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Article

Fecundity Study and Histological Analysis of the Gonads of the Sea Cucumber Holothuria tubulosa (Echinodermata: Holothuroidea) in the Central Aegean Sea, Greece: Insights into Reproductive Biology

by
Athina Balatsou
*,
Chrysoula Apostologamvrou
and
Dimitris Vafidis
Department of Ichthyology and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, GR-38446 Volos, Greece
*
Author to whom correspondence should be addressed.
Fishes 2025, 10(6), 283; https://doi.org/10.3390/fishes10060283
Submission received: 11 May 2025 / Revised: 3 June 2025 / Accepted: 6 June 2025 / Published: 8 June 2025
(This article belongs to the Section Biology and Ecology)
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Abstract

The Atlanto-Mediterranean sea cucumber Holothuria tubulosa is a species of great ecological and commercial importance, making it a primary target for collection in the Mediterranean region. This study investigated the reproductive biology of H. tubulosa (Gmelin, 1793) in a coastal area of the Central Aegean Sea (Eastern Mediterranean) over a one-year period, from June 2021 to May 2022. Monthly sampling was conducted via dives at depths up to 10 m, alongside the recording of environmental and biometric data. Histological analysis revealed a synchronous annual reproductive cycle, with gonadal maturation beginning in April and peak sexual maturity observed in July and August, followed by a single spawning event from August to September. The sex ratio was 1:1.31 (male: female), and the size at first maturity was 255.3 mm. These findings provide essential insights into the reproductive physiology of H. tubulosa and further contribute to the development of sustainable stock management strategies for sea cucumbers in the Hellenic Seas.
Keywords:
gametogenesis; oocytes; fecundity; histology; gonadosomatic index
Key Contribution: This study presents the first histological and fecundity-based analysis of H. tubulosa in the Central Aegean Sea, offering critical data for sustainable stock management in the region.

1. Introduction

Sea cucumbers, or holothurians (Echinodermata: Holothuroidea), are key members of benthic invertebrate communities inhabiting marine environments worldwide, spanning habitats from shallow coastal zones to the deep sea [1,2]. These benthic animals play a crucial ecological role in marine ecosystems, promoting habitat health, biodiversity, and productivity, through a variety of biological and behavioral mechanisms [3,4]. Due to their well-known bioturbation activity, sea cucumbers mix or rework the top sediment layer through ingestion and egestion, locomotion, respiration, and burrowing [5]. These activities improve sediment quality by oxygenating it and redistributing organic material at the sediment–water interface, thereby affecting physicochemical processes, maintaining water chemistry, and enhancing nutrient recycling, which contributes to the overall habitat health [1,3]. In addition to their ecological importance, sea cucumbers are a valuable commercial species that sustain fisheries in many coastal communities [6].
Nutritionally, sea cucumbers are valued for their high protein content and omega-3 fatty acids, with health-promoting properties and potential application as functional food ingredients [7,8,9]. Recent studies suggest that sea cucumbers, particularly those from Asia, are rich in bioactive compounds with various therapeutic properties. These include triterpene glycosides, carotenoids, bioactive peptides, vitamins, minerals, fatty acids, collagen, gelatin, chondroitin sulfates, and amino acids [9]. These compounds have been found to have potential health benefits, including wound healing, neuroprotection, antitumor [9,10], anticoagulant, antimicrobial, and antioxidant effects [9,10,11]. However, further clinical trials are required to fully understand the safety and efficacy of sea cucumber-derived products [12].
The Atlanto-Mediterranean sea cucumber species Holothuria tubulosa (Gmelin, 1793), commonly known as the brown sea cucumber, is one of the most abundant holothurian species in the Mediterranean Sea, serving an essential ecological role. This species is found at depths between 5 and 100 m, inhabiting soft sediment, rocky, and seagrass beds [13].
In the Mediterranean Sea, H. tubulosa has been extensively studied for its ecology [14,15], population dynamics [16,17,18], morphology and biometry [19,20], pharmacology [8,21], systematics and phylogeny [22,23], genetic diversity [24], and its potential as a new target species for exploitation and aquaculture [25,26]. The reproductive biology and gonadal development of this species have been studied in different regions of the Mediterranean Basin [19,27,28,29], from the Oran coast (Algeria) [30] to the Dardanelles Strait (Turkey) [31]. H. tubulosa is a gonochoric species, without external sexual dimorphism [26]. It reproduces through external fertilization and exhibits an annual reproductive cycle, with spawning mainly occurring during summer [27,28,32]. Across the Mediterranean, spawning typically coincides with the warm season [13]. In both sexes, the reproductive system consists of a single gonad formed by a tuft of tubules. During spawning, mature individuals adopt a characteristic L-shaped posture, releasing gametes into the water column via a gonopore located in the dorsal mesentery [32].
In recent years, interest in the economically valuable sea cucumber H. tubulosa has arisen from its abundance in the Mediterranean Sea, with a subsequent increase in its exploitation across Turkey, Greece, Italy, and Spain, prior to its export to Asian markets [33,34,35,36]. However, illegal and unregulated fishing poses significant challenges to sustainable management [37].
To effectively facilitate the scientific management of H. tubulosa, it is crucial to obtain fundamental information regarding its ecology, population dynamics, and reproductive patterns. The present study examines for the first time the reproductive biology of the H. tubulosa population in the Central Aegean Sea by describing the reproductive cycle over a 12-month period through histological examination of its gonads. The main objectives of this work were (1) the description of gametogenic development stages through monthly histological analysis of the gonads from June 2021 to May 2022); (2) the determination of the gonadosomatic index (GSI) and the possible correlation of them with environmental parameters (seawater temperature); (3) a detailed description of the reproductive cycle and structures (oocyte diameter measurements); (4) the determination of the size at first sexual maturity and the fecundity.

2. Material and Methods

2.1. Study Area and Sampling

The specimens of H. tubulosa were collected from seagrass beds of a coastal area of Paros Island (36°59′28.4″ N, 25°08′04.8″ E) (Cyclades, Central Aegean Sea, Greece) (Figure 1). This area represents a central location in the Aegean Sea, with a typical seasonal seawater temperature range in marine masses and dense populations of sea cucumbers. Given that H. tubulosa is primarily harvested in the area, the study of reproductive biology exclusively pertains to this species.
A total of 242 samples were collected from June 2021 to May 2022, during 12 monthly sampling events using scuba diving at depths of 3–10 m. Sampling was carried out by a trained researcher and a licensed sea cucumber fisherman using the surface air supply diving method [4]. Each collection followed a standardized 10 min dive protocol based on commercial fishing practices. Holothurians were identified to the species level onboard and stored in seawater containers. Sampling was performed seasonally and consistently throughout the study period [38].
After collection, the specimens were anesthetized in an isosmotic solution of magnesium chloride (7.5% MgCl2) in seawater for 20–30 min. This method is widely used and accepted in the international literature for mild anesthesia of echinoderms, including sea cucumbers [38,39,40]. All experimental procedures were approved by the Ethics Committee of the Department of Ichthyology and Aquatic Environment at the University of Thessaly. Subsequently, the specimens were stored in plastic bags and transported in a cool box to maintain a stable temperature [28], until arrival at the Marine Biology Laboratory of the University of Thessaly, Greece. The environmental parameter of the sea surface temperature (SST) was measured monthly with a CTD (conductivity, temperature, and depth) (Seabird-19plus, Washington, DC, USA). This parameter was employed to investigate its potential correlation with gonad maturation and to evaluate its impact on the reproductive cycle of H. tubulosa.

2.2. Biometric Measurements

In the laboratory, each specimen underwent measurements of total length (TL) to the nearest 0.1 mm, measured from mouth to anus by means of an ichthiometer; wet weight (W); drained weight (DW), following gently compression to eliminate excess water from the respiratory tree, as described by Costa et al. [14]; gonad weight (GW); and gutted body weight (GBW), obtained after removal of internal organs (gonads, alimentary canal, respiratory trees) according to Apostologamvrou et al. [38]. The biometric characteristic of weight was measured using a digital analytical balance (Ohaus PR64/E, OHAUS, Parsippany, NJ, USA) to the nearest 0.001 g.
The gonadosomatic index (GSI) was estimated based on morphometric measurements. The GSI is derived from the ratio of gonad weight (GW) to the total wet body weight (W) of the organism and is expressed as a percentage (%) [41,42]. The GSI was estimated as follows according to Conand [42]:
G S I = G W W × 100
where GW is the weight of the gonads (g), and W is the total wet body weight (g).

2.3. Fecundity

Absolute fecundity (mean number of oocytes/female) was determined for 18 individuals collected between June and August 2021 and from January to May 2022.
Due to the seasonal availability of mature individuals, the number of specimens analyzed per month ranged from two to four. Only females in early mature and mature gonadal stages (III–IV) with clearly visible oocytes were selected for analysis. Fecundity was estimated following the methodology described by Muthiga et al. [43], in which gonads were dissected, weighed, and homogenized in a known volume and oocytes were counted in subsamples under a stereomicroscope. Fecundity values were then extrapolated to the total gonad volume.
For this purpose, a small piece of gonad fixed in 10% formalin was weighed to the nearest 0.001 g and macerated using a mortar and pestle to extract oocytes from the tubules. Using the gravimetric method, the mixture was filtered through a coarse filter to remove tubule tissue, and the oocytes were suspended in 1000 mL of distilled water. Five aliquots (0.1 mL) were taken, and the mixture was stirred with a magnetic stirrer. The oocytes in each aliquot were counted, and absolute fecundity was calculated using the following formula:
Average number of oocytes per aliquot × dilution factor × proportion of the weight of gonad/total gonad weight.

2.4. Histological Analysis

The gonads underwent thorough processing for histological analysis. For each sample, a piece of gonad tissue (~1 cm long) was immediately fixed in Bouin’s solution for 2 h, followed by buffered 10% formalin solution for at least 48 h. Subsequently, the samples were processed using a Leica® TP1020 Automatic Tissue Processor (Leica Microsystems GmbH, Nussloch, Germany), involving sequential submersions in graded ethanol for dehydration, followed by xylene for clarification. The tissues were then embedded in paraffin wax at 60 °C using a Leica® EG1150 Modular Tissue Embedding Center (Leica Microsystems GmbH, Nussloch, Germany). After that, the samples were sectioned at 5 μm thickness using a SLEE rotary microtome series 5062 (SLEE Medical GmbH, Mainz, Germany). Slides were stained with Harris’ hematoxylin solution (Scharlab S.L., Sentmenat, Barcelona, Spain) and eosin Y (yellowish) (VWR International, Leuven, Belgium). Subsequently, the sections were dehydrated in graded ethanol (96–100%), cleared in xylene, and mounted in dibutylphthalate polystyrene xylene (DPX) [38,44]. Gonad tissues were then analyzed using a Zeiss light microscope (Axio Scope A1 pol, ZEISS, Jena, Germany) equipped with an Axiocam 305 color digital camera and the combined ZEN 3.6 (blue edition) imaging software (Zeiss, Germany).
Gametogenesis was classified into five stages of gonadal development according to previous studies [45]. In females, oocyte diameter was measured along the major axis, considering only those oocytes in which the nucleus was clearly visible. For each female, 60 oocytes were randomly selected and measured monthly. In males, the same five-stage classification was applied based on the presence and relative abundance of spermatogenic cells, including spermatogonia, spermatocytes, spermatids, and spermatozoa. Although morphometric measurements were not performed on male germ cells, qualitative evaluation of cell distribution and maturation allowed for reliable determination of testicular development throughout the reproductive cycle. Image processing was performed using the Fiji software (Version 2.15.0) (NIH, Bethesda, MD, USA).

2.5. Data Analysis

The potential deviation of the sex ratio (male: female) from the expected 1:1 ratio was assessed using the chi-square goodness-of-fit test [46]. Differences in monthly GSI between sexes were initially assessed using a parametric t-test; however, due to violations of the normality assumption, the non-parametric Mann–Whitney U test was ultimately applied. To assess differences in GSI across months, Welch’s ANOVA was used to account for heterogeneity of variances. Differences in oocyte diameter across maturity stages were analyzed using one-way ANOVA, followed by Games–Howell post hoc tests for pairwise comparisons, as this method is appropriate when variances are unequal and sample sizes differ. Seasonal differences in absolute fecundity were tested using the non-parametric Kruskal–Wallis test, followed by Dunn’s post hoc multiple comparisons with Bonferroni adjustment to identify pairwise differences among seasons.
Pearson correlation analysis was conducted to evaluate the relationship between mean monthly sea surface temperature and GSI values for both sexes. Prior to analysis, assumptions of linearity and a normal distribution of the variables were assessed using scatterplots and the Shapiro–Wilk test, respectively, confirming that Pearson’s correlation was appropriate.
Additionally, a linear regression analysis was performed to investigate the relationship between the mean monthly GSI values of males and females, and the slope, intercept, R2, and p-value were calculated to quantify the strength and significance of this association.
Statistical analyses were performed using Jamovi (Version 2.2.5) and RStudio (Version 2023.12.1+402) [47]. Data are presented as the mean ± standard deviation (s.d.). Statistical significance was set at a significance level of 5% (p < 0.05).
A Probit analysis was conducted, and a logarithmic curve was applied to the data to estimate the L50 value, which determines the size at the onset of maturity (SOM). The L50 represents the total length (TL) at which 50% of the female population is in a reproductive condition, following the approach outlined by Lolas and Vafidis [48].

3. Results

3.1. Sex Ratio

The study of the reproductive cycle of H. tubulosa was determined by microscopic observation of the gonads of adult individuals over 12 months, with an average total length of 159 ± 54 mm and an average wet weight of 118 ± 65.30 g. In total, 104 males (43.33%) and 136 females (56.67%) were sampled, with an average of approximately 10 males and 12 females collected each month. The sex ratio of H. tubulosa differed significantly from the 1:1 ratio, demonstrating a ratio of 1:1.31 (males: females) (χ2 = 4.27; p-value = 0.039 < 0.05), with a higher proportion of females than males in most months (Figure 2).

3.2. Reproductive Cycle

3.2.1. Microscopic Description

Based on histological observations, the gonadal development of both male and female individuals of H. tubulosa was categorized into five stages (Table 1), following the classification proposed by Slimane-Tamacha et al. [45]: recovery stage (I), growing stage (II), early mature stage (III), mature stage (IV), and spent gonad stage (V).
In males, during the recovery stage (I), the gonad wall attains its maximum thickness, featuring folds that increase the surface area for spermatogenesis. The lumen was nearly empty and primary spermatocytes were observed in the peripheral germinal layer (Figure 3A). In the growing stage (II), the gonad wall folds host spermatocytes arranged in columns along the germinal epithelium, while spermatozoa begin filling the lumen. The gonad walls become thinner as development progresses, with a reduction in germinal epithelium folds (Figure 3B). Rapid advancement in spermatogenesis is evident in the early mature stage (III), with spermatozoa centrally occupying the tubule lumen (Figure 3C). The mature stage (IV) was characterized by a thin gonad wall and the lumen filled with densely packed spermatozoa. Few spermatocytes were present along the germinal epithelium (Figure 3D). Following spawning, the final stage of spent gonads (V) was characterized by a thickened and wrinkled gonad wall. In the lumen, a few unspawned spermatozoa, debris, and phagocyte clusters appear (Figure 3E).
In females, during the recovery stage (I), the gonad wall exhibits its maximum thickness, and previtellogenic oocytes are embedded within the germinal epithelium of the tubule. Developing oocytes are arranged along the germinal epithelium of the gonad (Figure 3F). In the growing stage (II), the germinal layer is adorned with oocytes at various developmental phases, including developing and previtellogenic oocytes; however, the tubule lumen is not completely occupied by oocytes. The gonad wall remains thick, gradually narrowing over time (Figure 3G). The subsequent early mature stage (III) featured an ovarian wall with a minimal thickness and oocytes at diverse developmental stages. With the progression of vitellogenesis, fully grown oocytes started to occupy a central position in the lumen (Figure 3H). The mature stage (IV) is characterized by an abundance of fully matured oocytes featuring a well-defined nucleus, densely accumulated in the lumen and reaching their maximum size at this stage (Figure 3I). Following spawning, the final stage of the spent gonads (V) was characterized by a thicker and wrinkled ovary wall. The lumen is occupied by phagocytes and unspawned oocytes in atresia owing to phagocytic activity (Figure 3J).

3.2.2. Gonadosomatic Index and Monthly Variability of Sexual Maturity Stages

The GSI values differed significantly between male and female individuals (Mann–Whitney U, Statistic = 940, p = 0.002). An overall annual pattern was observed, with significant temporal variation in mean GSI values across sampling months (ANOVA: F = 6.85, p < 0.001). The highest GSI values were recorded during June–August 2021, corresponding to the peak reproductive period and leading to the late summer spawning event. A sharp decline in GSI was observed in September 2021, followed by persistently low values throughout autumn and reaching the lowest levels in winter. A gradual increase in GSI values was then recorded during spring (March–May 2022), suggesting renewed gonadal development (Figure 4).
As illustrated in Figure 4, four main reproductive phases were identified based on the GSI trends: (1) a growth phase characterized by a gradual increase in the gonadosomatic index (January–May 2022), (2) a maturation phase culminating in the peak of the gonadosomatic index (June–August 2021), (3) a spawning phase characterized by a drastic reduction in the gonadosomatic index (September 2021), and (4) a post-spawning phase characterized by a low and stable gonadosomatic index (October–December 2021). Within the span of these phases, differences in the gonadosomatic index and successions in gametogenesis stages were observed during the sampling months.
Throughout these phases, distinct but synchronous seasonal patterns of gonadal development were observed in both sexes. In females, GSI changes corresponded with the growth and release of oocytes, while in males, they reflected spermatogenic progression and sperm release. The parallel fluctuation in GSI values between sexes highlights coordinated reproductive timing within the population.
The monthly variability of reproductive maturity stages was analyzed separately for males and females to discern the reproductive cycle and spawning period of H. tubulosa. Observed over a one-year period, from June 2021 to May 2022, the reproductive cycle exhibited a distinct annual synchronous pattern in both sexes.
As depicted in Figure 4, which illustrates the relative frequencies of maturity stages, 80% of the females and 75% of the males reached complete reproductive maturity (stage IV) in June 2021. This percentage increased to 100% for both sexes in July and August 2021. The proportion of females in stage V exceeded 65% in September, increased to 75% in October, and reached 100% in November and December. In males, stage V was observed from September 2021 to January 2022 and was present in 20% of the gonads in the last month. For females, stage I was observed from January to February 2022, whereas in males, it was already evident in December 2021, with a presence of over 25%, persisting until March 2022, with a frequency exceeding 65% of gonads. Gonad growth occurred during stage II from March for both genders, lasting until May 2022 for females and until April of the same year for males. The early mature stage (stage III) was observed in females in June 2021, accounting for 20% of the gonads, and from April to May 2022, with a relative frequency exceeding 65%. In June 2021, 25% of male gonads were in the early mature stage, and this was also observed from April to May 2022, with relative frequencies of 83% and 100%, respectively.
The mean monthly sea surface temperature (SST) exhibited a distinct seasonal pattern, peaking in August and reaching a minimum in January, with an average of 19.18 ± 1.87 °C. Notably, the gonadosomatic index (GSI) values peaked simultaneously with the highest sea surface temperatures, whereas the GSI reached its lowest values during periods of low temperatures. Gametogenesis began at low temperatures in January and February. As temperatures increased in March and April, gonad development progressed, leading to the complete gonadal maturity observed during the peak temperatures in July and August. Spawning commenced during the period of sustained high temperatures, specifically from late August to early September. The temperature difference in the water, one month before and after the peak of the gonadosomatic index (GSI), was 0.61 °C.
Temperature exhibited a positive correlation with the mean monthly value of the gonadosomatic index (GSI) in both males (Pearson’s r = 0.712, p = 0.009) and females (Pearson’s r = 0.754, p = 0.005), as revealed by the Pearson correlation analysis.
A strong, significant linear relationship was also observed between the mean monthly GSI values of females and males (Figure 5). The regression model yielded a slope of 0.768, an intercept of 0.060, and an R2 value of 0.974, indicating that 97.4% of the variation in male GSI is explained by the female GSI (p < 0.001).

3.3. Size at First Sexual Maturity

The estimated size at first maturity (the size at which 50% of the female individuals in the population exhibited mature gonads) through the logistic approach was TL50 = 255.3 mm during the study period, and estimates were performed in terms of total body length (TL) (mm) (Figure 6).

3.4. Absolute Fecundity and Seasonal Variation

Fecundity ranged from 1.809 × 103 to 51.500 × 103, with a mean (±s.d.) fecundity of 19.057 ± 13.117 × 103. Fecundity and gonad weight were highly correlated (r = −0.722; p < 0.001) (Figure 7).
The absolute fecundity in monthly samples peaked at a mean of 32.694 ± 13.454 × 103 oocytes/female in winter (January–February 2022), followed by a notable decline to 17.228 ± 10.249 × 103 oocytes/female by spring (March–May 2022). During the summer period (June–August 2021), the absolute fecundity reached its lowest value of 9.826 ± 4.602 × 103 oocytes/female (Figure 8).
Statistical analysis using the Kruskal–Wallis test showed significant differences in absolute fecundity among seasons (χ2 = 7.54, df = 2, p = 0.023). Post hoc Dunn’s tests with Bonferroni correction indicated that fecundity in winter was significantly higher than in summer (p = 0.018), while differences involving spring were not statistically significant.

3.5. Oocyte Diameter

Oocyte diameter increased progressively through the developmental stages, peaking at full gonadal maturity (Figure 9). The measurements of oocyte diameter at each maturity stage provided a comprehensive overview of seasonal gonadal development in H. tubulosa female individuals (Figure 10A). Developing (non-vitellogenic, 12.6 ± 3.39 μm) oocytes were predominantly observed from December 2021 to February 2022, coinciding with the period of recovering gonads, while previtellogenic oocytes (32.7 ± 8.04 μm) were more frequent from March to April 2022, aligning with the period of gonadal growth. Vitellogenic oocytes were more prevalent from June to August 2021 and May 2022, during the early mature (77.6 ± 11.8 μm) and mature (114.0 ± 18.1 μm) stage of gonads. The maximum diameter was observed when the gonads were mature. One-way ANOVA indicated a significant difference in the diameter of oocytes across all recognized maturity stages (Games–Howell post hoc test, p < 0.01).
Furthermore, in the mature stage, the oocyte population exhibited a unimodal distribution with a peak at 110 μm, suggesting synchronized development of the ovaries of H. tubulosa females (Figure 10B).

4. Discussion

The reproductive biology of sea cucumbers plays a critical role in species conservation and the maintenance of ecological balance in marine ecosystems [49,50]. Despite their limited fecundity per reproductive cycle, reproductive processes in sea cucumbers remain fundamental to their survival and sustainability [28,49,51,52]. Several studies have examined the reproductive biology of H. tubulosa, contributing valuable insights into species-specific reproductive strategies [29,53].
The present study is the first to investigate the reproductive cycle of H. tubulosa in the Central Aegean Sea—an ecologically significant region characterized by moderate seasonal temperature variability and high marine biodiversity. Given that H. tubulosa is primarily harvested from this area, the focused examination of its reproductive biology underscores its ecological and commercial importance.
The population of H. tubulosa in the Central Aegean Sea exhibited a sex ratio of 1:1.31 (male:female), indicating an imbalanced sex ratio, with females outnumbering males in the studied population.
The sex ratio in the Central Aegean H. tubulosa population was 1:1.31 (male:female), indicating a significant female-biased ratio, particularly during winter and spring. This deviation from the expected 1:1 ratio was statistically significant (χ2 = 4.47, p = 0.035) and mirrors findings in other holothurian species, potentially arising from reproductive strategies, environmental sex determination, or sex-specific mortality rates [40,54]. While a female-skewed sex ratio may enhance the reproductive output under optimal conditions, sustained imbalances could compromise fertilization efficiency in broadcast spawners like H. tubulosa, where synchronized gamete release is vital [55]. These findings highlight the need for ongoing sex ratio monitoring to support informed resource management.
Comparable sex ratio patterns have been reported in other sea cucumber species, including H. mammata in the Atlantic Ocean [56,57], H. whitmaei in the Pacific Ocean [58], and H. leucospilota in the Western Indian Ocean [59]. Conversely, several studies have reported balanced sex ratios in H. tubulosa [16,27,28], suggesting potential variability across regions. Fishing pressure has been proposed as a contributing factor to sex ratio imbalances [43,56,58], possibly due to the formation of same-sex groups or increased activity during nocturnal fishing periods. However, the degree to which nocturnal activity differs between sexes remains unclear [60].
Comprehension of gonadal histomorphology is essential for elucidating reproductive cycles in sea cucumbers. This study provides the first histological description of gonadal development stages in H. tubulosa from the Hellenic Seas, with an emphasis on females due to the practicality of estimating fecundity. The reproductive cycle exhibited a clearly synchronized annual pattern, as inferred from histological analyses. Gametogenesis occurred from January to May, followed by a recovery phase in early winter. Regressed gonads contained predominantly oogonia and spermatogonia, indicating the onset of a new reproductive cycle. Gonadal development resumed in spring, culminating in peak maturity and spawning during late summer, coinciding with elevated sea temperatures. Similar temporal reproductive patterns have been reported in the Pagasitikos Gulf, Greece [28], and other western Mediterranean populations [18,26]. This seasonal strategy is also documented in other species, such as Holothuria atra, H. mexicana, H. nobilis, Actinopyga echinites, Stichopus variegatus, and Thelenota ananas [61].
Histological examination not only enhances the understanding of gonadal development at the tubular level but also provides critical insights into oocyte size across developmental stages. Oocyte diameter is a fundamental indicator of reproductive maturity in marine invertebrates such as sea cucumbers. The progressive increase in oocyte size reflects the advancement of oogenesis and is closely associated with the imminent spawning event. Furthermore, oocyte size distribution offers valuable information on the reproductive potential of populations and facilitates comparative analyses of reproductive performance among populations or geographic areas with differing environmental conditions. Monitoring seasonal variations in oocyte diameter throughout the annual cycle enables a more precise understanding of reproductive timing and intensity [28]. In the present study, oocyte diameters were categorized into size classes, revealing a predominance of smaller oocytes during winter, corresponding to females in the recovery phase. During the recovery (Stage I) and growing (Stage II) phases, oocyte diameters ranged from 8.6 to 52.5 μm. In early mature (Stage III) and mature (Stage IV) phases, oocytes measured between 55 and 150 μm, with spawning coinciding with oocytes exceeding 110 μm in diameter. These results are consistent with previous findings by Santos et al. [56] and Kazanidis et al. [28], who reported mature oocyte diameters ranging from 108 to 122 μm in H. mammata and exceeding 200 μm in H. tubulosa from the Pagasitikos Gulf.
Although both sexes were examined in the present study, a greater emphasis was placed on females due to the specific objective of fecundity estimation. Such assessments are only feasible in mature females, as they require oocyte counts to provide a direct and quantifiable measure of reproductive output and potential [50]. This focus does not diminish the biological relevance of male reproductive data but reflects the methodological constraints and standardized approaches commonly employed in echinoderm reproductive studies [62].
Gonadal tissues from both sexes were subjected to qualitative histological analysis across all reproductive stages, enabling the classification of gonadal maturity based on established morphological criteria, as widely practiced in holothurian research [28,38,63]. However, monthly quantification of individual germ cell types was not conducted, as such analysis would require advanced methodologies—such as flow cytometry, stereological analysis, or immunohistochemistry—which were beyond the logistical and methodological scope of the present study [62].
During the post-spawning recovery stage, gonads in both sexes were observed to be in a regressed state. In females, oogonia and early previtellogenic oocytes were present, while in males, early spermatogenic cells, predominantly spermatogonia, were detected. These findings suggest the initiation of a new gametogenic cycle, consistent with reproductive patterns reported in other holothurian populations [64].
This study provides novel insights into the reproductive biology of H. tubulosa by documenting, for the first time in the Central Aegean Sea, a fully synchronous and annual reproductive cycle characterized by distinct, sex-specific temporal patterns. Unlike previous studies conducted in other Mediterranean subregions [28,63,64], which primarily described general spawning periods or regional trends, our research offers a more detailed and integrative analysis. Specifically, we combine quantitative GSI measurements, histological staging of gametogenesis for both sexes, and statistical correlations with seabed temperature [38,50]. This comprehensive, multi-dimensional approach refines our understanding of reproductive seasonality and highlights SST as a key environmental driver, consistent with patterns observed in other echinoderms [3,65]. Moreover, by including fecundity estimates and environmental parameters, this study offers a more holistic picture of reproductive investment in H. tubulosa. Such an approach is essential for informing effective management and conservation strategies, especially in light of growing fishing pressure on sea cucumber populations across the Mediterranean [4,40,65].
In the Central Aegean Sea, the gonadosomatic index (GSI) of H. tubulosa demonstrated a significant correlation with sea surface temperature, corroborating earlier studies that identify temperature as a key abiotic factor influencing reproductive dynamics in H. tubulosa [16,27,31] and related holothurian species [42,66]. Accordingly, the GSI was employed to characterize the reproductive cycle of H. tubulosa following the methodology of Kazanidis et al. [16]. Peak GSI values were observed in August 2021, with spawning occurring between August and September 2021. This reproductive timing aligns with previous reports for the northwestern Aegean Sea [16,28] and Mediterranean populations of H. tubulosa [19,27] based on gonadosomatic index assessments.
The observed synchronization of peak spawning with the highest recorded sea surface temperatures at the sampling site reflects a well-documented pattern among sea cucumber species [16,19,27,57,65,67].
Sea temperature is a principal determinant of reproductive periodicity and exerts a considerable influence on reproductive processes [68]. This association emphasizes the critical role of temperature fluctuations, particularly during gonadal maturation and spawning phases, and is consistent with observations from other holothurian species [42,69,70,71].
Size at first sexual maturity is a pivotal parameter for establishing minimum legal catch sizes, aimed at sustaining adequate spawning biomass and ensuring that individuals contribute at least one reproductive event before harvest [72]. Our results indicate that H. tubulosa attains sexual maturity at approximately 255 mm total length. The estimated size at first sexual maturity (TL50 = 255.3 mm) indicates that more than half of the female H. tubulosa individuals attain their reproductive capability only after reaching relatively large body sizes. This highlights the importance of implementing size-based regulatory measures to safeguard reproductive output, especially in exploited populations. Similar management approaches have been recommended for other holothurian species in the Mediterranean and beyond [1,38,50]. Currently, H. tubulosa is not subject to species-specific minimum size limits in Greek waters [4], which raises concerns about premature harvesting. Considering our findings, we propose the introduction of a minimum legal catch size exceeding the TL50 threshold (e.g., ≥260 mm) to ensure that individuals reproduce at least once before collection.
Comparable maturity sizes have been reported in related studies; Kazanidis et al. [28] reported a size at first maturity of H. tubulosa, expressed as drained weight (DW50), of 220 g in the Pagasitikos Gulf, while Navarro et al. [50] estimated the size at first sexual maturity of H. sanctori from Gran Canaria to range between 201 and 210 mm.
This study presents the first estimates of fecundity for H. tubulosa, with values ranging from 1.809 × 10³ to 51.5 × 10³ oocytes per female. To our knowledge, this study provides the first fecundity estimates for H. tubulosa in the Central Aegean Sea. While our findings offer a baseline understanding of the reproductive potential of the local population, it is important to acknowledge that fecundity may vary substantially among individuals due to internal factors such as body size, age, and physiological condition. These variables were not explicitly assessed in the present study, as our primary objective was to obtain representative fecundity estimates during the peak reproductive period.
Nevertheless, previous research on holothurian species has shown that fecundity often correlates positively with body size and age [50], indicating that individual variability may influence the reproductive output within populations. Future studies incorporating biometric data and age-class analyses would contribute to a more comprehensive understanding of fecundity variation and its ecological implications.
These fecundity estimates are substantially lower than those reported for Red Sea H. atra, which range from 12.11 × 10³ to 1342.278 × 10³ oocytes per female [73]. It is important to note that absolute fecundity in this study was estimated from a total of 18 mature females sampled over a six-month period, with approximately 3 individuals analyzed per month. While this sample size is relatively limited, it reflects the seasonal availability of reproductively active females and is consistent with sample sizes employed in comparable studies on holothurian species [50,74]. Nevertheless, this limitation may constrain the statistical power to detect inter-individual variability. Future research should aim to incorporate larger sample sizes spanning multiple reproductive cycles to improve the resolution of fecundity patterns at the population level. Such discrepancies likely reflect interspecific biological differences, as reproductive physiology varies among species [75,76]. Moreover, environmental factors such as water temperature, food availability, and water quality can influence reproductive output [53,77,78,79].
While temperature was examined as a principal abiotic driver of reproductive timing and fecundity, other environmental parameters—such as food availability and nutrient concentrations—were not assessed. Future research should integrate biotic and physicochemical variables to provide a more comprehensive evaluation of the ecological determinants of reproductive performance in H. tubulosa populations. This approach is consistent with prior research underscoring the multifactorial nature of environmental influences on holothurian reproduction, including regional studies within the Aegean Sea [1,4].
Previous studies have demonstrated that anthropogenic factors, such as fishing pressure and pollution, significantly impact sea cucumber reproduction [1,4,38]. Although these factors were not directly measured in the present study, the observed inter-individual variation in fecundity likely reflects broader ecological dynamics, highlighting the need for integrative, multifactorial research frameworks.
Genetic diversity has also been proposed as a potential influence on reproductive success in holothurians [80], though genetic analyses were beyond the scope of this study. Nonetheless, future studies should explore population genetics to assess intraspecific variability and resilience. Moreover, anthropogenic impacts such as fishing and pollution can impair reproductive performance, further increasing species vulnerability [49].
The observed variability in fecundity may also result from environmental heterogeneity and population structure. For example, regional differences in size, gonadal output, and maturity profiles have been documented in H. arguinensis and H. mammata [75]. Thus, differences in fecundity between H. tubulosa in the Central Aegean and H. atra in the Red Sea likely reflect complex interactions among environmental, biological, and anthropogenic factors. This study did not assess intraspecific fecundity variation due to limited availability of mature females and restricted sampling locations. Future research should involve larger sample sizes and incorporate biometric data (e.g., body size, age, weight) to better understand the relationship between ecological factors and reproductive capacity in H. tubulosa.

5. Conclusions

The present study provides the first comprehensive insights into the reproductive biology of H. tubulosa in the Hellenic Seas, employing histological analysis and fecundity assessments that can serve as a valuable reference for future comparative research. Our findings indicate that the reproductive cycle of H. tubulosa in the Central Aegean Sea is characterized by an annual, synchronous pattern, with a concise spawning period occurring at the end of summer. The strong association observed between spawning activity and elevated seawater temperatures suggests that temperature acts as a critical environmental cue triggering reproduction.
Considering the increasing exploitation of H. tubulosa populations in the Mediterranean Sea [16], driven by the expanding demand and high profitability of processed sea cucumber products in Asian markets [34,35], it is imperative to deepen biological and ecological knowledge of wild populations.
The results obtained from the present study, including the detailed characterization of the reproductive cycle and spawning timing, provide a foundational framework for developing evidence-based regulatory measures. Such measures are essential for the sustainable management of H. tubulosa stocks and the preservation of marine biodiversity within the Eastern Mediterranean Basin.

Author Contributions

Conceptualization, A.B., C.A., and D.V.; methodology, A.B., C.A., and D.V.; software, A.B. and C.A.; validation, A.B., C.A., and D.V; formal analysis, A.B.; investigation, A.B., C.A., and D.V.; resources, C.A. and D.V.; data curation, A.B., C.A., and D.V.; writing—original draft preparation, A.B., C.A., and D.V.; writing—review and editing, A.B., C.A., and D.V.; visualization, A.B., C.A., and D.V.; supervision, C.A. and D.V.; project administration, D.V.; funding acquisition, C.A. and D.V. All authors have read and agreed to the published version of the manuscript.

Funding

This work was implemented in the framework of the project entitled “Exploitation and management of sea cucumber fisheries (Holothuria spp.): Processing (food and biotech products) and safeguarding of stocks” with grant number MIS 5010720, which was funded from the European Union, European Maritime and Fisheries Fund, in the context of the Operational Programme “Maritime and Fisheries 2014–2020”.

Institutional Review Board Statement

Ethical review and approval were waived for this study due to the absence of a relevant framework for the usage of holothurians in marine research; Holothuria spp. is a fishery resource legally allowed to be collected from the wild, and the collected specimens were immediately preserved without performing any experimental treatment that could torture the animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets analyzed in the current study are available from the corresponding author upon reasonable request. The data are not publicly available for privacy reasons.

Acknowledgments

The authors would like to thank the captains and crews for their help in sampling, providing their fishing vessels involved in field surveys. We also gratefully acknowledge the insightful and constructive comments provided by the two anonymous reviewers, whose careful evaluation and thoughtful suggestions significantly enhanced the clarity, structure, and scientific rigor of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Map of the study area.
Figure 1. Map of the study area.
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Figure 2. Monthly variation in sex ratio (%) of the H. tubulosa population in the Central Aegean Sea, Greece. Dashed line indicates the 1:1 equilibrium. Dots positioned above the dashed line (orange) indicate female dominance, those below the dashed line (blue) indicate male dominance, and those aligned with the dashed line (grey) represent an equal ratio of females to males.
Figure 2. Monthly variation in sex ratio (%) of the H. tubulosa population in the Central Aegean Sea, Greece. Dashed line indicates the 1:1 equilibrium. Dots positioned above the dashed line (orange) indicate female dominance, those below the dashed line (blue) indicate male dominance, and those aligned with the dashed line (grey) represent an equal ratio of females to males.
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Figure 3. Macroscopic depiction and histological characterization of maturity stages in male and female H. tubulosa using hemoxylin and eosin staining. (A,F) Recovering gonads with thick walls (gw), developing spermatocytes (sc) and developing oocytes (do) lining the germinal epithelium (ge). (B,G) Growing gonads with increasing abundance of spermatozoa (S) in the lumen (L), and active vitellogenesis with previtellogenic oocytes (po). (C,H) Early mature gonads with various stages of gamete development, spermatocytes (sc) and spermatozoa (S) in testes and previtellogenic oocytes (po) and mature oocytes (mo) in ovaries. (D,I) Mature gonads, with the lumen completely filled with spermatozoa (S) and large mature vitellogenic oocytes (mo). (E,J) Spent gonads, with relict spermatozoa (rs) and oocytes (ro), and phagocytes (ph) degrading unspawned gametes. Scale bars: (A,B,D–I) = 200 μm, (C,J) = 500 μm.
Figure 3. Macroscopic depiction and histological characterization of maturity stages in male and female H. tubulosa using hemoxylin and eosin staining. (A,F) Recovering gonads with thick walls (gw), developing spermatocytes (sc) and developing oocytes (do) lining the germinal epithelium (ge). (B,G) Growing gonads with increasing abundance of spermatozoa (S) in the lumen (L), and active vitellogenesis with previtellogenic oocytes (po). (C,H) Early mature gonads with various stages of gamete development, spermatocytes (sc) and spermatozoa (S) in testes and previtellogenic oocytes (po) and mature oocytes (mo) in ovaries. (D,I) Mature gonads, with the lumen completely filled with spermatozoa (S) and large mature vitellogenic oocytes (mo). (E,J) Spent gonads, with relict spermatozoa (rs) and oocytes (ro), and phagocytes (ph) degrading unspawned gametes. Scale bars: (A,B,D–I) = 200 μm, (C,J) = 500 μm.
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Figure 4. Monthly distribution of gonad developmental stages in H. tubulosa from the Central Aegean Sea: (A) males, (B) females. Sea surface temperature (SST, °C) and mean gonadosomatic index (GSI) are plotted for each month. Gonadal development stages: I—Recovery; II—Growing; III—Early Mature; IV—Mature; V—Spent.
Figure 4. Monthly distribution of gonad developmental stages in H. tubulosa from the Central Aegean Sea: (A) males, (B) females. Sea surface temperature (SST, °C) and mean gonadosomatic index (GSI) are plotted for each month. Gonadal development stages: I—Recovery; II—Growing; III—Early Mature; IV—Mature; V—Spent.
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Figure 5. Relationship between the mean monthly gonadosomatic indexes (GSIs) of female and male H. tubulosa during the sampling period (June 2021–May 2022).
Figure 5. Relationship between the mean monthly gonadosomatic indexes (GSIs) of female and male H. tubulosa during the sampling period (June 2021–May 2022).
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Figure 6. Logistic curve describing the proportion of mature H. tubulosa females according to total length (mm). TL50 indicates the size at which a randomly chosen individual has a 50% probability of being mature.
Figure 6. Logistic curve describing the proportion of mature H. tubulosa females according to total length (mm). TL50 indicates the size at which a randomly chosen individual has a 50% probability of being mature.
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Figure 7. Relationship between absolute fecundity (×103 oocytes/female) and gonad weight (g) in H. tubulosa.
Figure 7. Relationship between absolute fecundity (×103 oocytes/female) and gonad weight (g) in H. tubulosa.
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Figure 8. Absolute fecundity (×103 oocytes/female ± s.d.) in different seasons of samples collected in 2021–2022 (winter: January–February; spring: March–May; summer: June–August). The boxes illustrate the interquartile range, with the median value indicated by the horizontal line and the ‘+’ sign indicating the mean, while whiskers show the range.
Figure 8. Absolute fecundity (×103 oocytes/female ± s.d.) in different seasons of samples collected in 2021–2022 (winter: January–February; spring: March–May; summer: June–August). The boxes illustrate the interquartile range, with the median value indicated by the horizontal line and the ‘+’ sign indicating the mean, while whiskers show the range.
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Figure 9. The oocyte diameter measured using light microscopy.
Figure 9. The oocyte diameter measured using light microscopy.
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Figure 10. (A) Box plot of the oocyte diameter (μm) at different maturity stages. Boxes represent the interquartile range, the horizontal line indicates the median, and the black square denotes the mean. Whiskers show the full range of values. Individual points represent outliers. (B) Histogram of the size–frequency distribution of oocyte diameter (μm) in mature ovaries.
Figure 10. (A) Box plot of the oocyte diameter (μm) at different maturity stages. Boxes represent the interquartile range, the horizontal line indicates the median, and the black square denotes the mean. Whiskers show the full range of values. Individual points represent outliers. (B) Histogram of the size–frequency distribution of oocyte diameter (μm) in mature ovaries.
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Table 1. Description of macroscopic and microscopic gonadal characteristics of male and female H. tubulosa specimens at different stages of gametogenesis. Description of macroscopic and microscopic gonadal characteristics of male and female H. tubulosa specimens at different stages of gametogenesis.
Table 1. Description of macroscopic and microscopic gonadal characteristics of male and female H. tubulosa specimens at different stages of gametogenesis. Description of macroscopic and microscopic gonadal characteristics of male and female H. tubulosa specimens at different stages of gametogenesis.
StageMacroscopic DescriptionMicroscopic Description
MalesFemales
Recovery (I)Short and thin blind-ended tubules.
Color: Translucent to whitish.		Thick gonadal walls (gw) with developing spermatocytes (sc) and oocytes (do) (12.6 ± 3.39 μm) lining the germinal epithelium (ge). Empty lumen.
Growing (II)Thick and long tubules.
Color: Whitish in males, light pink in females.		Spermatocytes lining the numerous invaginations of the germinal epithelium, and increasing of spermatozoa in the lumen.Developing oocytes lining the germinal epithelium, with pre-vitellogenic oocytes (32.7 ± 8.04 μm) progressively occupying the lumen of the tubule.
Early mature (III)Long, numerous, elongated, and branching tubules.
Color: Cream in males, light pink to orange in females.		Spermatogenesis advances, and spermatocytes are present along the germinal epithelium. Spermatozoa centrally located in the lumen.Presence of oocytes at various stages of vitellogenesis (77.6 ± 2.158 μm). With the progression of vitellogenesis, fully developed oocytes, surrounded by follicular cells, commence occupying a central position in the lumen.
Mature (IV)Maximum length and width of blind-ended tubules with thin walls.
Color: Cream to beige in males, orange to reddish orange in females.		Lumen completely filled with densely packed mature spermatozoa.Lumen completely filled with mature oocytes, surrounded by the follicle membrane, reach their maximum size (114.0 ± 18.1 μm).
Spent (V)Shrunken gonadal walls.
Color: Medium-brown to semitransparent with brownish blotches.		Presence of phagocytes and relict gametes within the lumen.
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Balatsou, A.; Apostologamvrou, C.; Vafidis, D. Fecundity Study and Histological Analysis of the Gonads of the Sea Cucumber Holothuria tubulosa (Echinodermata: Holothuroidea) in the Central Aegean Sea, Greece: Insights into Reproductive Biology. Fishes 2025, 10, 283. https://doi.org/10.3390/fishes10060283

AMA Style

Balatsou A, Apostologamvrou C, Vafidis D. Fecundity Study and Histological Analysis of the Gonads of the Sea Cucumber Holothuria tubulosa (Echinodermata: Holothuroidea) in the Central Aegean Sea, Greece: Insights into Reproductive Biology. Fishes. 2025; 10(6):283. https://doi.org/10.3390/fishes10060283

Chicago/Turabian Style

Balatsou, Athina, Chrysoula Apostologamvrou, and Dimitris Vafidis. 2025. "Fecundity Study and Histological Analysis of the Gonads of the Sea Cucumber Holothuria tubulosa (Echinodermata: Holothuroidea) in the Central Aegean Sea, Greece: Insights into Reproductive Biology" Fishes 10, no. 6: 283. https://doi.org/10.3390/fishes10060283

APA Style

Balatsou, A., Apostologamvrou, C., & Vafidis, D. (2025). Fecundity Study and Histological Analysis of the Gonads of the Sea Cucumber Holothuria tubulosa (Echinodermata: Holothuroidea) in the Central Aegean Sea, Greece: Insights into Reproductive Biology. Fishes, 10(6), 283. https://doi.org/10.3390/fishes10060283

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