The effect of temperature and sand substrate in tanks on the growth and survival of the warty sea cucumber Neostichopus grammatus

ABSTRACT

Water temperature and the presence of sand substrate are considered critical environmental factors affecting the growth of cultured sea cucumbers. This study investigated the effects of water temperatures and the presence or absence of sand substrate in tanks on the growth of N. grammatus fed an equal ration of abalone waste for eight weeks. Sea cucumbers with a mean weight of 8.34 g were used for the study. The treatments used in the study were Ambient water temperature + sand (A + S), Ambient water temperature without sand (A-S), Cold water temperature + sand (C + S), and Cold-water temperature without sand (C–S). The cold/constant water temperature was 16°C and was representative of winter conditions while the ambient/fluctuating water temperature fluctuated between 16.4 and 18.7 °C and was representative of spring conditions. At the end of the study, the sea cucumbers in A + S and C + S treatment groups exhibited an overall weight increase of 21.08% and 6.18%, respectively. The sea cucumbers reared in ambient water temperature exhibited significantly higher mean weight than those reared in cold water (F_{1, 66} = 5.69; p = 0.02), and sea cucumbers reared with sand substrate had significantly higher mean weight compared to those without sand substrate (F_1,66 = 19.06; p = <0.001). This study has shown that growth rates of N. grammatus in tanks were higher in ambient temperatures and that stress was reduced in the presence of sand, a habitat that mimics its natural environment. This implies that sand substrate may be necessary for the farming of N. grammatus.

KEYWORDS:

• Growth
• Neostichopus grammatus
• survival
• sand substrate
• water temperature

Introduction

Sea cucumbers are prized delicacies in many parts of Asia and are perceived as a luxury food (Fabinyi 2012; Purcell 2014). Sea cucumbers are in high demand, leading to the enlargement of sea cucumber wild stocks and the over-exploitation of sea cucumber fisheries globally (Anderson et al. 2011; Purcell et al. 2013). Consequently, areas where sea cucumbers are naturally abundant, are overfished, making it challenging to obtain a reasonable catch of sea cucumbers (Purcell et al. 2013). Sea cucumber species of commercial importance, such as Holothuria scabra, Holothuria lessoni, and Holothuria nobilis, are sold dried for US$373–1237kg⁻¹, US$503–635 kg⁻¹ and US$106–338 kg⁻¹ respectively (Purcell et al., 2023). Consequently, sea cucumber aquaculture has been developed to supplement catch from sea cucumber fisheries, thereby reducing pressure on sea cucumber fisheries while making financial gains (Han et al. 2016). Recent years have seen a rise in studies on growth improvement/ enhancement in sea cucumbers of commercial interest and, increasingly, even those of less commercial importance (Cutajar et al., 2022; Neofitou et al., 2019; Onomu et al. 2023; Onomu et al. 2024; Sun et al., 2020and Tolon et al., 2017).

Water temperature and the presence of sand substrate are considered critical environmental factors affecting the growth of cultured Apostichopus japonicus and Holothuria scabra (Battaglene et al. 1999; Dong et al. 2008; Ji et al. 2008; Robinson et al. 2013). Sea cucumbers are ectothermic, i.e. their body temperature conforms to the ambient water temperature. The ambient water temperature regulates their biochemical and physiological processes affecting their feeding behaviour, metabolism, growth, and survival (Hamel and Mercier 1998; Shao et al. 2015; Sin et al. 2019). Sea cucumbers stop feeding when the water temperature becomes very high; for example, at a high temperature (above 20.1 °C), juvenile A. japonicus aestivates (Yang et al. 2005). At temperatures above 30 °C, the burying cycle of H. scabra is altered, causing them to remain on the surface, while at reduced temperatures, H. scabra buries itself for extended periods (Mercier et al., 1999; Purcell and Kirby, 2006). Aestivation has been linked to poor functioning of digestive enzymes (Jiang et al. 2007). Also, when sea cucumbers stop feeding and remain inactive in the case of aestivation, growth becomes stunted, and the animals begin to lose weight (Ji et al. 2008).

Sand-muddy sediment substrate is a requirement in the culture of some species of sea cucumbers, especially H. scabra (Robinson et al. 2013). The type of substrate needed by a species of sea cucumber for growth optimisation may be influenced by the substrate on which they are found naturally. Studies have reported an increase in the growth of H. scabra when sand is placed at the bottom of tanks compared to bare tanks (Hamel and Mercier 1998; Battaglene et al. 1999; Robinson et al. 2013). This may be because most sea cucumbers inhabit sandy/ muddy habitats with sand as part of their natural diet (Mercier et al. 1999; Dissanayake and Stefansson 2012; Liu et al. 2013). The sea cucumber in tanks with sand substrate benefits from the presence of sand, which provides shelter and acts as additional food (Battaglene et al. 1999). The presence of sand substrate leading to improved growth of sea cucumber has been attributed to sand aiding in the food digestion (Watanabe et al. 2012). It has also been suggested that sand substrate provides a large surface area for the proliferation of bacteria /microphytobenthic primary producers, which are diets of sea cucumber in their natural environment (Robinson et al. 2013).

Neostichopus grammatus is a species of sea cucumber endemic only to South Africa; where it is found from Cape Agulhas in Cape Town (34°50′0″S, 20°00′5″E) to Cape Vidal in Kwazulu Natal (28°7′26″S, 32°33′26″E) (Thandar 1987). They are red in colour, though their colour may vary from light pink to pale brown (Thandar 1987) . The maximum length recorded for N. grammatus is 150 mm (Deichmann 1948), with body size at first maturity recorded as 3-3.9 cm^3, approximately 2.5–4.0 g drained wet weight. Gametogenesis takes place from July to September with spawning occurring within October to December (Foster and Hodgson, 1995). In Port Elizabeth (one of the places N. grammatus are found in South Africa), the seawater temperature ranges from 16 to 24°C.

Interest has recently been aroused in the potential of N. grammatus as an aquaculture candidate (Onomu et al. 2023; Onomu et al., 2024). Not much literature is available for N. grammatus neither is there information on the temperature suitable for its culture, growth, and thermal tolerance. Also, it is unknown if sand substrate will be beneficial to N. grammatus, as they are found partly covered with sand underneath rocks in their natural habitat, and a high percentage of the gut content is made up of sand (Foster and Hodgson 1996; Onomu et al. 2023). This study investigates the effects of ambient and cold-water temperatures and the presence or absence of sand substrate in tanks on N. grammatus growth, providing insight to farming of this species and to the coculture of abalone with N. grammatus.

Materials and methods

Collection of animals

Sea cucumbers of about 8.34 g mean weight were collected in July 2021 by divers from Nahoon's reef (32°59′40″ S, 27°55′57″ E), East London, Eastern Cape South Africa. The water temperature at the site of collection was 17.6 °C. They were transported (within one hour) to the study site (Wild Cost Abalone (Pty) Ltd, 32°45′06″S, 28°16′28″E) in buckets containing seawater. Sea cucumbers were acclimatised in holding tanks for about two weeks before commencing the experiment; the sea cucumbers were fed on abalone (Haliotis midae) faecal waste. The temperature of the seawater was not controlled during this period. Abalone faecal waste was fed to the sea cucumbers as this diet is acceptable to the sea cucumber based on ingestion rates reported by (Onomu et al. 2023). Before the commencement of the experiment, the sea cucumbers were starved for 48 hours for the gut to be utterly voided of food, according to Zamora and Jeffs (2012).

Experimental design

The experiment was conducted over eight weeks with an experimental design consisting of four treatments and three replicates.

The treatments used in the study are:

• Ambient water temperature + sand (A + S),

• Ambient water temperature without sand (A-S),

• Cold water temperature + sand (C + S), and

• Cold-water temperature without sand (C–S).

A cold-water temperature of 16 °C was selected for the study; this was achieved by chilling the seawater using a chiller (SIRAC heat pump, South Africa). The cold-water temperature was chosen to be representative of winter conditions, as the average seawater temperature in East London (the study site) falls as low as 16 °C during winter. The experiment was conducted during spring when the ambient water temperature fluctuated between 16.4 and 18.7 °C. Therefore, the ambient water temperature was representative of the spring conditions.

Twelve tanks were used for the experiment. Each tank measures 24.5 cm × 23.3 cm × 23.2 cm (L × B × H). Each tank had a separate water supply, drainage, airline, and dome-shaped PVC pipe. The dome-shaped PVC served as a shelter and substrate for attachment and hide-out for the sea cucumber (Onomu et al. 2023). Each tank contained approximately 13 L of water, and 580 ml of water flows into the tank per minute. The total water volume in each tank was renewed approximately every 22 min. Six sea cucumbers with an average weight of 8.34 g were stocked per tank. The individual mean tank weights ranged from 47.74 to 51.55 g (wet weight), leading to a stocking density of 837.5–850 g m^-2 of the tank floor. Dry sand from the beach was placed at the bottom of the tanks, which acted as a substrate for the sea cucumbers according to the treatment. The sand was untreated, i.e. not washed, acid-washed, or sieved (as the grain size particle was consistent) before use, according to Onomu et al. (2023) and Robinson et al. (2013). The mean dissolved oxygen over the duration of the experiment was 8.05 ± 0.56 mg L⁻¹.

Feeding and maintenance of tanks

During the experimental period, sea cucumbers were fed abalone waste ad libitum with 50 mL of abalone waste (20 g dw per tank) weekly, which is approximately 5% of their body weight per day. The amount of food supplied is in line with Onomu et al. (2023) who reported that N. grammatus consumed 3.4% of their body weight when fed abalone waste.

The water inlet and airflow were interrupted before the feed was placed in tanks and 30 minutes after feeding so as to allow the feed settle at the bottom of the tank. Tanks were cleaned once every two weeks. Abalone waste was used as feed in this study as it is reported to be acceptable and palatable by the sea cucumber (Onomu et al. 2023). The sand was changed fortnightly while cleaning tanks as it was impossible to clean tanks without flushing out sand.

Measurement of weight

Sea cucumbers in each tank were measured every four weeks (g ± 0.01) with a weighing balance (Mettler PE 3600). Before the sea cucumbers were weighed, they were kept on a sponge for about two minutes to get rid of surface water and water from the cloaca and coelomic cavities.

Measurement of growth

Weight gain was calculated as $\mathrm{Mean}\mathrm{weight}\mathrm{gain}(\text{\%})={\displaystyle \frac{w_f-w_i}{w_f}\ast 100}$Specific growth rate was calculated as daily growth increase according to Bell et al. (2010) $\mathrm{Mean}\mathrm{SGR}(\text{\%}{\mathrm{d}}^{-1})={\displaystyle \frac{(\ln w_1-\ln w_0)}{\mathrm{days}\mathrm{of}\mathrm{trial}}\ast 100}$Where w_f and w_i are the mean final and initial weights of the sea cucumbers

Where w_o and w₁ are the mean initial and final body weight of the sea cucumbers.

Statistical analysis

At the beginning of the experiment, the sea cucumber's initial weight in each tank was analysed with a one-way ANOVA to ensure there was no bias in the weight of sea cucumbers per tank. At the end of the experiment, a two-way ANOVA was used to analyse the effect of temperature and sand substrate on the growth of animals. Tukey's post hoc test was used for pairwise comparison when a significant ANOVA result was obtained.

Results

Survival

The survival of sea cucumbers was 100% in all treatment groups. One sea cucumber was lost as an escapee (down the outflow pipe) in the C + S treatment group close to the end of the study and was not replaced or recovered during the study. The temperatures used and the presence/ absence of sand substrate had no effect on the survival of the sea cucumber.

Growth

The SGR of sea cucumber was 0.34 ± 0.08; – 0.07 ± 0.17; 0.09 ± 0.13 and – 0.38 ± 0.13% d⁻¹ for A + S, A-S, C + S, and C–S respectively. At the end of the study, both factors, individually influenced the growth of sea cucumbers temperature (F_1,66= 5.69; p = 0.02) and sand (F_1,66 = 19.06; p = <0.001). The interaction term between both factors was insignificant (F_1,66 = 2.61; p = 0.11) (Figure 1). The sea cucumbers in A + S and C + S treatment groups exhibited an overall weight increase of 21.08% and 6.18%, respectively. While sea cucumbers in A-S and C–S exhibited a reduction of 5.18 and 22.94%, respectively. However, post-hoc pair-wise comparison showed no significant difference between the mean weight of sea cucumbers in the A + S and A – S treatment groups (p = 0.055) (Figure 2). Sea cucumbers in the C + S treatment group had significantly higher mean weight than those in the C–S treatment group (p < 0.001). While, sea cucumbers in the A – S treatment group had significantly higher mean weight than those in the C–S treatment group (p = 0.005). However, the mean weight of sea cucumbers in the A + S treatment group was similar to those in the C + S treatment (p = 0.599). (Figure 2).

Figure 1. Interaction plot of final mean weight (± SE) of sea cucumbers reared in ambient or cold-water temperature with/without sand substrate over eight weeks.

Figure 2. The mean weight (g) of sea cucumbers over eight weeks. Data are presented as mean (±SE; n = 15). Different letters beside each line represent a significant difference between treatments (Tukey, p < 0.05). A + S = Ambient water temperature + sand; A − S = ambient water temperature – sand; C + S = cold water temperature + sand; C-S = cold water temperature – sand.

Discussion

Sea water temperature influences the feeding behaviour, metabolism, growth, and survival of sea cucumbers (An et al. 2007; Dong et al. 2008; Ji et al. 2008; Meng et al. 2009). The temperature range at which sea cucumbers perform best varies from species to species and geographical location (Yanagisawa 1998). The thermal tolerance range in A. japonicus is between 0 and 30 °C, but growth occurs from 12 to 21 °C (Yang et al. 2005). However, the optimal temperature for juvenile growth in A. japonicus ranges from 15 to 18 °C (Dong et al. 2006). At a temperature above 21 °C, juvenile A. japonicus aestivates (Yang et al. 2005). The present study shows that N. grammatus reared in ambient temperature had significantly higher growth than those reared in cold water, reflecting that N. grammatus perform better in warmer temperatures than cold. This is in agreement with several studies report that the growth of A. japonicus is reduced during the winter season (Yu et al. 2014; Ren et al. 2017).

As with A. japonicus, water temperature fluctuation between the thermal tolerance of sea cucumbers appears to enhance growth compared to constant temperature in N. grammatus (Dong et al. 2006).

The presence of sand substrate in N. grammatus sea cucumber tanks also led to significantly increased growth compared to bare tanks. It could be that sand was beneficial in trapping food and organic matter thus making them available to the sea cucumbers for longer periods. It could also be that the sand in the tank simulated the natural habitat of the sea cucumber; as such, the sea cucumbers were less stressed. In nature, N. grammatus are cryptic and are found underneath rocks covered with sand for camouflage, just as the tropical H. scabra. It could be that N. grammatus are able to sense the presence of sand and become stressed when they feel exposed. Although sand in the sea cucumber tank has been shown to improve growth, the mean weight of sea cucumbers in the A + S treatment was higher but not significantly different from those in the A – S treatment. It could be that the ambient water, which was a bit warm, compensated for the effect the absence of sand is expected to have on the sea cucumbers in the short term. Over a long term, the effect of the absence of the sand may become evident as there was a reduction in weight in A-S treatment. Also, the statistical value (p = 0.055) only deviated slightly from being significant during the short period and could be considered biologically significant.

In the present study, sea cucumbers in C + S treatment had significantly higher growth than those in C–S at the end of the study. The result suggests that sea cucumbers in cold water temperatures need sand for growth. However, no significant difference in mean weight was observed in the case of sea cucumbers in the A + S and C + S treatments. This means that when sand was present in tanks, the effect of the cold-water temperatures on the sea cucumber was subdued. The exact effect the cold-water temperature used in this study has on the physiological and metabolic response of N. grammatus is unknown. Based on the result, it could be deduced that sand has some beneficial effects on sea cucumbers, especially those in cold water temperatures. This finding is supported by the result of the A-S and C–S treatments, where sea cucumbers in A – S had significantly higher growth than those in the C–S treatment since the C–S treatment was free of sand to mask the effect of the cold-water temperature.

Although the sea cucumbers in this study were not starved to empty gut content at the end of the study, the gut content of all sea cucumbers used in the study was observed to be empty (as they were dissected after weight measurement). As such, the weight recorded at the end of the study was not due to the content of the gut. The empty gut content of the sea cucumbers without being deliberately starved may be due to significantly reduced feed in the tanks (as no feed was added to the tanks prior to weight measurement). It may also be that the sea cucumbers had excreted all gut content and had not ingested additional feed prior to weight measurement.

The SGR of sea cucumbers in the A + S treatment group (0.34% d⁻¹) in this study outperformed those of N. grammatus (−0.31% d⁻¹) reared in tanks void of sand substrate and fed with abalone waste (Onomu et al. 2023). It similarly outperformed those of Yuan et al. (2006) who reported an SGR of – 0.7% d⁻¹ when A. japonicus was fed on dried bivalve faeces. It also outperformed those of Holothuria tubulosa (0.07 g d⁻¹) and Holothuria poli (−0.20 g d⁻¹) that received mussel waste as feed beneath long-line mussel farms (Grosso et al. 2023). The SGR similarly compares well with Slater et al. (2009) who reported an SGR of 0.32% d⁻¹ when Australostichopus mollis was fed a mussel waste diet. However, Zhou et al. (2006) reported an SGR 0f 0.5% d⁻¹ for A. japonicus cocultured in tanks with scallop Chlamys farreri and received faeces of the scallop as feed.

The result of this study implies that sand substrate may likely be necessary for land-based rearing of N. grammatus and coculture with abalone (Haliotis midae) for optimal growth. This is in line with Onomu et al. (2024), who reported that sea cucumber, N. grammatus cocultured with abalone (Haliotis midae) led to a reduced frequency of tank cleaning at the expense of the sea cucumber growth because they were reared in tanks void of sand substrate. However, incorporating sand in commercial land-based operations may not be realistic as this may clog the drainage system. It may be cumbersome in terms of labour, as the sand substrate requires regular cleaning and maintenance to prevent the accumulation of organic debris, which may negatively impact water quality and sea cucumbers’ health. Ambient water temperature best supports the growth of N. grammatus, so during winter, when water temperatures become low, heating up of the rearing water may be required to sustain growth. However, heating rearing water may be expensive, adding to the cost of production. Generally, it is necessary that the temperature tolerance of sea cucumbers be ascertained as they differ in species and geographical location and play a role in growth and development. Temperate sea cucumber species may require chilling of rearing water, especially in land-based rearing. Regarding the coculture of abalone and sea cucumber, both species must be compatible, requiring similar water temperatures. Heating/ chilling of rearing water may be required for sea cucumbers in coculture with abalone (depending on the species and geographical location), which may be detrimental to the growth and well-being of abalone if they do not require a similar temperature range.

Conclusion

This study has shown that N. grammatus is affected by water temperature just like other species of sea cucumber. N. grammatus thrives in warmer water temperatures than cold water temperatures, and the presence of sand substrate in tank seems to be a necessity which supports the growth of N. grammatus. To improve the culture success of N. grammatus, future research should investigate the thermal tolerance range of N. grammatus and the optimum temperature at which N. grammatus would grow best as a narrow thermal range was evaluated in this study.

Acknowledgement

The authors would like to appreciate the management of Wild Coast Abalone farm and staff, including Sanet Petschel, Noel Marshal, and Johnathan Philander, for the provision of experimental animals, infrastructure, and facilities. Immense appreciation also goes to Daphne Taylor, for her support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data set used in the study is available from authors upon request.

Additional information

Funding

This project is part of Aqua Vitae and was partly funded by European Union’s Horizon 2020 Research and Innovation Programme under [grant number 818173]. The University of Fort Hare’s Govan Mbeki Research and Development Center (GMRDC) and the National Research Foundation’s (NRF) Research Technology Fund [grant number 135443] are acknowledged for the provision of a bursary and funding.