1 INTRODUCTION

Capitella is an elongate, thread-like marine benthic annelid, which is globally known in ecological research as an indicator to marine pollution (Grassle and Grassle, 1976; Dean, 2008). Capitella species are common in the Chinese marine regions, and often dominant in high densities of up to 127 200 inds./m² (Wu, 1964; Yang and Sun, 1988). Wu (1964) reported the first species of Capitella in China, C. capitata (Fabricius, 1780), based on the specimens of the Yellow Sea and the East China Sea. However, Capitella specimens collected from different locations of Bohai Sea, Yellow Sea, and the East China Sea exhibit various types of chaetal arrangement, developmental patterns, and ecological characters, which might represent a series of different species (Wu et al., 1988; Lin et al., 2008). Although several sibling species were reported with provisional names in China (Wu et al., 1988), so far, all the specimens of Capitella have been identified as C. capitata (Fabricius, 1780) without a detailed taxonomic study.

Capitella capitata was formerly considered as a cosmopolitan species and had been widely used as an indicator of environmental pollution for a long time. However, various studies over the past 40 years suggested the nominal "C. capitata" include approximately 12–13 sibling species (Grassle and Grassle, 1976; Gamenick et al., 1998; Méndez, 2006). In order to clarify the species, Blake (2009) provided a detailed re-description of C. capitata based on a large collection of specimens and indicated C. capitata is distributed in Arctic and subarctic areas. Therefore, previous records of C. capitata from boreal and temperate localities, including Chinese waters, should be re-evaluated.

Regeneration is a process of re-growing a partial or whole body after being damaged. It is a wide-spread phenomenon in the animal kingdom. However, the regeneration ability is significantly different depending on the organism groups. For example, some animals such as planarian and hydra can regenerate their whole body from a fraction (Wagner et al., 2011; Buzgariu et al., 2018), while zebrafish and salamander are able to regenerate a partial tissue only (Mierzwa et al., 2020; Pronobis and Poss, 2020; Sibai et al., 2020). Capitella has been recognized as a model in regeneration study for long years as its remarkable regeneration abilities (Seaver, 2016).

Based on the examination of the specimens of Capitella from the coastal water of Shandong, a newly recorded species in China, C. teleta Blake et al., 2009 was verified through morphological and molecular analysis. Being one of the original sibling species of C. capitata, C. teleta has been commonly used as an experimental species under the provisional name Capitella sp. Ⅰ for over 40 years. It was described as a new species Capitella teleta by Blake et al. (2009). The species is widely distributed along the east and west coasts of North America, Korea, Japan, and the Mediterranean, and considered as a highly opportunistic species. Furthermore, the regeneration process of C. teleta was studied through whole mount immunohistochemistry and chemical staining (Hill and Boyer, 2001; Meyer et al., 2015). The muscle and nervous tissue of C. teleta regenerated rapidly, more than 15 segments regenerated at 16 dpa. Capitella teleta is expected to be an excellent model in the functional study and regeneration evolution study of marine invertebrate due to its high regenerative ability, easy to culture in lab settings, and wide distribution in coastal waters. This is for the first time that the species have been recorded in China seas. We described the morphology of C. teleta in this study, and displayed the phylogenetic analysis of Capitella species based on the partial sequences of COI gene. We also investigated the rapid regeneration ability of this species.

2 MATERIAL AND METHOD 2.1 Collection, laboratory culture, and morphological examination

Specimens were sampled from the Weifang Port adjacency (119°3'53.54"E, 37°12'20.91"N) and Qingdao Zhanqiao Pier (120°19'15"E, 36°3'40"N), Shandong Province, China on October 30, 2019 and September 9, 2020, respectively (Fig. 1). All specimens were taken back to laboratory and cultured in a 16–19 ℃ temperature incubator, where they were kept in fresh sea water and fed with sea bottom mud once a week.

Specimens examined were anesthetized with 7.5% magnesium chloride and then fixed in 70% ethanol solution (Lewbart and Mosley, 2012). Details of prostomium, nuchal organs, segments, parapodia, and chaetae were observed through a ZEISS DiscoveryV12 stereomicroscope (ZEISS, Germany) and a ZEISS Axio Imager Z2 compound microscope (ZEISS, Germany) according to Blake et al. (2009). Digital images were taken through an AxioCam 512 color camera mounted on the two microscopes. Images of different focal planes were stacked through the extended depth of field by the software Helicon Focus 7 (Helicon, Ukraine). The collected specimens were stained with methyl green to increase the contrast and to observe the staining pattern according to Blake et al. (2009). Specimens for SEM (scanning electron microscope) observation were fixed in 2.5% glutaraldehyde solution at 4 ℃ for 1 h, dehydrated with gradient concentrations of ethanol ending up with 100% ethanol, followed by drying at critical point, coated with gold, and finally, observed and photographed using the HITACH S-3400N scanning electron microscope (HITACH, Japan).

2.2 DNA extraction and sequencing

Five specimens, consisting of three Weifang worms and two Qingdao worms, were embedded in 0.5% cornmeal agarose plate for 2 h to remove as much gastrointestinal tract matter as possible. Afterwards, the genomic DNA was extracted from the posterior segments using Fastpure^® Cell/Tissue DNA Isolation Mini kit (VazymeDC102, Nanjing, China) (Tomioka et al., 2016). The primer pair was jgLCO 1490 (5'-TITCIACIAAYCAYAARGAYATTGG-3') and jgHCO 2198 (5'-TAIACYTCIGGRTGICCRAARAAYCA-3') (Geller et al., 2013). The 25-μL PCR (Polymerase Chain Reaction) mix contained 8-μL sterile water, 2.5-μL DNA template, 1-μL each primer of 10-μmol/L, 12.5-μL (10 μmol/L) 2X Phanta Master Mix (Vazyme P511, Nanjing, China). PCR was conducted on a T100TM Thermal cycler (Bio Rad, USA) with the following thermal conditions: 95 ℃ for 3 min (initial denaturation); 35 cycles of 95 ℃ for 15 s (denaturation), 60 ℃ for 15 s (annealing), 72 ℃ for 1 min (extension), and a further 5 min elongation at 72 ℃. Subsequently, the PCR products were detected by 1.0% agarose gels (BBI Life Sciences 9012-36-6, Shanghai, China) electrophoresis. Amplified DNA was sequenced in Single directions using 3730xl ABI by Sangon Biotech (Shanghai) Corporation.

2.3 Barcoding and phylogenetic analysis

Five sequences of C. teleta in our study were performed for barcoding and phylogenetic analysis. Related sequences of Capitella for phylogenetic analysis were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The outgroup comprised species of Nereis and Sipunculus. The sequences were aligned using MEGA6 with the ClustalW program (Tamura et al., 2013). Genetic distances of mitochondrial cytochrome c oxidase subunit I (COI) within and among the clades of Capitella were calculated using MEGA6 with Kimura's two-parameter model (Tamura et al., 2013). Maximum likelihood (ML) analysis was carried out using MEGA6 with 1 000 bootstrap replicates.

2.4 Antibody staining and phalloidin staining

After anesthesia and amputation, the whole mount immunohistochemistry for C. teleta in China was performed according to the method of de Jong and Seaver (2016), followed by fixation with 4% paraformaldehyde (PFA) (Sigma-Aldrich P6148, Darmstadt, USA) in filtered sea water (FSW) for at least 40 min at room temperature (RT) for target samples. Samples were subsequently washed with washing solution (PBT: phosphate buffer saline (PBS)+0.1%Triton X-100, Sigma-Aldrich T8787, Darmstadt, USA) for several times. After that, samples were blocked in PBT with 10% normal goat serum for 60 min at room temperature. Then the worms were incubated in primary antibody overnight at 4 ℃ while gently rocking, followed by secondary antibody incubation for 4 h at RT while rocking. After rinsing for several times, samples were labeled by 1꞉400 phalloidin (Thermo Fisher A12379, Massachusetts, USA) in PBT with 10% normal goat serum for 60 min at room temperature while rocking. Finally, all samples were incubated in Hoechst33342 (Thermo Fisher H3570, Massachusetts, USA) overnight at 4 ℃. Then images were taken by confocal microscopy LSM710 (ZEISS). Primary antibody was 1꞉400 mouse anti-acetylated-α-tubulin (Sigma-Aldrich 6-11b-1, Darmstadt, Germany USA), and second antibody was 1꞉250 donkey antimouse secondary antibody (Invitrogen A10036, Carlsbad, CA, USA). All figures were edited in Adobe Photoshop CS6 and Adobe Illustrator CS6.

3 RESULT 3.1 Taxonomy

Family Capitellidae Grube, 1862

Genus Capitella Blainville, 1828

Capitella teleta Blake et al., 2009

Figs. 2a–j, 3a–i; Tables 1–3

Material examined: Samples were collected in the intertidal zone (119°3'53.54"N, 37°12'20.91"E) in Weifang Port adjacency, Weifang, Shandong, China, collected by Qian LI and Yongnan LI on Oct. 30, 2019 (WF200726.1–24, 24 specimens); and the intertidal zone (120°19'15"E, 36°3'40"N) in Zhanqiao Pier, Qingdao, Shandong, China, collected by Xiaofei ZHU, Jieyang WENG, and Jie LI, on Sep. 9, 2020 (ZQ200909.1–18, 18 specimens). Molecular analysis was conducted for WF200726.22–24 and ZQ200909.14–15; micrographs were taken for WF200726.7–10, 17–21, and ZQ200909.17–18; SEM was done on WF200726.11–16.

Description: Living specimens bright red or light yellow, preserved animals with pale color (Fig. 2c–j). 5.0–11.0-mm long with 39–60 chaetigers (based on collected material). 0.5–1.1-mm wide. Body slender, slightly wider in anterior segments, gradually narrow posteriorly.

Prostomium flat, triangular and narrow (Fig. 3a). Eyes present in vivo, located in lateral side of prostomium, not observed after preserved. Peristomium and prostomium bounded by nuchal organs. Head composed of peristomium and prostomium, with no appendages, usually longer than the 1^st chaetiger (Fig. 3a & b). Proboscis not exposed.

Nine thoracic segments with small folds (Figs. 2c–e, 3b). Chaetal arrangement of thoracic segments on 9 segments varies. Chaetigers 1–7 with capillary chaetae on both notopodia and neuropodia, 3–8 per fascicle (Fig. 3b). Capillaries slender, tapering posteriorly (Fig. 3d). Chaetigers 8–9 of females with neuropodial hooded hooks, 3–8 per fascicle. Chaetigers 8–9 of males with 2 pairs of genital spines on notopodia (Figs. 2a, d, f, j, 3e). Anterior end of hooded hooks curve to hood shape (Fig. 3f). Hooded hooks with a robust main fang (Fig. 3i) and small teeth around main fang (Fig. 3h & i). Genital spines with a sharp and pointy tip. They are different in chaetigers 8 and 9. Chaetiger 8 with six or eight shorter spines, chaetiger 9 with four robust genital spines (Fig. 3e). Genital spines on chaeigers 8 and 9 oppositely arranged (Figs. 2f & 3e). Thoracic setigers usually wider than abdominal setigers (Fig. 3b & c).

Abdominal region with many narrower segments, gradually thinner posteriorly. Abdominal segments with ventral and lateral grooves, more wrinkled than thoracic segments (Fig. 3c). Abdominal chaetae all hooded hooks, similar to thoracic hooded hooks; anteriorly 2–4 hooks per fascicle, reduced posteriorly to 1–2 hooks. 1–4 per fascicle (Fig. 3g & h). Pygidium with rounded lobes (Fig. 3c).

Most C. teleta are gonochoristic. Mature females with pairs of ovaries on some abdominal segments (Fig. 2i). Mature males with genital spines on chaetigers 8 and 9 (Fig. 2d & j). The average number of embryos produced per female less than one hundred within a brood tube. Embryos firstly develop in a brood tube and then release until the late stage of larvae. It will take 7–9 days to finish a transition from single-cell embryo to late trochophore. In addition, no food is required during this period, and then followed by living benthic life, become reproductive approximately 8 weeks after fertilization.

Methyl green staining: Males were dyed blue except for the chaetiger 10 (Fig. 2a). The thoracic staining of the female was not obvious, only light staining was observed on chaetigers 5–8. Abdominal part from chaetiger 15 to few chaetigers before pygidium were stained blue (Fig. 2b). Four worms were treated with methyl green dye, including 3 females and 1 male. In 3 females, methyl green staining pattern (MGSP) was consistent.

Remark: Although some Capitellidae species have been found (Lin et al., 2019a, b), C. capitata was the only recorded species of Capitella in the whole China Seas according to the description given by Yang and Sun (1988), "C. capitata" is characterized by the following chaetal arrangement: chaetigers 1–7 of both females and males with capillaries; chaetigers 8–9 of females with hooded hooks on notopodia and neuropodia, and notopodial hooded hooks replaced by genital spines in males. However, these characters are consistent with C. teleta in this study. It indicates that the specimens of C. teleta were formerly misidentified as C. capitata by Chinese authors. According to Blake (2009), C. capitata s. str. is considered Arctic and subarctic in distribution, therefore early records of C. capitata from the China should be re-examined.

Morphologically, C. teleta collected from Shandong exhibited some differences from American, Korean, and Japanese populations in chaetal number per fascicle, methyl green staining pattern, and number of genital spines of males (Table 1), which represents intraspecific variations among different populations of C. teleta.

3.2 Barcoding and phylogenetic analyses

The genetic distances and phylogenetic analyses assessed from COI (Fig. 4; Table 2) were performed to clarify the status of C. teleta in this study. Capitella species were grouped into 6 clades based on the ML phylogenetic tree (Fig. 4). Populations of C. teleta from China, Japan, Korea, America, and Italy were clustered together in Clade 1 with high node support (Tomioka et al., 2016; Jeong et al., 2018); Clade 2 consisted of a species closely related to C. teleta from Gamo of Japan with an inconclusive taxonomic identity; Clade 3 included an unknown species of Capitella from Japan; members of both Clade 4 and Clade 5 were identified as C. capitata, collected from Canada and India, respectively. Strangely, the worms, both from Canada, were grouped into 2 clades, this suggested one of populations of C. capitata might represent another species and warrant reinvestigation. Clade 6 contained two Capitella species from the United States.

K2P distances within and among the 6 clades were calculated (Table 3). The COI genetic distances among the species of Capitella ranged from 3.7% to 23.7% (12.4%–23.7%, excluding Clade 2), while the intraspecific distances within 6 clades were very low (0.2%–1.9%). Excluding Clade 2, there was a clear barcoding gap (1.9%–12.4%) for the species of Capitella. The K2P distances among the Clade 1 ranged from 0.000 to 0.017 (mean 0.003), indicating that our specimens from Shandong represent the same species as in Japan, Korea, America, and Mediterranean, and should be identified as C. teleta with no doubt.

3.3 Regeneration feature of C. teleta in China

To investigate the regeneration ability of C. teleta in China, the whole mount immunohistochemistry and phalloidin staining were performed. At 0-h post amputation (hpa), cells labelled evenly distributed through the whole body, and sizes were uniform and round (Fig. 5a). The longitudinal muscles seemed to disappear at the cut site, while only circular muscles existed (Fig. 5a'). The ventral nerve cord (VNC) was arranged in the trunk part, attaching to the anterior brain (Fig. 5a"). VNC likely to have an obvious termination at the cut site, which was viewed as a symbol of amputation location (de Jong and Seaver, 2018). At 3 hpa and 12 hpa, no epithelium to seal the wound was observed at the surgery site, which meant that the wound healing process might not have been completed (Fig. 5b & c). Similarly, muscles and neurites seemed to stop at the cut site (Fig. 5b', b", c', c"). From 12 to 24 hpa, the wound completely healed. At 24 hpa, a group of labeled cells gathered around the wound, which meant the regeneration of a small new blastema (Fig. 5d). However, no extension of muscles or nerves were found (Fig. 5d', d"). At 3 dpa, a larger regenerated blastema was observed, which consisted of a group of cells (Fig. 5e). The muscles had some messy extension growth (Fig. 5e'). VNC displayed slight increases well (Fig. 5e"). At 5 dpa, muscles obviously regenerated (Fig. 5f'), but only a few nerve fibers regenerated (Fig. 5f"). At 7 dpa, a mass of cells gathered at the wound site (Fig. 5g), and both longitudinal muscles and multiple rings of circular muscles near the pygidium were observed (Fig. 5g'). Although the extension and growth of VNC was remarkable, the mature ganglia had not yet formed (Fig. 5g"), which indicated that it failed to regenerate a complete chaetiger at present. Re-grew segment numbers were counted with mature ganglion labeled by anti-acetylated α-tubulin. Up to 16 dpa, approximately 17 segments regenerated (Fig. 5h') and the muscle tissue completely re-grew (Fig. 5h). The muscle architecture of C. teleta consists of circular, longitudinal, and helical muscles (Fig. 5h).

4 DISCUSSION

In this study, we verified the taxonomic status of C. teleta from Shandong through morphological examination and phylogenetic analysis. Capitella teleta from Shandong is the second reported species of Capitella in Chinese waters. It is consistent with C. teleta from Korea, Japan, and the United States in most of the characters. However, there are some morphological differences among these populations. For example, there are 6(8) and 4 genital spines on chaetigers 8 and 9 in Shandong population, respectively, while 8 and 6 genital spines are found in American, Japanese, and Korean populations. Besides, the methyl green staining patterns vary among different populations. In the American population, the internal organ in chaetiger 7 of males was stained, chaetigers 6–9 of females remain stained. In the Japanese population, both females and males were dyed all over the body. In the Korean population, chaetigers 5–9 were stained in females but not in males. Almost the whole body of the males was stained except for the chaetiger 10, while abdominal segments of the female was significantly stained in the Chinese population. We assumed that the morphological differences among C. teleta from different areas probably resulted from intraspecific variations. Capitella teleta is considered as a cosmopolitan species that is known to be distributed in the Mediterranean, the United States, Japan, Korea, and China (Blake et al., 2009; Tomioka et al., 2016; Jeong et al., 2018; this study). The worldwide distribution might be related to anthropogenic dispersal, as proposed by Tomioka et al. (2016). C. teleta is gonochoristic in a normal situation, embryos would first develop in a brood tube that is around the pregnant female, and would release from the tube until they develop into the late stage of larvae. C. teleta in Shandong has a relative short reproductive cycle of about 2 months, with relatively fewer embryos produced each time compared to C. teleta in USA, which can produce 100–250 embryo once.

Lin et al. (2008) collected numerous Capitella specimens with various kinds of thoracic chaetal arrangements along the Chinese coastal waters. It indicates that there might be many unknown species of Capitella in the China seas. However, all the specimens of Capitella have been identified as C. capitata (Fabricius, 1780) since Wu (1964) firstly reported the species in China. The chaetal arrangement of "C. capitata" reported in China is the same as C. teleta (chaetigers 1–7 with capillaries, chaetigers 8–9 with hooded hooks and genital spines). However, C. teleta can be easily distinguished via the absence of neuropodial capillaries on chaetigers 8 and 9 (vs. present in C. capitata). Besides, C. capitata s. str. is considered to be Arctic and subarctic in distribution according to Blake (2009), while most specimens of Capitella in China are collected in lower latitude regions from 20°N to 40°N. Therefore, it seems that records of C. capitata in China might represent other species and need to be re-investigated.

The regeneration of C. teleta in China conforms to a strict timeline and process, and posterior part can be regenerated. In a few days, the different tissues could regrow. Besides, Giani et al. (2011) described the regeneration process of C. teleta, at 1 dpa, the wound was completely close, and with a small blastema. Within 3 dpa, the blastema became bigger, and at 5 dpa, axons extended into the blastema, likely entering into the regeneration tissue. About 20 segments regenerated at 18 dpa. The regeneration process is similar to our worms, with no significant differences and some subtle differences that may be related to the environment. Many members of polychaeta are able to regenerate, some species can regenerate both head and tail, or only tail. For example, the fireworm Eurythoe (Annelida) can complete the anterior and posterior regeneration within 50 days (Yáñez-Rivera and Méndez, 2014). Regeneration ability is benefic to the benthic worms and can improve the survival rate of its population. It is reported that some asexually reproductive worms can autotomize to regenerate (Ribeiro et al., 2018); however, the relative between asexual reproduce and post-injury regeneration is controversial. Although autotomy seems similar to regeneration, as far as we concern, regeneration should be initiated by injury, which is a kind of passive behavior rather than active.

The process in polychaeta regeneration usually include wound healing, formation of a regeneration blastema, cell differentiation, and segments addition. However, some worms regenerate without a blastema, such as Spirobranchus lamarcki, the opercular regenerate is based on non-blastema (Bubel et al., 1985). The blastema is a regeneration specific structure fulling with the mass of cells. In many cases, regeneration generally involves a regeneration blastema, such as planarian (Tanaka, 2016; Planques et al., 2019). Exceptionally, the regeneration of ctenophore is based on non-blastema (Ramon-Mateu et al., 2019). However, the cellular sources and potential fates of source-cells of blastema need to further address.

5 CONCLUSION

In conclusion, the worms found in Qingdao and Weifang are Capitella teleta, which was identified by morphological descriptions and genetic evidence. Surprisingly enough, this worm is likely to be a model for function study and regeneration evolution study due to its rapid regeneration ability.

6 DATA AVAILABILITY STATEMENT

The data generated and/or analyzed in this study are available from the authors on reasonable request.

7 ACKNOWLEDGMENT

We thank Jieyang WENG, Jie LI, Xuegang WANG, and Xiaofei ZHU for their assistance in sampling specimens, and are very grateful to the staff of the SEM room for their help. We also express our gratitude for the support by Oceanographic Data Center, IOCAS.