Inspection of Gonadal Maturation in Mediterranean Sea Cucumber (Holothuria tubulosa Gmelin 1791) Under a Constant Temperature Regime ^‡

Abstract

This preliminary study explored the effect of a constant thermal regime on gonadal maturation and spawning readiness in the temperate sea cucumber Holothuria tubulosa Gmelin, 1791, under aquaculture conditions. Adult sea cucumbers were conditioned at a constant mean temperature of 23.4 ± 0.4 °C for 30, 60, 90, and 120 days following gonad regression, and gonadal development was assessed using the gonad index (GI). Survival and growth performance were used as references for the tolerance and suitability of sea cucumbers. The effective accumulated temperature (EAT) in the thermally treated groups ranged from 339.9 to 1370.4 °C·d, significantly exceeding that of the wild controls (17.7–247.5 °C·d). While no significant GI changes occurred at 30 and 60 days, individuals exposed for 90 and 120 days exhibited marked gonadal development, with a mean GI of 6.5% at day 120 compared to 3.45% in wild counterparts. Despite accelerated gonad growth, full maturation and spawning were not achieved within the experimental timeframe, indicating that additional environmental and nutritional cues are required. The specific growth rates up to 0.32% d⁻¹ and 100% survival suggest good thermal tolerance of this species. These findings demonstrate that constant thermal regimes may shorten the gonadal development period in H. tubulosa, offering the potential to advance broodstock conditioning. Future studies integrating optimised diets and photoperiod manipulation may enable off-season spawning and improve aquaculture efficiency.

1. Introduction

The notable market value, and global demand of sea cucumbers recently exceeding 110,000 metric tons [1] have generated interest in the aquaculture of multiple species prominently in East and Southeast Asia, including China, Japan, South Korea, and Vietnam, as well as emerging regions such as USA, Turkey, Italy, and Portugal [2,3,4,5,6,7].

The region-specific reproductive biology of commercial sea cucumbers is a key determinant of the economic feasibility of aquaculture. In tropical regions, species such as Holothuria scabra (sandfish) have become the leading species in commercial aquaculture because of their favourable reproductive traits and high market value. The year-round asynchronous but continuous gonadal development and multiple spawning ability of H. scabra contribute to the rapid advancement and economic viability of tropical sea cucumber aquaculture [8,9]. In contrast, temperate species, such as Apostichopus japonicus, Holothuria tubulosa, Holothuria poli, Holothuria mammata, Holothuria forskali, Holothuria arguinensis, Cucumaria frondosa, Cucumaria japonica, Parastichopus californicus, and Apostichopus parvimensis, which are native to the Northern Hemisphere, exhibit a seasonal reproductive cycle, typically spawning once per year, with gonad development closely tied to region-specific environmental conditions [2,4,10,11,12,13,14,15,16]. The seasonal availability of gametes restricts the timeframe for hatchery activities, a constraint that is further complicated by asynchronous gonad maturation between wild males and females, which necessitates collecting larger numbers of broodstock to ensure successful synchronised spawning and ultimately reduces overall production efficiency [17].

H. tubulosa, a temperate sea cucumber species widely distributed along the northeastern coasts of the Mediterranean Sea and the North Atlantic Ocean, has emerged as a promising candidate for high-value aquaculture [18,19]. It is in high demand in East Asian markets for culinary purposes and has been identified as a potential species for integrated multi-trophic aquaculture (IMTA) systems [20,21] in the Mediterranean Sea. In terms of reproductive biology, H. tubulosa exhibits seasonal patterns of gonadal development and spawning similar to those of other temperate species. In the Mediterranean, gonadal maturation typically occurs from February to July, spanning approximately 150–180 days and coinciding with a gradual increase in seawater temperature. Peak reproductive activity, indicated by a maximum gonadosomatic index, generally occurs in July and August, resulting in spawning events [2,22,23,24]. Gonads remain in a resting phase lasting approximately six months, during which no reproductive activity occurs between October and March. This extended period of inactivity, consistent with that observed in other temperate sea cucumber species, poses a significant limitation for hatchery operations, leading to prolonged production interruptions and reduced economic efficiency in aquaculture settings.

Numerous studies investigating gonadal development in sea cucumbers have reported a strong and direct correlation between gametogenesis (including vitellogenesis and spermatogenesis) and various environmental factors, such as seawater temperature, photoperiod, salinity, pH, and the quantity and type of nutrients available in the environment. Temperature is generally considered the primary environmental factor controlling gametogenesis [25]. Peak gonadal maturation and spawning events are typically observed during periods of elevated seawater temperatures [9,26,27]. Moreover, several studies have demonstrated that seasonal fluctuations in seawater temperature, which vary across regions, significantly influence the timing and duration of the reproductive cycle in sea cucumbers [2,16,25,28,29].

There are a limited number of studies focusing on thermal manipulation to accelerate gonadal development in sea cucumbers as part of broodstock management strategies. One of these studies reported that the broodstock of the tropical species H. scabra, maintained under controlled thermal conditions in tanks, exhibited earlier gonadal development and spawning induction than their natural reproductive cycles [30]. However, comparable evidence for temperate species is lacking. Existing applications of broodstock conditioning are limited in number and highly species-specific, highlighting the need for further research to optimise protocols tailored to different species, particularly those inhabiting temperate regions.

The main objective of this study was to examine the influence of a constant thermal regime on gonadal maturation and spawning readiness in H. tubulosa over four experimental durations (30, 60, 90, and 120 days) in aquaculture systems. Moreover, the tolerance and adaptability of the species to sustained thermal conditions were evaluated based on their survival rates and specific growth performance.

2. Materials and Methods

2.1. Wild Broodstock Collection, Acclimation, and Gonad Regression

Mature individuals of H. tubulosa, approximately over 100 g of wet weight that exceeded the reported size at first sexual maturity [2,31], were hand-picked by SCUBA diving from the depths of 5–10 m in coastal waters of the Aegean Sea (Mediterranean) near the Urla district of İzmir, Türkiye (38°21′55.5″ N, 26°46′14.2″ E). The specimens were transported to the Ege University, Faculty of Fisheries, Aquaculture laboratories located in the Urla district within approximately 30 min using aerated containers. The collected sea cucumbers were transferred to fibreglass acclimation tanks (200 × 200 × 100 cm), where the water was continuously exchanged with 10-µm filtered ambient seawater, and constant aeration was provided to maintain optimal water quality. The mean seawater temperature in the acclimation tanks was 12.1 ± 0.6 °C. Dissolved oxygen, salinity, and pH levels were maintained at an average of 7.19 ± 0.26 mg/L, 38.70 ± 1.82 ppt, and 7.69 ± 0.14, respectively. Sea cucumbers were kept in acclimation tanks for one week prior to the gonad regression procedure, until no observable signs of stress, such as impaired crawling and movement, delayed righting response, or altered foraging and defecation, were detected. Sea cucumbers were fed ad libitum once a day using dried and powdered macroalgae (Ulva sp.) during this period.

Sea cucumbers were subjected to a gonad regression protocol following the acclimation period, as outlined by Andriyono et al. [32], to decrease the gonadosomatic index (GI) to near zero prior to the constant thermal regime experiment. This procedure involved exposing the wild-collected and acclimated broodstock to thermal stimulation, increasing the water temperature by 3–5 °C with aquarium heaters to induce the release of residual gametes and initiate gonadal regression into the resting phase. Subsequently, the sea cucumbers were maintained in filtered seawater at a mean temperature of 12.0 ± 1.9 °C, compatible with the ambient seawater conditions associated with gonadal resting in H. tubulosa [2] for 15 days before the experiments. No feed was provided for 15 days during the gonad regression procedure, as food scarcity in sea cucumbers has been reported to induce gonadal regression by redirecting energy away from the development of the reproductive tissue [33,34]. Water quality parameters were closely monitored, and the sea cucumbers were observed daily to ensure the absence of stress indicators. The completion of gonad regression was confirmed through dissection and visual inspection of randomly selected individuals (n = 10), which verified the absence of mature gonads.

2.2. Experimental Design

Trials under a constant thermal regime were conducted in triplicate using polyester tanks measuring 200 × 200 × 100 cm (length × width × depth), with a bottom surface area of 4 m², over a 120-day period from 18 January to 17 May 2022. The experimental timeline was aligned with the natural gonadal maturation period observed in the wild [13,35]. Natural marine sand, which was disinfected with a 10% sodium hypochlorite solution for six hours, rinsed with freshwater, and sun-dried, was evenly distributed at the bottom of the treatment tanks to a uniform depth of approximately 10 cm. The trial tanks were filled with filtered seawater, and approximately 50% of the water volume in each tank was replaced daily with pre-heated seawater to match the selected temperature during the experimental period. Water temperature was monitored daily using a digital thermometer (HI98501, Hanna, RI, USA) with an accuracy of ±0.2 °C. Continuous aeration and effective water circulation were achieved by evenly positioning three air stones in each tank to ensure adequate dissolved-oxygen levels and uniform temperature distribution. A timer-controlled daylight illumination system positioned above the tanks provided a light intensity of 250 lx to simulate the natural light conditions of sea cucumbers’ shaded habitats, operating under a 12L:12D photoperiod.

Twenty randomly selected adult sea cucumbers from the acclimation tanks, with a mean initial wet weight of 132.05 ± 23.55 g, were stocked in each treatment tank at an optimal stocking density of 500 g/m² [36] (n = 60). The initial wet weights (W_i) of the adult sea cucumbers were measured using a precision balance (Scout Pro 4000, OHAUS, Nänikon, Switzerland; accuracy: 0.1 g) following the wet weighing method described by Dong et al. [37]. The water in the experimental tanks with sea cucumbers was gradually heated by 1–2 °C per day (7 days), from 12.0 ± 1.9 °C to the target temperature of 22–24 °C which is in the range temperature of gonad maturation and spawning period in nature [2,14,38]. The experimental period was initiated after the water temperature in the tanks reached the target level, and the target mean temperature stability was maintained using a laboratory climate-control system throughout the duration of the trials [30].

Dried and powdered macroalgae (Ulva sp.) was supplemented daily at a rate of approximately 2% of the total biomass [39,40,41] and uneaten feed was not removed, allowing the accumulation of natural benthic food sources within the sediment.

A comparative analysis of gonadal development between thermally treated and wild H. tubulosa individuals (control) was conducted by sampling 15 individuals from the natural habitat that were identical to the wet weights of the trial specimens at each time point. Wild samples served as references for natural gonadal development under fluctuating ambient conditions.

2.3. Gonadal Maturation and Growth Performance

Gonadal maturation was quantitatively assessed using the gonad index (GI) of 15 randomly selected individuals on days 0 (D₀), 30 (D₃₀), 60 (D₆₀), 90 (D₉₀), and 120 (D₁₂₀) of the experiment. Each individual was ventrally dissected from the anus to the mouth, allowing for the careful removal of the gonads, viscera, and respiratory trees to ensure consistency in calculating reproductive indices, as viscera and respiratory trees can vary significantly in mass due to feeding status or water content, potentially confounding comparisons of gonad investment across individuals. The excised gonads and body wall were gently blotted dry with a clean towel and weighed separately using a precision balance to obtain gonad weight (GW) and gutted body weight (GBW) measurements. The gonadosomatic index was calculated using the following formula:

$$\mathrm{G}\mathrm{I}={\displaystyle \frac{\mathrm{G}\mathrm{W}}{\mathrm{G}\mathrm{B}\mathrm{W}}} \times 100$$

(1)

The survival rate (SR) of sea cucumbers in each group was calculated using the following formula:

$$\mathrm{S}\mathrm{R}\left(\mathrm{\%}\right)={\displaystyle \frac{{\mathrm{N}}_{\mathrm{f}}}{{\mathrm{N}}_{\mathrm{i}}}} \times 100$$

(2)

where N_i represents the initial number of individuals at the beginning of the experiment, and N_f denotes the number of individuals remaining at the end of the experimental period.

The growth performance of the individuals in the treatment tanks was evaluated by calculating the specific growth rate (SGR) using the following formula:

$$\mathrm{S}\mathrm{G}\mathrm{R}\left(\mathrm{\%}{\mathrm{d}}^{-1}\right)={\displaystyle \frac{\ln {\mathrm{W}}_{\mathrm{f}}-\ln {\mathrm{W}}_{\mathrm{i}}}{\mathrm{t}}} \times 100$$

(3)

where W_i is the initial wet weight (g), W_f is the final wet weight (g), and t is the duration of the trial in days.

Effective Accumulative Temperature (EAT) values were calculated for both tank-reared and wild sea cucumber broodstock using the following formula:

$$\mathrm{E}\mathrm{A}\mathrm{T}=\mathrm{N}\left(\mathrm{T}-\mathrm{C}\right)$$

(4)

where N is the duration of the experiment (days), T is the average water temperature during the trial period, and C is the biological zero, which is the threshold temperature at which gonadal development is initiated. The biological zero for H. tubulosa was determined to be 12 °C, which corresponds to the average seafloor temperature in February-March along the Aegean Sea coast, when gonad development typically begins [2].

Seafloor water temperatures at the control group sampling site were obtained as daily averages between 18 January and 17 May 2022 using remote-sensing data provided by the Copernicus Marine Environment Monitoring Service [42]. The mean temperature data were grouped into 30, 60, 90, and 120 days, corresponding to the respective thermal exposure periods.

2.4. Statistical Analysis

Normality and homogeneity of variance were evaluated using Kolmogorov–Smirnov and Levene tests, respectively, and where necessary, sqrt (x) and arcsin (x) transformations of data were performed before the analyses. In cases where heterogeneity of variances occurred after data transformation, the Mann–Whitney U test (two groups) and Kruskal–Wallis test (three or more groups) were used. Significant differences between treatments were determined using One-way ANOVA (parametric) or Kruskal–Wallis (non-parametric) tests, followed by post hoc multiple comparisons with Tukey’s HSD test or Dunn’s test with Bonferroni correction. Student’s t-test was used to compare the differences between the means of the two groups. Seafloor temperature data were analysed using the Kruskal–Wallis test to assess temporal changes across four defined periods over the 120-day experimental duration. Descriptive statistics, including means and standard deviations, were calculated for each period. Statistical significance was set at p < 0.05. All analyses were performed using SPSS v27 software.

3. Results

Mean water temperatures during the thermal exposure periods ranged narrowly from 23.3 ± 0.5 °C to 23.5 ± 0.4 °C (D30–D120), with no statistically significant differences observed among the time points (p > 0.05) (Figure 1).

The seafloor temperatures at the sampling site of the control group ranged from 11.5 to 19.6 °C during the 120-day experiment, showing a consistent upward trend (Figure 2a). The mean seawater temperatures to which the wild specimens were exposed in nature during the respective trial periods D₃₀, D₆₀, D₉₀, and D₁₂₀ were 12.6 ± 0.5 °C, 12.8 ± 0.5 °C, 12.9 ± 0.9 °C, and 14.1 ± 2.2 °C, respectively. Post hoc pairwise comparisons with Bonferroni correction identified that the increase in mean temperature that the wild sea cucumbers were exposed to during the 30-, 60-, and 90-day periods was not statistically significant (Kruskal–Wallis test, df = 2; H = 4.062; p > 0.05). In contrast, a significant difference was observed at 120 days compared to the preceding periods (Kruskal–Wallis test, df = 3; H = 21.172; p < 0.001) (Figure 2b). Overall, the results demonstrated a consistent and significant warming trend in seafloor temperatures over the monitored periods; however, the bottom seawater temperatures remained significantly below the mean water temperatures of the thermally treated group (p < 0.05).

The EAT in the constant thermal regime tanks ranged from a minimum of 339.9 °C·d in D₃₀ to a maximum of 1370.4 °C·d in D₁₂₀. Based on the seafloor temperatures at the control site, the EAT values ranged from 17.7 °C·d (30 days) to 247.2 °C·d (120 days) (

Table 1. Effective accumulative temperature for H. tubulosa broodstock in constant thermal regime tanks and in nature for 120 days between 17 January and 18 May 2022.

		D30	D60	D90	D120
Effective accumulative temperature (°C·d)	Trial tanks	339.9	684.6	1031.4	1370.4
	Control	17.7	45.9	82.5	247.5

). Statistically significant differences in EAT values were detected between the control and experimental groups during each period (p < 0.05).

The survival rate of individuals during the trial period was 100%. At the end of the 60-, 90-, and 120-day thermal exposure periods, the final mean wet weights were significantly higher than the initial values (p < 0.05), with the exception of D₃₀. Furthermore, the final wet weights of the sea cucumbers differed significantly among all exposure durations (p < 0.05). The specific growth rates ranged from 0.03% d⁻¹ to 0.32% d⁻¹ across the various thermal exposure periods, indicating growth in all individuals (

Table 2. Mean final wet weights (±SD) and specific growth rate (SGR, % day⁻¹) of H. tubulosa broodstock maintained under a constant thermal regime for 30, 60, 90, and 120 days (D₃₀–D₁₂₀). Superscript letters indicate statistically significant differences among groups within the same row (p < 0.05).

	D30	D60	D90	D120
Mean final wet weight (g)	133.60 ± 24.51 a	158.16 ± 35.18 b	168.53 ± 35.99 c	176.18 ± 33.23 d
SGR (% d−1)	0.03 a	0.32 b	0.27 b	0.24 b

).

At the initial sampling (D₀), all individuals had either empty or unpigmented gonad tubules or lacked observable gonadal structures. The mean initial gonad weight was 0.08 ± 0.03 g, and the average gonad index was 0.12 ± 0.04%, which confirms the absence of developed gonads and indicates a baseline reproductive condition prior to constant thermal exposure. The final mean gonad index of the thermally treated sea cucumbers were 0.18 ± 0.12% (D₃₀), 0.43 ± 0.11% (D₆₀), 2.03 ± 0.87% (D₉₀), and 6.50 ± 1.94% (D₁₂₀). No statistically significant changes were observed between the initial and final gonad indices at D₃₀ and D₆₀ (p > 0.05). In contrast, the sampled sea cucumbers at D₉₀ and D₁₂₀ showed significant increases in gonad index values over the experimental period (p < 0.05), reflecting measurable gonad development (Figure 3).

The mean gonad index of wild sea cucumber specimens was 0.20 ± 0.11%, 0.39 ± 0.12%, 1.06 ± 0.61%, and 3.45 ± 0.58% at 30, 60, 90, and 120 days, respectively. No statistically significant differences were observed in gonad index values between the initial measurement and those taken at 30, 60, and 90 days (p > 0.05), with a significant difference observed only at D₁₂₀ (p < 0.05). Similarly, the mean gonad index values of thermally treated sea cucumbers and their wild counterparts did not significantly differ at D₃₀ and D₆₀ periods (p > 0.05). However, a statistically significant difference was identified between thermally treated sea cucumbers and their wild counterparts at the D₉₀ and final time point of the experiment (p < 0.05) (Figure 4).

4. Discussion

The annual and seasonally limited spawning of economically valuable temperate sea cucumber species presents a major constraint to aquaculture potential and commercial viability. The ability to accelerate gonadal development under controlled aquaculture conditions would represent a significant advancement in overcoming this limitation and enhancing the efficiency of sea cucumber aquaculture.

Water temperatures across all treatment tanks were consistently maintained at a mean of 23.4 ± 0.4 °C, with no statistically significant differences detected among the experimental periods. In contrast, seafloor temperatures at the control site demonstrated a gradual increase from 11.5 °C to 19.6 °C over a 120-day period, with a statistically significant increase noted only after the 90th day. Such divergence in thermal exposure resulted in significantly higher cumulative temperature loads in sea cucumbers in the trial tanks than in the wild specimens, underscoring the effectiveness of the thermal regime treatments. There is limited information in the literature regarding the influence of effective accumulated temperature on gonadal development in marine organisms, with only two studies addressing this relationship in A. japonicus [43,44]. The main contribution of this study is the explicit quantification of EAT for H. tubulosa in captivity and in the field and its association with GI dynamics. Broodstock held at average 23.4 °C exposed to an EAT range of 339.9–1370.4 °C·d across 30–120 days, far exceeding the 17.7–247.5 °C·d exposed by wild individuals over the same intervals, and showed significant GI elevation after the 90th day. However, the GI threshold of ~10%, previously reported for spawning in related species [45], was not reached in the thermally treated broodstock. On the other hand, the concept of a fixed GI threshold should be interpreted with caution. For instance, in Holothuria sanctori, Navarro et al. [46] reported a maximum seasonal GI of approximately 4% at spawning during a 24-month field study, highlighting interspecific and population-level variability that limits the applicability of absolute GI benchmarks across taxa.

The findings of this study indicate that the EAT required for gonadal development in captive H. tubulosa likely exceeds 1370 °C·d. In contrast, raw data from a study conducted in the northeastern Aegean Sea [2] suggests that an EAT of approximately 360 °C·d, accumulated between February (12.3 ± 0.7 °C) and June (19.0 ± 4.2 °C), was sufficient for H. tubulosa to reach the spawning stage under natural conditions. This discrepancy underscores that although sustained elevated temperatures accelerate gonadal growth in captivity, additional environmental or endogenous cues are essential to achieve full maturation and induce spawning. Similarly, Smiley et al. [47] and Mercier and Hamel [23] highlighted that, beyond water temperature, environmental factors such as light intensity, photoperiod, and food availability significantly influence the hormonal processes that initiate gonadal maturation in sea cucumbers. Among these factors, the significance of nutritional input in facilitating reproductive development has been extensively documented by Thongbuakaew et al. [48] and Venâncio et al. [49], with evidence highlighting the essential role of consistent feeding in the reproductive success of sea cucumbers.

In this study, the basic diet consisting solely of powdered macroalgae supported a high survival rate and yielded a specific growth rate of 0.03–0.32% per day, which falls within the expected range for adult temperate species, such as H. tubulosa and A. japonicus, under controlled aquaculture conditions [21,50,51]. González-Durán et al. [52] noted that tropical and temperate holothurians exhibit vulnerability to elevated temperatures, with potential declines in growth and reproductive success under climate change scenarios. Nevertheless, the observed growth rates of H. tubulosa under the experimental conditions in this study provide positive insights into its thermal adaptability. Furthermore, Setiawati, Widiastuti, Sembiring and Giri [34] and Bai et al. [53] reported that dietary protein levels between 24% and 30% are optimal for promoting gonadal development and maturation in sea cucumbers, with levels closer to 30% being particularly effective in enhancing reproductive outcomes across several species. Therefore, in future studies a nutritionally balanced diet specifically formulated to support gonadal development, combined with a constant thermal environment, should be tested to accelerate gonadal maturation and reduce the time required for broodstock to achieve spawning readiness.

Wang [54] reported that some sea cucumber farmers in China successfully induced gametogenic maturation and initiated spawning in A. japonicus within as little as two months using thermal regimes; however, details were not provided regarding the initial gonadal stage at which thermal treatment began and whether additional supportive factors were employed during the process. In the present study, under a constant thermal regime, the mean gonad index of H. tubulosa broodstock increased significantly by day 90 compared to baseline values, reaching an average of 6.5% by day 120, approaching the threshold commonly associated with spawning readiness. Although full reproductive maturity was not achieved within the experimental timeframe, macroscopic observations on the presence of well-developed and pigmented gonads in all male and female individuals suggest that the thermal protocol effectively stimulated gonadal development in both sexes. Considering that the gonadal development phase in temperate sea cucumber species typically span approximately 5–6 months from February to July in nature [2,13,14,31,35,46,55], the present findings demonstrate that this duration can be significantly reduced to about 3–4 months under a constant thermal regime in aquaculture. This acceleration of the reproductive cycle is a key advancement for hatchery operations, as it enables more frequent spawning events, improves production efficiency, and potentially increases the annual yield of juveniles. Such time compression offers a strategic advantage in commercial sea cucumber farming by optimising resource use and shortening generation intervals.

5. Conclusions

Operationally, holding H. tubulosa at a constant temperature range of 23–24 °C for approximately 90 days is a viable way to advance or extend gonad development relative to wild phenology, effectively doubling the GI by late spring compared with field controls. These findings suggest that further research integrating gonad-supportive nutritional strategies and variable photoperiods in combination with constant thermal conditions may offer promising approaches for accelerating gonadal maturation in captive broodstock. Additionally, the application of a constant thermal regime before the natural gonad maturation period may provide valuable insights into methods to promote off-season gonadal development and spawning of H. tubulosa in aquaculture systems. Such advancements would significantly improve broodstock management and contribute to the sustainable production of this ecologically and economically important species.