Morphological and molecular features of early regeneration in the marine annelid Ophryotrocha xiamen

Regeneration capability varies in the phylum Annelida making them an excellent group to investigate the differences between closely related organisms. Several studies have described the process of regeneration, while the underlying molecular mechanism remains unclear, especially during the early stage (wound healing and blastema formation). In this study, the newly identified Ophryotrocha xiamen was used to explore the early regeneration. The detailed morphological and molecular analyses positioned O. xiamen within ‘labronica’ clade. We analyzed the morphological changes during regeneration process (0–3 days post amputation) and molecular changes during the early regeneration stage (1 day post amputation). Wound healing was achieved within one day and a blastema formed one day later. A total of 243 DEGs were mainly involved in metabolism and signal transduction. Currently known regeneration-related genes were identified in O. xiamen which could help with exploring the functions of genes involved in regeneration processes. According to their conserved motif, we identified 8 different Hox gene fragments and Hox5 and Lox2 were found to be absent in early regeneration and during regular growth. Our data can promote further use of O. xiamen which can be used as an experimental model for resolving crucial problems of developmental biology in marine invertebrates.

Annelids are an excellent group to investigate regeneration abilities, as they exhibit various regeneration capacities [1][2][3][4] . The anterior and posterior regeneration are proved to be ancestral for Annelida 5 . Numerous species, including Enchytraeus japonensis, Pristina leidyi and Cirratulus cirratus, and Eurythoe complanata, have been reported to regenerate both anterior and posterior segments to form an entire individual 3,6-9 . Posterior regeneration is more widespread than anterior regeneration. Others, such as Alitta virens and Capitella teleta and species in Ophryotrocha, can regenerate segments posteriorly but not anteriorly 4,10,11 . Although several studies describing the process of regeneration in both anterior and posterior regeneration annelids are available, investigating the molecular basis of species with the posterior regeneration ability only can help to distinguish the differences between anterior and posterior regenerative mechanisms in annelids.
For annelids, epimorphosis and morphallaxis are two different processes leading to regeneration. Early regeneration, mainly including wound healing and formation of blastema, involves muscular contraction and tissue autolysis at the site of the wound in annelids 12 . Several types of migrating cells are involved in early regeneration. Some of them are thought to phagocytize damaged tissues, while others help to regenerate new tissues. Previous studies have mainly focused on morphological changes and cell division, while comparative transcriptome analysis can help in understanding the regeneration process [6][7][8]10 . Numbers of genes, including Hox genes and several genes of the germline multipotency program (GMP), are demonstrated to be involved in early regeneration in annelids 10,[13][14][15][16][17] .
Annelids of the genus Ophryotrocha Claparède & Mecznikow, 1869 (Errantia: Dorvilleidae) are distributed in a wide range of habitats from shallow water to the deep sea [18][19][20] . To date, approximately 86 species in this genus have been described according to the GBIF data but records from the coastal zones of China are scarce 21 . Molecular characters support four clades within Ophryotrocha, the 'labronica' , 'hartmanni' , 'lobifera' , and an undefined clade, identified with 16S, cytochrome c oxidase I (COI), and histone 3 (H3) sequences 22,23 .
Due to their capability of laboratory maintenance, high fecundity, short generation time, and rapid individual growth rate, some species of Ophryotrocha have been used as model organisms of marine invertebrates in the fields of genetics, reproduction, development, and regeneration 19,24 . Morphological descriptions illustrate that RNA extraction, library construction and Illumina sequencing. For RNA extraction, about 60 individuals with 12-15 segments were amputated in the midbody (6-8 post-pharynx segments; Fig. 1). Both anterior and posterior parts were merged and collected 1 day after amputation. The control group (60 individuals) without amputation was cultured under the same conditions as the regeneration group. Total RNA was extracted using MiniBEST Universal RNA Extraction Kit (TaKaRa, 9767) according to the manufacturer's instructions. The integrity and size distribution of two RNA samples were verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Total RNA with RNA integrity numbers (RINs) ≥ 7.5 was used for cDNA library preparation. The mRNA-seq libraries were performed at Beijing Berry Genomics Co., Ltd. (Beijing, China). The mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads. The cleaved RNA fragments were reverse transcribed into first-strand cDNA using random primers and then synthesized into double-stranded cDNA. From the cDNA, paired-end libraries were synthesized for both control and 1 day post amputation. Short fragments were purified with the QIA quick PCR Purification Kit (Qiagen), which was also used for continued end repair and ' A' base addition. These fragments were then ligated to adapters and purified through gel separation. Finally, the adaptor-ligated libraries were amplified by PCR for sequencing. Illumina sequencing was performed using the NovaSeq 6000 platform, and 150 bp paired-end reads were generated. Raw sequences were deposited in the NCBI Short Read Archive (SRA) database (http:// www. ncbi. nlm. nih. gov/ Traces/ sra/) under accession numbers: SRR12074689 and SRR12074688. www.nature.com/scientificreports/ Assembly sequencing and functional annotation. After removing the adaptor sequences, ambiguous 'N' nucleotides (with the ratio of 'N' greater than 10%) and low-quality sequences (with a quality score of less than 5) using Trimmomatic, the remaining clean reads were assembled using Trinity for transcriptome assembly without a reference genome. The longest transcript of each single gene was selected as a unigene. For annotation analysis, unigenes were BLASTX-searched against seven databases, including the National Center for Biotechnology Information (NCBI) nonredundant protein sequence (Nr) database, nonredundant nucleotide   28 , and the Swiss-Prot, using a cutoff E-value of 10-5. Differentially expressed genes (DEGs) between control and regeneration groups (1 day post amputation) were identified with DEGseq analysis from the adjusted read count data 29 . The Benjamini & Hochberg method was used to adjust the P-values. Significantly differential expressed genes were determined by setting the threshold of corrected P-value of 0.05 and log2 (Fold change) of 1. Unigenes were annotated based on the BLASTX results, and the best alignments were used for downstream analyses. GO and KEGG databases were both used to predict the functions of unigenes.
Analysis of the Hox genes during the early regeneration. Hox genes have been implicated in wound healing and the dedifferentiation process during the early stage of regeneration 10,[13][14][15] . We performed a local BlastP search against the predicted amino acid database with an E-value cutoff at 1e−3 by using the highly conserved homeodomain (60 amino acid residues) and ten flanking positions of the Hox protein. We used published sequences that were only full or near full length accessible from the GenBank database at NCBI (Supplementary Table S2). As mentioned above (Phylogenetic analysis section), the alignments of protein sequences were generated using ClustalW with the BLOSUM matrix, gap-opening penalty of 10 and gap-extension penalty of 0.1. The datasets for ML analysis were combined and run in the Jones Taylor Thornton (JTT) model.

Scanning electron microscopy (SEM) for the morphology of O. xiamen.
The morphological characters of whole worms, anterior and posterior parts were investigated by using SEM, and the preparation was conducted according to a previous method 30 . Living specimens were washed thrice using sterilized seawater and suspended in 2.5% glutaraldehyde at 4 °C for 24 h and then transferred to a mixture of a saturated solution of HgCl2 and 1% OsO4 (4:1) at 4 °C for 10 min. All solutions mentioned above were diluted in sodium cacodylate buffer (pH 7.2) followed by specimen dehydration in a graded ethanol series, critical point drying, setting on aluminum stubs, and sputter-coating with platinum. The prepared samples were examined with a JSM-6380LV SEM (JEOL, Tokyo, Japan) at the Fujian Academy of Agricultural Sciences.

Results
Systematics. Ophryotrocha  Description. The Specimens (n = 15) with 24 segments (maximum number of segments) measured 3.10 ± 0.44 mm. Body shape dorso-ventrally flattened, narrow, tapering gently to pygidium, color opaque white in alcohol (Fig. 2a,b). Jaws and paired light-reflecting eyes in the enlarged head could be observed under microscope (Fig. 2a). The head displaying paired digitiform antennae surmounted with the curved cilia was similar to O. labronica and O. japonica (Fig. 2c). Dorsal and ventral bundles of cilia were present throughout the body, interrupted by parapodia (Fig. 2d). The pygidium was observed bearing two pygidial cirri and a median stylus. The median stylus only appeared in larval stage and disappeared in adult stage (Fig. 2b). Rosette glands (Fig. 2e), one per segment, presented mid-dorsally on the posteriormost segments of the mature animals. Glands appeared in adults of 10 to 12 chaetigers. The life history events are given in Supplementary Table S3. Tube-shaped eggcocoons (Fig. 2f, Supplementary Fig. S1) were found protected by the female and each cocoon contained approximately 150-230 zygotes. Seven days after egg-laying, larvae with a short pygidial stylus were released from the cocoon as two-segmented individuals at 25 °C and further growth was achieved by adding new segments before the pygidium. They moved around the bottom or the seawater surface using both parapodia and rings of cilia on their surface. We also notice that the adults can produce a network of mucous trails that may be recognized by conspecifics. The mandibles showed no significant changes during the life-cycle while the maxillae in worms with 12 segments started to change to K-type maxillae (Fig. 2g). The molting time was different between males and females, the change occurred early in males. Many oocytes were concentrated in the middle and posterior regions of the coelom. The first spawning was observed at 28 days.
Etymology. Ophryotrocha xiamen sp. nov. was first discovered in Xiamen, hence the name. Phylogenetic analysis. The combined alignment consisted of 804 bp, of which COI had 524 bp and H3 had 280 bp. Phylogenetic analyses resulted in similar tree topologies regardless of which tree reconstruction methods were used, therefore, only the results from the maximum likelihood analysis were discussed and shown (Fig. 3). www.nature.com/scientificreports/  Morphological characterization of early regeneration events. The anterior and posterior regeneration abilities were investigated using worms with 12-15 segments. A rapid regeneration process was observed in posterior growth (Fig. 4), in contrast, anterior growth was a slow process. The internal organs and coelomic fluid were oozed out immediately after amputation (Fig. 4a,e,i,m,o). In general, the morphological description of the posterior regeneration stages (n = 30) were as follows: day 1, after amputation, the edges of the cut gut fused with the edges of the cut body wall, reforming a posterior opening. This process was similar to typical healing by fusion with tissues, indicating that wound healing was already achieved (Fig. 4b,f,j,n,r); day 2, a posterior protuberance had already been observed indicating that blastema formation might have started at day  www.nature.com/scientificreports/ 1 ( Fig. 4c,g,k,o,s); and day 3, as the regeneration proceeded, the growing protuberance increased in size, and a complete pygidium bearing two pygidial cirri could be restored (Fig. 4d,h,l,p,t).
Worms were amputated at post-pharynx segments 0, 2-4, 6-8, and 10-12 to assess the influence of different amputation sites. During the first 3 days after amputation, the worms, except those amputating at post-pharynx segment 0, were able to form a complete regenerate pygidium with two pygidial cirri (Fig. 4t). The survival rates of different position of the amputation plane were showed in Supplementary Fig. S2.
Interestingly, we observed that O. xiamen could only partially restore the anterior end, but no new segment was observed. This happened only when part of the prostomium remained. The anterior regeneration process was completed within two weeks (Supplementary Fig. S3). Although worms without a peristomium could survive for months, they eventually starved to death.  (Table 2).
Among them, 19,574, 3048, and 13,755 unigenes were matched in the NR, NT, and SwissProt databases, respectively. More than 62.5% of the unigenes possessed an E-value of more than 1e−30. Among the database proteins that matched predicted proteins, O. xiamen unigenes had the highest number of hits to Capitella teleta (20.9%), a polychaete worm, followed by Lingula anatina (16.2%) (Fig. 5). www.nature.com/scientificreports/ For functional predictions and categories, all 8788 unigenes were assigned to three functional GO terms, including cellular component (452 subcategories), molecular function (180 subcategories), and biological process categories (2114 subcategories). Cell (4100 unigenes) and cell part (3958 unigenes) were the main subcategories in the cellular component category, while the main subcategories in molecular function were catalytic activity (3344 unigenes) and binding (3271 unigenes), and the major biological process was cellular process (4944 unigenes).

Comparative gene expression during regeneration. To identify the differentially expressed genes
(DEGs) involved in regeneration processes, gene expression in the treated (1 day post amputation) to the control group was compared and selected using a statistical cutoff of fold change > 2 and FDR < 0.05. A hierarchical clustering heatmap was generated to represent the up-and down-regulated genes. A total of 243 genes could be detected; 50 were upregulated and 193 were downregulated (Fig. 6). Among them, 15 significantly up-regulated genes were annotated as neurotrypsin, nitric oxide synthase 2 (Nos2), deleted in malignant brain tumors 1 (DMBT1), SCO spondin, endotubin, 18S protein and 28S protein, which suggest these genes might be important during early regeneration and thus candidates for further research ( Table 3). Numerous of unknown genes were also found to have significantly different expression levels, which indicated that the process of regeneration might involve some new genes. Besides, lack of well-annotated genomes/transcriptome or technical artifacts would also increase the number of new genes.
After the data analysis, the DEGs between the treated (1 day post amputation) and control groups were classified into 124 GO subcategories (78 subcategories for biological process, 26 subcategories for molecular function, and 20 subcategories for cellular component, p < 0.01). Translation, structural constituent of ribosome, ribosome and intracellular ribonucleoprotein complex subcategories contained the most DEGs and were treated as the focus of the analysis (Fig. 7). To further explore the mechanisms of regeneration, the DEGs were mapped to 132 KEGG pathways. DEGs were mainly involved in material metabolism and signal transduction, such as pathogenic Escherichia coli infection, arrhythmogenic right ventricular cardiomyopathy (ARVC), Salmonella infection, Huntington's disease, Alzheimer's disease, and Hippo signaling pathways were related to structure and signal transduction. The top 20 most abundant differentially expressed signaling pathways are listed (Fig. 7b).

Candidate genes involved in regeneration processes.
To better understand the molecular changes during early regeneration, the putative genes that have been implicated in other regeneration models were identified using BLAST searches (Supplementary file Table S4). A total of 130 transcripts were found in this study. Among these genes, brachyury, cyclin B, Hox A2, Indian hedgehog, myc, notch1, notch4, PRDM8, and PRDM9 were up-regulated, while notch2, neurogenin, matrix metalloproteinase, and glutamine synthetase were detected to be down-regulated (|logFC|> 1). Only cyclin B showed significantly increased expression during early regeneration and no significantly down-regulated genes were detected (|logFC|> 1, FDR < 0.05).
Hox genes and the preliminary expression pattern. In the phylogenetic analysis, 8 out of 43 homeodomain-like fragments were assigned to the anterior class Hox gene Hox1, Hox2; a Hox3 gene; central class genes Hox4, Lox5 and Lox4; and the posterior genes Post1 and Post2 (Fig. 8, Supplementary Fig. S4). No support for Hox5 or Lox2 orthologs was found. All genes except Post1 were up-regulated in the early regeneration stage (|logFC|> 1, FDR < 0.05).

Discussion
In the present study, we first describe O. xiamen sp. nov. collected from the Baicheng Bay of Xiamen. To establish a confirmed taxonomic position, the identity of this species is revised based on molecular tools combined with morphological characters 19,31 . Using morphological characters and phylogenetic analyses based on the mitochondrial gene COI and nuclear gene H3, we describe a new species, Ophryotrocha xiamen. Members of the genus Ophryotrocha are similar to each other in external morphology. For 'labronica' clade, all members are We observe a single rosette gland in the median position of posterior segments which mostly start from segment 12. The jaws, molting from the initial larval jaws to P1and P2 and finally the K-maxillae, develop at an earlier age in males than in females. In addition, females are observed to take care of their egg-cocoon until www.nature.com/scientificreports/ hatching and this phenomenon seems universal in Ophryotrocha. Within Annelida, mucus constitutes key factors in tube building, egg protection, and the prevention of proliferation of pathogenic microorganisms making it a particularly attractive class of biocidal agents 34 .
In some studies, it has already been advocated that new models that are amenable to molecular, cellular, and functional analyses are required to better understand the mechanisms of regeneration 35,36 . Based on their rapid individual growth rates and high regeneration efficiency, O. xiamen seemed to be a well-suited model to study the mechanisms of regeneration.
A rapid posterior regeneration process that followed a reproducible path and timeline allowed us to explore changes at different time points. Similar to Platynereis dumerilii (P. dumerilii) 15 , the whole process in O. xiamen was also subdivided into two continuous phases, regeneration per se (first 3 days) and post-regenerative growth. In annelids, epimorphosis is common mode of posterior regeneration and often involves the formation of a blastema comprised of an outer sheet of epithelial cells and an inner mass of mesenchyme-like cells that finally formed a complete posterior part. Our results showed that the amputated part could add new segments posteriorly and then grow to its original size with a similar growth rate even after multiple amputations. This phenomenon has been explained by the fact that the worm is able to 'sense' the site that has been cut and adjust its growth accordingly 15 . www.nature.com/scientificreports/ No anterior segment regeneration was observed unless part of the prostomium remained intact. Through the release of hormones, the brain promoted or inhibited regeneration in some annelids 12,37 . It was previously reported that no further replacements occurred after K-type jaws formed in Ophryotrocha 1,38,39 . Thus, more details about the molecular mechanisms between posterior and anterior regeneration in O. xiamen is needed to better understand the essential foundation for future mechanistic and comparative studies of regeneration.
The regeneration process involves complex morphological changes, but only 243 transcripts with significantly different transcript levels were found in O. xiamen. It seemed that genes involved in the regeneration and regular growth process largely overlapped and had been demonstrated to have similar expression patterns 15,17,40 . On the other hand, lack of replication could also affect the detection of DEGs 41,42 . During regeneration processes, neurotrypsin and SCO-spondin were thought to be crucial for cognitive brain functions and they regulated the balance between neuroepithelial proliferation and differentiation, respectively 43,44 . Both DMBT1 and endotubin were required for enterocyte morphogenesis and differentiation 45,46 . DMBT1 was also known as a protein that functions in innate immunity, inflammation, and angiogenesis by influencing the proliferation, migration, and adhesion of endothelial cells 46,47 . A previous study showed that genes involved in the Wnt/β-catenin signaling pathway were crucial for early regeneration 48,49 .
In the early stages of regeneration, inflammation and apoptosis factors initiated the downstream process of development 50,51 . All of them were reported to play important roles in early embryonic development, involving several genes with integral roles in the re-specification of regenerated structures in several annelid species 2 . Due to the scarcity of annelids genomic data used for annotation, a large set of potentially novel DEGs in O. xiamen were identified. Previous studies revealed that unknown genes that showed similar expression trends with key regulators might be important and were required during regeneration processes. Thus, well annotated genome data of O. xiamen, which is already in progress, are needed to explain the exact gene functions and regeneration mechanisms.
To further understand the regeneration mechanisms in annelids, it will be of particular importance to identify genes that are specifically related to this process. By using BLAST searches, we found regeneration-related or putative genes that have been implicated in regeneration in other regeneration models 52 . All of these genes were reported to be involved in regeneration processes, including wound healing, blastema formation, cell proliferation control and morphogenesis 15,17,53 . Among these candidate genes, only cyclin B was significantly up-regulated which needed to be further confirmed. Several studies indicated that the function of cyclin B protein in invertebrates might have a dual role as cyclin B1 and cyclin B2 which was suggested to participate in the reorganization of different aspects of the cellular architecture at mitosis and have different functions in the cell cycle 54,55 . The early regeneration stage is a rapid process in annelids that consists of muscular contraction and epithelium formation 12 . Thus, cyclin B in O. xiamen was supposed to have the same functions in regulating the cell cycle during early regeneration.
Most of the Hox genes identified from A. virens, P. dumerilii, and C. teleta were detected in early blastema 13 . In the present study, 8 Hox genes were identified, but not Hox5 or Lox2. In P. dumerilii 14 , Hox5 and Lox2 were also not to be expressed in the components of the regenerating nervous system or in the posterior growth zone. However, the expression of Hox5 in A. virens was detected in the whole body and downregulation occurred at 10 h post amputation 10 . In this study, the absence of Hox5 and Lox2 in the transcriptome of both early regeneration and normal adult growth process of O. xiamen might be due to low transcription level during these stages. The down regulated Post1 which was reported to be associated with the formation of new chaeta seemed to have a limited function in early regeneration 14 . Other homeodomain fragments clustered with the genes, such as Cdx, even-skipped and engrailed, were also identified (Supplementary Table S4). In addition to cellular-level studies, molecular-level studies are needed to provide a complete understanding of regeneration processes.

Conclusions
In this study, the newly identified O. xiamen is suitable for studying the mechanism of regeneration. Comparative transcriptome analysis provides the expression changes during early regeneration in this species. Genes  www.nature.com/scientificreports/ involved in regeneration, especially Hox genes, have also been investigated to reveal the similarity in regeneration mechanisms among related species. We hope that further investigations based on O. xiamen as a new model organism will provide deep insights into the regeneration process at the morphological and molecular levels and will stimulate interest in evolutionary research.