A deposit-feeder sea cucumber also ingests suspended particles through the mouth

Sea cucumbers are epibenthic animals (Burton and Burton, 2002) that play important roles in marine ecosystems, influencing the activity of certain benthic organisms (Uthicke, 2001), improving water chemistry (MacTavish et al., 2012) and reducing algal blooms (Michio et al., 2003), and also have potential utility remediating aquacultural waste (Zamora et al., 2018). Moreover, sea cucumbers have attracted attention on account of their nutritional and medicinal value (Bordbar et al., 2011). Worldwide, approximately 1717 sea cucumber species have been identified (Paulay and Hansson, 2013), among which Apostichopus japonicus, belonging to the order Aspidochirotida, is considered the most commercially important sea cucumber species (Yang et al., 2015). Members of the order Aspidochirotida are recorded as deposit feeders (Conand, 2006), and although during the early stages of life A. japonicus is pelagic, it subsequently undergoes metamorphosis into a benthic form (Zhang et al., 2015), with the adults being characterised as deposit feeders (Yang et al., 2015). The digestive tract of the sea cucumber comprises the mouth, pharynx, oesophagus, stomach, intestines, rectum, cloaca and anus. The mouth of adult individuals is surrounded by tentacles that push sediment particles into the mouth (Sun et al., 2015). The accumulated ingested sediment is subsequently ground in the pharynx, oesophagus and stomach, and is thereafter pushed into the intestines, wherein it is progressively compacted and coated with mucus, and then fragmented into faecal pellets, presumably in the posterior section of the intestines (Féral and Massin, 1982).

The clearance of suspended particles through the mouth has been described in the swimming larvae of sea cucumbers (Hart, 1991; Zhang et al., 2015), which have been observed to clear small plastic particles, namely microplastics (MPs), from the water column (Hart, 1991). When feeding, adult A. japonicus individuals move towards sediments, and the tentacles expand to capture and transfer sediment particles into the mouth (Sun et al., 2015). Additionally, previous studies have determined the food sources of A. japonicus based on the analysis of gut contents, and have established that nutrients sourced from the water column can be found in the intestines of sea cucumber (Sun et al., 2013; Zhang et al., 2016). Furthermore, A. japonicus has been found to reduce algal blooms by grazing on the upper surface of sediments (Michio et al., 2003), and it has also been demonstrated that suspended particles can be transferred to the coelomic fluid via uptake through the anus (Mohsen et al., 2020). However, the mechanisms whereby nutrients in seawater can enter the intestines of A. japonicus, and whether other suspended particles (other than nutrients) can enter the intestines, have yet to be determined.

We have detected synthetic particles in the intestines of A. japonicus when study animals were exposed to a suspension of these particles. However, it was unclear whether these particles entered the intestines from the mouth or the anus. Therefore, in the present study, we sought to clarify how these particles enter the intestinal tract of A. japonicus. Additionally, given that the use of synthetic particles can enable an accurate estimation of clearance rates (Hart, 1991), we used such particles to examine the clearance rate of suspended particles. Furthermore, we examined whether the ingestion of suspended particles changes over time under relatively constant concentrations of the suspension.

All procedures used in this study, including animal collection, rearing and euthanasia, were performed according to the Guidelines of Ethical Regulations of Animal Welfare of the Institute of Oceanology, Chinese Academy of Sciences (IOCAS 2013.3).

Sea cucumbers A. japonicus (Selenka 1867) (n=90) were collected from a local farm in September 2019, and thus no specific permission was required for the purposes of collection. The sea cucumbers were artificially bred in a laboratory in Rushan, China (36°46′40.7″N 121°34′34.7″E). For 1 week prior to conducting experiments, the animals were acclimated to the laboratory conditions in a recirculation unit at a temperature of 18±0.5°C, salinity of 30–32 ppt and pH of 7.8–8.5. Animals were fed using a formulated diet (crude protein 6.23%, crude lipid 0.96%, moisture 1.16%, energy 2.68 kJ g⁻¹ and ash 76.15% dry matter; Shandong Aquatic Products Technology Co., Ltd, Yantai, China), which was supplied when needed, and faeces were collected daily by siphoning. The mean (±s.d.) body mass of the study animals after fasting for 2 days was 32.25±13.8 g.

Synthetic particles were prepared using polyester threads, with a mean (±s.d.) length of 57±14 µm, which were initially cut using scissors, then using a cutting machine, and once again using scissors. The particles thus generated were subsequently scattered into a container of seawater, followed by thorough mixing to produce a suspension (Fig. S1). The particles were maintained in suspension via a stream of bubbles released from air stones placed in the centre of each beaker (2 l; see Movie 1). The air stones were connected to an air pump operating at a power of 10 W and output of 10 l min⁻¹ (Hailea, Guangdong, China). The particles were coloured red, and therefore their suspension was clearly visible and could be monitored in the beakers with the aid of a powerful headlight (DL-200LED, Dian Lang, Shanghai, China).

The following method was used to examine the uptake of suspended particles by A. japonicus. A suspension of synthetic particles and Spirulina powder (a dried blue–green alga) was prepared, and a blue dye (Coomassie Brilliant Blue, Solarbio, Beijing, China) was added to the water as a tracer. The suspension of the particles was maintained, and having allowed the feed to settle on the bottom of each beaker, the study animals were placed in the beakers. We used both starved and fed animals, which allowed us to determine whether both starved and non-starved sea cucumbers ingest the suspended particles. This meant we were able to negate the effect of hunger on the observed behaviour. Furthermore, using starved animals allowed us to detect and confirm the pathway of the particles because only the suspended particles were observed in the digestive tract of the starved animals. An opening in the ventral surface of each study animal was made, and the synthetic particles, algae and blue dye in the intestines were observed using a dissecting microscope (SMZ-161-BLED, Motic, Xiamen, China). The faeces of the animals were also collected by siphoning, and their contents were examined under the dissecting microscope.

In order to determine the route(s) via which particles enter the intestines of sea cucumbers, we used rubber bands to close the anus or the mouth of study animals (Newell and Courtney, 1965). The band was expanded to cover the anus (Fig. S2). The animals were placed in 2 l beakers (Fig. S2) and were able to move freely throughout the beakers, i.e. they were not confined to the base of the beaker (Fig. S2). Furthermore, to confirm suspension uptake by the sea cucumbers, the feed was placed in each beaker. When the sea cucumbers started to feed, suspended particles were added to the beaker and their suspension was maintained via the airflow generated by air stones (Movie 1). To record the mechanism of particle capture, we used a digital camera (Leica, 40 megapixels) placed in front of the beakers.

SPSS Statistics 20 (SPSS Inc., Chicago, IL, USA) was used to conduct statistical analyses. The normality of the data was examined using the Shapiro–Wilk normality test, with a 95% confidence level. Where necessary, natural logarithmic transformation was used to meet the assumption of normality. Pearson correlation analysis was used to examine the relationships between variables.

Apostichopus japonicus sea cucumbers are known as deposit feeders that use their tentacles to collect sediment in the mouth, from which nutrients are extracted (Sun et al., 2015). Although the pelagic larvae of A. japonicus have been shown to capture suspended MPs via their ciliated bands (Hart, 1991), the uptake of suspended particles by the adults of this species has not previously been demonstrated. Previous studies focused on determining the food sources of A. japonicus by analysing their gut contents (Gao et al., 2014; Sun et al., 2013; Zhang et al., 2016). In the present study, however, we unexpectedly detected synthetic particles in the intestines of A. japonicus adults when study animals were placed in a suspension of these particles. It was unclear whether the particles had entered the intestines via the anus or the mouth as an earlier study had shown that particles could be transferred from the water to the coelomic fluid via the anus (Mohsen et al., 2020). Therefore, in addition to ingestion via the mouth, it was necessary to take into consideration the possible transfer of particles from the water to the intestines via the anus.

In the present study, we observed that A. japonicus adults extend their tentacles in the water to capture suspended particles, after which the tentacles are retracted to transfer the particles into the mouth (Fig. 1; Movie 2). We also detected synthetic particles, algae and blue dye derived from the water in the digestive tract and faeces of A. japonicus (Figs S3 and S4). Furthermore, when passage via the anus was blocked, we observed red-coloured particles, or the blue dye, in the oesophagus and stomach of the sea cucumbers (Fig. S3), and when the sea cucumbers started to feed on the sediment and the suspended particles were added, they were observed to climb the wall of the container and capture particles from the water (Fig. S5). Collectively, these observations thus revealed that A. japonicus ingest particles from the water via the mouth and that consumption continued as long as the tentacles were able to capture the particles (Movie 2). Moreover, A. japonicus adults were shown to take up any suspended non-nutrient particles, as long as they were of a suitable nature for capture by the tentacles and ingestion via the mouth (Movie 2).

To ensure that the ingested particles were not taken up after settlement, the suspended particles were prevented from settling at the base of the beaker (Movie 1). We also observed sea cucumbers capturing particles with their tentacles while moving freely along the walls of the beakers, but not on the beaker floor (Fig. 1; Fig. S5 and Movie 2). Furthermore, it is noteworthy that if the starved sea cucumbers found the particles only after settlement on the bottom of the beaker, they preferred to stay in this position in the beaker. However, the sea cucumbers were not confined to the bottom of the beaker; they also climbed and moved along the walls of the beaker. Additionally, in this regard, the findings of a previous field study have indicated that the guts of A. japonicus contained organic matter derived from levels in the water column that were above the sediment (Zhang et al., 2016). Accordingly, our collective observations confirmed that A. japonicus can ingest suspended particles, which may further explain the previous findings of food source studies. Our findings may also explain the observed seasonal variation in the gut contents of sea cucumbers, which reflect the contents of particles in the water in the respective habitats (Zhang et al., 2016), as well as how A. japonicus can reduce algal blooms (Michio et al., 2003), and why some water-borne nutrients can be found in the guts of A. japonicus (Zhang et al., 2016). We assume that uncertainties regarding the mechanism whereby A. japonicus ingests suspended particles can be attributed to methodological limitations associated with the identification and quantification of particulate matter, and tracking particle uptake by sea cucumbers. In the present study, using a relatively simple methodology, we were able to gain valuable insights into this mechanism.

Our analysis of particle clearance by A. japonicus revealed clearance rates ranging from 0.3 to 0.9 l h⁻¹ (Fig. 2A). Furthermore, we determined that the particle clearance rate of starved sea cucumbers (i.e. without feed) ranged from 0 to 0.9 l h⁻¹ (Fig. S6). The difference in the clearance rates of echinoderm larvae is assumed to be attributable to the activity of the larvae, which had maximum clearance rates of 1–2 µl min⁻¹ (Hart, 1991). Therefore, high clearance rates are presumably typical of A. japonicus with high activity levels. Furthermore, given that the particle ingestion rate increased with an increase in the concentration of suspended particles (Fig. 2B; Pearson correlation: r=0.808) and the clearance rate of particles decreased over time when the particle concentration was not restored (Fig. 2D; Fig. S6C), we assume that particle ingestion by the sea cucumbers is also dependent on the abundance of particles in the water, i.e. the likelihood of encountering particles in the water. Additionally, we found that only 0.1–0.6% of the particles removed from the water were transferred to the coelomic fluid of A. japonicus via the anus, thereby indicating that the uptake of suspended particles occurs predominantly through the mouth (Mohsen et al., 2020).

Our results indicate that sea cucumbers might contribute to the removal of synthetic particles of micrometre size (e.g. MPs, small plastic particles <5 mm) from the aquatic environment (see Fig. 3; Figs S3 and S4). MPs are among the most widespread pollutants in marine habitats, and their removal is one of the major challenges facing the world today (Suran, 2018). Burying MPs in the sediment may potentially protect marine biota from exposure to suspended MPs, particularly those organisms at the base of the trophic chain (Suran, 2018). In the current study, we observed that the abundance of suspended synthetic particles declined over time (Pearson correlation: r=−0.972; Fig. 2D), and clearance rates ranged from 0.3 to 0.9 l h⁻¹. These observations accordingly highlight the bioremediation potential of sea cucumbers with respect to removing MPs from the water column, particularly in regions in which sea cucumbers may be exposed to high loads of suspended MPs. However, the efficiency with which sea cucumbers remove MPs from the water column requires further investigation. For example, it would be helpful to assess whether particles that have been mixed with the sediment by sea cucumbers can subsequently become resuspended in the water, and also to evaluate the ability of sea cucumbers to remove MPs of differing shapes and sizes from the water.

A further consideration in this regard is that certain components of some MPs are considered hazardous, such as the monomers of polyvinyl chloride (Lithner et al., 2011), the consumption of which can have detrimental physical effects; for instance, damaging the gut via disruption of intestinal cells (Jiang et al., 2020). Additionally, some ingested MPs may cause adverse biological effects in numerous species, including increased mortality (Aljaibachi and Callaghan, 2018), reduced growth (Naidoo and Glassom, 2019), and induced immunity and oxidative stress (Limonta et al., 2019). In contrast, some species appear to experience little or no adverse effects from ingesting MPs. For example, exposing the crustacean Gammarus pulex to MPs did not affect its survival, moulting, metabolism or feeding activity (Weber et al., 2018). Accordingly, further investigations will be necessary to establish whether the uptake of suspended MPs by sea cucumbers via the mouth is potentially hazardous to them and whether the ingestion of these MPs has implications for human health given that the intestines of sea cucumbers are consumed by humans in some countries (Mao et al., 2015).

We anticipate that the findings of the present study will provide a solid basis for further determination of the nutritional requirements that might influence the growth rate of sea cucumbers. These include the potential influence of differences in the type and concentration of particles and/or nutrients, and the length of time to which A. japonicus individuals are exposed to such particles suspended in the water.

In conclusion, in this study, we established that the commercially important sea cucumber A. japonicus takes up suspended particulate matter from the surrounding water via the mouth, a mechanism that, to the best of our knowledge, has not previously been reported. Our observations revealed that A. japonicus can ingest not only suspended nutrients (without preferential selection) but also non-nutrient suspended particles via the mouth, and that the amount of particles ingested is positively correlated with the concentration of suspended particles. However, further investigation will be necessary to establish whether differences in the size and shape of the suspended particles affect the ingestion rate of A. japonicus. The findings of this study will, nevertheless, provide a basis for further investigations regarding the influence of nutrients or particles in the water environment on the distribution (i.e. whether sea cucumbers show habitat preference for regions characterised by high concentrations of suspended particles/nutrients) and growth performance of this sea cucumber.

We would like to thank Jinchun Sun for her assistance in obtaining the materials required for the experiment.