teleta, a new species designation for the opportunistic and experimental Capitella sp. I, with a review of the literature for confirmed records. Zoosymposia. 2009;2:25–53.

    Article  Google Scholar 

  26. Dill KK, Seaver EC. Vasa and nanos are coexpressed in somatic and germ line tissue from early embryonic cleavage stages through adulthood in the polychaete Capitella sp. I Dev Genes Evol. 2008;218:453–63.

    Article  PubMed  Google Scholar 

  27. de Jong DM, Seaver EC. A stable thoracic Hox code and epimorphosis characterize posterior regeneration in Capitella teleta. PLoS One. 2016;11:e0149724.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Neal S, de Jong M, Seaver EC. CRISPR/CAS9 mutagenesis of a single r-opsin gene blocks phototaxis in a marine larva. Proc R Soc B Biol Sci. 2019;286:20182491.

    Article  CAS  Google Scholar 

  29. de Jong DM, Seaver EC. Investigation into the cellular origins of posterior regeneration in the annelid Capitella teleta. Regeneration. 2018;5:61–77.

    Article  PubMed  CAS  Google Scholar 

  30. Audouin JV, Milne EH. Classification des Annélides et description de celles qui habitent les côtes de la France. Ann des Sci Nat Paris. 1833;1:195–269.

    Google Scholar 

  31. Prud’homme B, Rosa R, Arendt D, Julien JF, Pajaziti R, Dorresteijn AWC, et al. Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation. Curr Biol. 2003;13:1876–81.

    Article  PubMed  CAS  Google Scholar 

  32. de Rosa R, Prud’homme B, Balavoine G. Caudal and even-skipped in the annelid Platynereis dumerilii and the ancestry of posterior growth. Evol Dev. 2005;7:574–87.

  33. Kerner P, Zelada González F, Le Gouar M, Ledent V, Arendt D, Vervoort M. The expression of a hunchback ortholog in the polychaete annelid Platynereis dumerilii suggests an ancestral role in mesoderm development and neurogenesis. Dev Genes Evol. 2006;216:821–8.

    Article  PubMed  Google Scholar 

  34. Rebscher N, Zelada-González F, Banisch TU, Raible F, Arendt D. Vasa unveils a common origin of germ cells and of somatic stem cells from the posterior growth zone in the polychaete Platynereis dumerilii. Dev Biol. 2007;306:599–611.

    Article  CAS  PubMed  Google Scholar 

  35. Gazave E, Lemaître QIB, Balavoine G. The notch pathway in the annelid Platynereis: insights into chaetogenesis and neurogenesis processes. Open Biol. 2017;7:160242.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Allen EJ. Regeneration and reproduction of the syllid Procerastea. Philos Trans R Soc B Biol Sci. 1923;211:131–77.

    Article  Google Scholar 

  37. Boilly B, Thibaut N. Etude histologique de la regeneration pharyngienne de Syllis gracilis Grube (Annelide polychete). Can J Zool. 1974;52:169–77.

    Article  Google Scholar 

  38. Paulus T, Müller MC. Cell proliferation dynamics and morphological differentiation during regeneration in Dorvillea bermudensis (Polychaeta, Dorvilleidae). J Morphol. 2006;267:393–403.

    Article  PubMed  Google Scholar 

  39. Weidhase M, Bleidorn C, Helm C. Structure and anterior regeneration of musculature and nervous system in Cirratulus cf. cirratus (Cirratulidae, Annelida). J Morphol. 2014;275:1418–30.

    Article  PubMed  Google Scholar 

  40. Weidhase M, Helm C, Bleidorn C. Morphological investigations of posttraumatic regeneration in Timarete cf. punctata (Annelida: Cirratulidae). Zool Lett. 2015;1:20.

    Article  Google Scholar 

  41. Weidhase M, Beckers P, Bleidorn C, Aguado MT. On the role of the proventricle region in reproduction and regeneration in Typosyllis antoni (Annelida: Syllidae). BMC Evol Biol. 2016;16:196.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Weidhase M, Bleidorn C, Beckers P, Helm C. Myoanatomy and anterior muscle regeneration of the fireworm Eurythoe cf. complanata (Annelida: Amphinomidae). J Morphol. 2016;277:306–15.

    Article  PubMed  Google Scholar 

  43. Lindsay SM, Jackson JL, Forest DL. Morphology of anterior regeneration in two spionid polychaete species: implications for feeding efficiency. Invertebr Biol. 2008;127:65–79.

    Article  Google Scholar 

  44. Coulon J, Diano M, Arsanto J-P, Thouveny Y. Remodeling processes during anterior regeneration of Owenia fusiformis (Polychaeta, Annelidae): a morphological and immunocytochemical survey. Can J Zool. 1989;67:994–1005.

    Article  Google Scholar 

  45. Kulakova MA, Cook CE, Andreeva TF. ParaHox gene expression in larval and postlarval development of the polychaete Nereis virens (Annelida, Lophotrochozoa). BMC Dev Biol. 2008;8:1–18.

    Article  CAS  Google Scholar 

  46. Planques A, Malem J, Parapar J, Vervoort M, Gazave E. Morphological, cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii. Dev Biol. 2019;445:189–210.

    Article  CAS  PubMed  Google Scholar 

  47. Özpolat BD, Sloane ES, Zattara EE, Bely AE. Plasticity and regeneration of gonads in the annelid Pristina leidyi. Evodevo. 2016;7:22.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Özpolat BD, Bely AE. Gonad establishment during asexual reproduction in the annelid Pristina leidyi. Dev Biol. 2015;405:123–36.

    Article  PubMed  CAS  Google Scholar 

  49. Okada YK. Regeneration and fragmentation in the syllidian polychaetes. Wilhelm Roux Arch Entwickl Mech. 1929;115:542–600.

    Article  Google Scholar 

  50. Malmgren AJ. Annulata Polychaeta Spetsbergiæ, Grœnlandiæ, Islandiæ et Scandinaviæ. Helsink: Ex Officina Frenckelliana; 1867.

  51. Boilly B. Contribution a l’étude de la régénération antérieure chez Eusyllis blomstrandi Malmgren (Annélide Polychète). Bull la Société Zool Fr. 1961;86:216–29.

  52. Grube AE. Actinien, Echinodermen und Würmer des Adriatischen- und Mittelmeers, nach eigenen Sammlungen beschrieben von Adolph Eduard Grube. Königsberg: Verlag von J. H. Bon; 1840.

    Book  Google Scholar 

  53. Mesnil MMF. Sur un cas de régénération de la partie antérieure du corps et de la trompe chez un syllidien. C R Seances Soc Biol Fil. 1901;53:268–70.

    Google Scholar 

  54. Mesnil MMF, Caullery M. Sur un processus normal de fragmentation, suivie de régénération, chez un Annélide polychéte Syllis gracilis Gr. Comptes rendus l’Académie des Sci. 1919;169:926–9.

    Google Scholar 

  55. Parapar J, Caramelo C, Candás M, Cunha-Veira X, Moreira J. An integrative approach to the anatomy of Syllis gracilis Grube, 1840 (Annelida) using micro-computed X-ray tomography. PeerJ. 2019;7:e7251.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Claparède ARÉ. Beobachtungen über Anatomie und Entwicklungsgeschichte wirbelloser Thiere: an der Küste von Normandie angestellt. Leipzig: W. Engelmann; 1863.

    Book  Google Scholar 

  57. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31:3210–2.

    Article  CAS  PubMed  Google Scholar 

  58. Walter W, Sanchez-Cabo F, Ricote M. GOplot: an R package for visually combining expression data with functional analysis. Bioinformatics. 2015;31:2912–4.

    Article  CAS  PubMed  Google Scholar 

  59. Demilly A, Steinmetz P, Gazave E, Marchand L, Vervoort M. Involvement of the Wnt/β-catenin pathway in neurectoderm architecture in Platynereis dumerilii. Nat Commun. 2013;4:1911–5.

    Article  CAS  Google Scholar 

  60. Cho S-J, Lee MS, Tak ES, Lee E, Koh KS, Ahn CH, et al. Gene expression profile in the anterior regeneration of the earthworm using expressed sequence tags. Biosci Biotechnol Biochem. 2009;73:29–34.

    Article  CAS  PubMed  Google Scholar 

  61. Kitakoshi T, Shimizu T. An oligochaete homologue of the Brachyury gene is expressed transiently in the third quartette of micromeres. Gene Expr Patterns. 2010;10:306–13.

    Article  CAS  PubMed  Google Scholar 

  62. Kozin VV, Kostyuchenko RP. Vasa, PL10, and piwi gene expression during caudal regeneration of the polychaete annelid Alitta virens. Dev Genes Evol. 2015;225:129–38.

    Article  CAS  PubMed  Google Scholar 

  63. Takeo M, Chou WC, Sun Q, Lee W, Rabbani P, Loomis C, et al. Wnt activation in nail epithelium couples nail growth to digit regeneration. Nature. 2013;499:228–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Bakalenko NI, Poznyak AV, Novikova EL, Kulakova MA. Effect of retinoids on Post2 Нох gene expression in nereid polychaetes. Russ J Dev Biol. 2017;48:211–8.

    Article  CAS  Google Scholar 

  65. Malaquin A. Recherches sur les syllidiens: morphologie, anatomie, reproduction, développement. Lille: L. Danel; 1893.

    Google Scholar 

  66. Mesnil F, Caullery M. Sur un processus normal de fragmentation, suivie de regeneration, chez un annelide polychete, Syllis gracilis Gr. Comptes Rendus l’Academie des Sci Paris. 1919;169:926–9.

    Google Scholar 

  67. Sureda-Gómez M, Martín-Durán JM, Adell T. Localization of planarian β-CATENIN-1 reveals multiple roles during anterior-posterior regeneration and organogenesis. Development. 2016;143:4149–60.

    Article  PubMed  CAS  Google Scholar 

  68. Srivastava M, Mazza-Curll KL, Van Wolfswinkel JC, Reddien PW. Whole-body acoel regeneration is controlled by Wnt and bmp-Admp signaling. Curr Biol. 2014;24:1107–13.

    Article  CAS  PubMed  Google Scholar 

  69. Gurley KA, Rink JC, Alvarado AS. β-catenin defines head versus tail identity during planarian regeneration and homeostasis. Science. 2008;319:323–7.

    Article  CAS  PubMed  Google Scholar 

  70. Hill EM, Petersen CP. Positional information specifies the site of organ regeneration and not tissue maintenance in planarians. eLife. 2018;7:e33680.

    Article  PubMed  PubMed Central  Google Scholar 

  71. Peiris TH, Weckerle F, Ozamoto E, Ramirez D, Davidian D, García-Ojeda ME, et al. TOR signaling regulates planarian stem cells and controls localized and organismal growth. J Cell Sci. 2012;125:1657–65.

    Article  CAS  PubMed  Google Scholar 

  72. Kao D, Felix D, Aboobaker A. The planarian regeneration transcriptome reveals a shared but temporally shifted regulatory program between opposing head and tail scenarios. BMC Genomics. 2013;14:797.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. Kenny NJ, de Goeij JM, de Bakker DM, Whalen CG, Berezikov E, Riesgo A. Towards the identification of ancestrally shared regenerative mechanisms across the Metazoa: a Transcriptomic case study in the Demosponge Halisarca caerulea. Mar Genomics. 2018;37:135–47.

    Article  PubMed  Google Scholar 

  74. Ponz-Segrelles G, Bleidorn C, Aguado MT. Expression of vasa, piwi, and nanos during gametogenesis in Typosyllis antoni (Annelida, Syllidae). Evol Dev. 2018;20:132–45.

    Article  PubMed  Google Scholar 

  75. Heikkinen LK, Kesäniemi JE, Knott KE. De novo transcriptome assembly and developmental mode specific gene expression of Pygospio elegans. Evol Dev. 2017;19:205–17.

    Article  CAS  PubMed  Google Scholar 

  76. Boilly B. Étude ultrastructurale de l’évolution des tissus impliqués dans la régénération céphalique et caudale de Syllis amica Q. (Annélide Polychète). I. La dédifférenciation. J Microsc. 1968;7:865–76.

    Google Scholar 

  77. Boilly B. Étude ultrastructurale de l’évolution des tissus impliqués dans la régénération céphalique et caudale de Syllis amica Q. (Annélide Polychète). II. L’activation et la différenciation. J Microsc. 1968;7:877–95.

    Google Scholar 

  78. Tasaki J, Shibata N, Sakurai T, Agata K, Umesono Y. Role of c-Jun N-terminal kinase activation in blastema formation during planarian regeneration. Develop Growth Differ. 2011;53:389–400.

    Article  CAS  Google Scholar 

  79. Almuedo-Castillo M, Crespo X, Seebeck F, Bartscherer K, Salò E, Adell T. JNK controls the onset of mitosis in planarian stem cells and triggers apoptotic cell death required for regeneration and remodeling. PLoS Genet. 2014;10:e1004400.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  80. Juliano CE, Swartz SZ, Wessel GM. A conserved germline multipotency program. Development. 2010;137:4113–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Giani VC, Yamaguchi E, Boyle MJ, Seaver EC. Somatic and germline expression of piwi during development and regeneration in the marine polychaete annelid Capitella teleta. Evodevo. 2011;2:10.

    Article  PubMed  PubMed Central  Google Scholar 

  82. Papageorgiou IE, Valous NA, Lahrmann B, Janova H, Klaft ZJ, Koch A, et al. Astrocytic glutamine synthetase is expressed in the neuronal somatic layers and down-regulated proportionally to neuronal loss in the human epileptic hippocampus. Glia. 2018;66:920–33.

    Article  PubMed  Google Scholar 

  83. Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Goodman CS, et al. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell. 1999;96:795–806.

    Article  CAS  PubMed  Google Scholar 

  84. Koushika SP, Lisbin MJ, White K. ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform. Curr Biol. 1996;6:1634–41.

    Article  CAS  PubMed  Google Scholar 

  85. Petersen CP, Reddien PW. Smed-β-catenin-1 is required for anteroposterior blastema polarity in planarian regeneration. Science. 2008;319:327–30.

    Article  CAS  PubMed  Google Scholar 

  86. Dubuc TQ, Stephenson TB, Rock AQ, Martindale MQ. Hox and Wnt pattern the primary body axis of an anthozoan cnidarian before gastrulation. Nat Commun. 2018;9:1–12.

    Article  CAS  Google Scholar 

  87. Álvarez-Campos P, Giribet G, Riesgo A. The Syllis gracilis species complex: a molecular approach to a difficult taxonomic problem (Annelida, Syllidae). Mol Phylogenet Evol. 2017;109:138–50.

    Article  PubMed  Google Scholar 

  88. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Kück P, Meusemann K. FASconCAT : convenient handling of data matrices. Mol Phylogenet Evol. 2010;56:1115–8.

    Article  PubMed  CAS  Google Scholar 

  90. Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006;22:2688–90.

    Article  CAS  PubMed  Google Scholar 

  91. Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst Biol. 2008;57:758–71.

    Article  PubMed  Google Scholar 

  92. Liu Y, Zhou J, White KP. RNA-seq differential expression studies: more sequence or more replication? Bioinformatics. 2014;30:301–4.

    Article  CAS  PubMed  Google Scholar 

  93. Schurch NJ, Schofield P, Gierliński M, Cole C, Sherstnev A, Singh V, et al. How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use? RNA. 2016;22:839–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. von Reumont B. Studying smaller and neglected organisms in modern evolutionary venomics implementing RNA-seq (Transcriptomics)—a critical guide. Toxins. 2018;10:292.

    Article  CAS  Google Scholar 

  95. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Philip D, Bowden J, et al. De novo transcript sequence reconstruction from RNA-seq using the trinity platform for reference generation and analysis. Nat Protoc. 2013;8:1–43.

    Article  CAS  Google Scholar 

  98. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.

    Article  CAS  PubMed  Google Scholar 

  99. Petersen TN, Brunak S, Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8:785–6.

    Article  CAS  PubMed  Google Scholar 

  100. Lagesen K, Hallin P, Rødland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35:3100–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305:567–80.

    Article  CAS  PubMed  Google Scholar 

  102. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, et al. WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 2006;34:293–7.

    Article  Google Scholar 

  103. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. McCarthy DJ, Chen Y, Smyth GK. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012;40:4288–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26:139–40.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Guillermo Díaz-Agras, Marcos Abad, María Candas, and Xela Cunha-Veira for helping in sampling and for providing facilities to perform the experiments at the Marine Biological Station of A Graña (Ferrol, Spain); Alexa Bely and one anonymous reviewer for their valuable suggestions to improve this manuscript.

Funding

This research was supported by MINECO/FEDER, UE funds (Grant: CGL2015–63593-P, “Macroevolutionary transitions in Syllidae” project, PI: MTA). RPR is supported by the program “Contratos predoctorales para Formación de Personal Investigador, FPI-UAM,” Universidad Autónoma de Madrid. GPS is supported by the “Contratos Predoctorales para la Formación de Doctores 2016” program of the MINECO, Spain (code: BES-2016-076419), cofinanced by the European Social Found. We acknowledge support by German Research Foundation and the Open Access Publication Funds of the Georg-August-Universität Göttingen.

Author information

Authors and Affiliations

  1. Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049, Madrid, Spain

    Rannyele Passos Ribeiro, Guillermo Ponz-Segrelles & Maria Teresa Aguado

  2. Animal Evolution & Biodiversity, Georg-August-Universität Göttingen, 37073, Göttingen, Germany

    Christoph Bleidorn & Maria Teresa Aguado

  3. German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, 04103, Leipzig, Germany

    Christoph Bleidorn

  4. Centro de Investigación en Biodiversidad y Cambio Global (CIBC-UAM), Universidad Autónoma de Madrid, Madrid, 28049, España

    Maria Teresa Aguado

Authors
  1. Rannyele Passos Ribeiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guillermo Ponz-Segrelles
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christoph Bleidorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Teresa Aguado
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RPR, CB, and MTA conceived and designed experiments. RPR, GPS and CB collected the specimens. RPR conducted regeneration experiments and transcriptome analyses. GPS performed the transcriptome assembly and analyses. RPR wrote the first draft of the manuscript. MTA supervised the whole study. All authors have written, reviewed, and approved the final manuscript.

Corresponding authors

Correspondence to Rannyele Passos Ribeiro or Maria Teresa Aguado.

Ethics declarations

Ethics approval and consent to participate

Sampling was conducted under the supervision of the staff at Estación de Bioloxía Mariña da Graña, USC, and with permission and in agreement with the requirements by the Galician regional government (Consellería de Medio Ambiente, Territorio e Vivenda; Xunta de Galicia).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1.

Results of functional annotation of the transcriptomes of Sphaerosyllis hystrix and Syllis gracilis. a Results against all metazoan database. b Results within Annelida. c Gene ontology distribution of the annotated genes grouped in the three main functional categories (cellular component, molecular function, and biological process). GO terms with percentage of genes > 4% were plotted.

Additional file 2.

Differential gene expression and functional enrichment results for Sphaerosyllis hystrix.

Additional file 3.

Differential gene expression and functional enrichment results for Syllis gracilis.

Additional file 4.

BLAST results of candidate genes including differential gene expression results. ns: not significant.

Additional file 5.

Syllis gracilis phylogeny including sequences identified from the transcriptome assembly of this study. The lineages are in agreement with previous study [87].

Additional file 6.

GenBank accession numbers of sequences used for phylogenetic reconstruction. Codes used for S. gracilis sequences by Alvarez-Campos et al. [87] were maintained here.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ribeiro, R.P., Ponz-Segrelles, G., Bleidorn, C. et al. Comparative transcriptomics in Syllidae (Annelida) indicates that posterior regeneration and regular growth are comparable, while anterior regeneration is a distinct process. BMC Genomics 20, 855 (2019). https://doi.org/10.1186/s12864-019-6223-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12864-019-6223-y

Keywords

BMC Genomics

ISSN: 1471-2164

Contact us