Reproductive biology of the sea cucumber Holothuria arguinensis (Echinodermata: Holothuroidea) and its relationship with the environment in the coastal waters of Morocco

Abstract

This study investigates the reproductive biology of Holothuria arguinensis over one year (December 2016 to December 2017) from two locations on the Atlantic coast of Morocco: Skhirat (North Atlantic) and Souiria K'dima (Central Atlantic), including the sex ratio, size at sexual maturity, and reproductive cycle. Monthly sampling was carried out, along with the collection of environmental and biometric data at each site. The sex ratio did not differ significantly from 1:1 at either study site. The gonadosomatic index (GSI) reached its peak during spring (April and May) for both sexes, and the spawning period was observed from summer to autumn (June–October) at both locations, suggest annual spawning. GSI was correlated with increasing or high chlorophyll-a concentrations, salinity, and spawning with rainfall periods. The estimated size at sexual maturity was 15.9 cm body length, 189.1 g total body weight, and 92.5 g gutted body weight. The minimum mean oocyte diameter was recorded during the recovery stage (31 ± 11 μm (mean ± SD) at Skhirat and 34 ± 23 μm (mean ± SD) at Souiria K'dima), while the highest values were observed in the partial spawning stage (112 ± 27 μm (mean ± SD) at Skhirat and 104 ± 19 μm (mean ± SD) at Souiria K'dima). This study highlights the need for further research to expand our knowledge of the biological and ecological characteristics of H. arguinensis populations, to facilitate the cultivation of juveniles in captivity, and to develop fishing regulations based on scientific findings. As a fishery management strategy, we suggest a closed season from April to October to protect the reproductive period of H. arguinensis on the Moroccan coast.

Introduction

Sea cucumbers are generally gonochoristic and reproduce sexually, while some species show asexual reproduction (Conand et al., 2002). They are occasionally hermaphrodites and are primarily broadcast spawners (Hyman, 1955; Smiley, 1988, 1990). In most holothurians, the gonadal tubules of males and females can be distinguished by their different colours when gonads become mature, and the thickness of these gonadal tubules is used as an indicator of sexual maturation (thicker tubules hold higher numbers of oocytes). In holothurians, the gametogenesis process appears to be affected by either exogenous (temperature, photoperiod, lunar cycle, tidal flux, chlorophyll-a concentrations, rainfall, phytoplankton bloom, and food availability) or endogenous factors (neuroendocrine activity). These different environmental, chemical, and hormonal factors acting directly or indirectly, separately or in combination, have been suggested to prompt, support, or moderate reproductive performance from the commencement of gametogenesis to spawning (Hamel and Mercier, 1996, 1999; Wigham et al., 2003; Mercier et al., 2007; Katow et al., 2009; Dissanayake and Stefansson, 2010; Omar et al., 2013; Marquet et al., 2017; Venâncio et al., 2022), but still, the role of each of these factors in modulating the reproduction is far from to be fully realized.

The sea cucumber Holothuria arguinensis is widely spread around the Northeastern (NE) Atlantic from the Berlengas Islands (Portugal) to Morocco and Mauritania, including the Canary Islands (Costello et al., 2001; Rodrigues, 2012). In the Mediterranean, it was reported for the first time on the eastern coast of Spain and the Algerian coast (González-Wangüemert and Borrero-Pérez, 2012; Mezali and Thandar, 2014). In response to the increasing Chinese market demand for sea cucumbers, it has become a target species in the NE Atlantic sea cucumber fisheries (González-Wangüemert and Borrero-Pérez, 2012; González-Wangüemert et al., 2013, 2016). Thus, in these regions, information on biological attributes mostly unknown for this class of echinoderms is extremely important for initiating management and conservation action plans for ecosystems, implementing minimum size limits and maximum catch limits in fisheries, and establishing practical approaches to aquaculture for commercial production and for restocking purposes.

The sea cucumber H. arguinensis in which sexes are separate but cannot be differentiated externally is a common, large species of ecological and economic importance and attracted the attention of researchers many years ago. However, information on its biology and ecology is very limited. Marquet et al. (2017) conducted the first study on reproductive biology of H. arguinensis from southern Portugal. Later, other studies have addressed the aquaculture and fisheries of this species (Domínguez-Godino et al., 2015; Domínguez-Godino and González-Wangüemert, 2018, 2019).

On the Moroccan coast, the H. arguinensis population structure was first studied by Haddi et al. (2022) and revealed that H. arguinensis were commonly comprised of individuals ranging in size from 12 to 13 cm with an average length of 13.3 ± 3.1 (±SD). The growth performance index (Ф) value within the scale of other commercially important species of sea cucumber. In Morocco, the sea cucumber fishery falls into the broader category of echinoderm fisheries and is not recorded separately in national fishing reports. It has been acknowledged as belonging to the Echinoderms class. From 2010 to 2018 an average of 50 tons of echinoderms landed with a peak of 112 tons in 2015 (National Office of Fisheries, 2015). Therefore, the Moroccan government has proposed a ban on sea cucumber fishing after 2011 to protect this resource and is committed to regulate the illegal fishing of these species.

One of the principal causes of the collapse of several sea cucumber stocks can also reflect serious knowledge gaps in basic biological and ecological characteristics and their reproductive biology. In fisheries management, awareness of the reproductive biology of sea cucumbers is a principal component of developing sustainable management actions. For example, the initiation of a closed season during spawning and a minimum capture size can help to restore and conserve wild stocks (Mercier and Hamel, 2009; Wang et al., 2015). Therefore, the current study aims to investigate and overcome the lack of baseline knowledge of the species related to reproduction, for example, macro and microscopic characteristics of gonadal tubules, different gonadal development stages (GDS), and reproductive cycle. This study also assesses the gonadosomatic index (GSI) and correlates it with different environmental variables (temperature, salinity, and chlorophyll-a) and considers the population sex ratio, size, and weight at the first maturity of this species. The results of this study will be used to improve the management of this marine resource along the Moroccan Atlantic coast.

Materials and Methods

Sampling strategy and environmental parameters

To study the reproduction of Holothuria arguinensis along the Moroccan Atlantic coast, two sampling sites, Skhirat (North Atlantic) (33°52′14″N, 7°03′44″W) and Souiria K'dima (Central Atlantic) (32°03′2.936″N, 9°20′30.415″W) were selected (Figure 1). The distance between these two sites is 340 km. The selection was made based on the abundance of the species and its current importance as a commercial resource (National Fisheries Research Institute (INRH) conducted prospection surveys of different sites along the Atlantic to choose the best sites). At Skhirat, H. arguinensis occurs in the subtidal zone (according to the previous prospecting survey by the INRH), while at Souiria K'dima it is in the intertidal zone (information provided by local fishermen). Both study sites are characterized by the presence of rocky patches with boulders that intersperse sandy beaches (Bayed, 2003). The study areas are rich in biodiversity, with a variety of seaweed, bivalve, and echinoderm species (Haddi et al., 2022).

Figure 1. Geographical location of the sampling stations (Source: Ocean Basemap, customized in ArcGIS software 10.8.2).

Holothuria arguinensis was collected monthly between December 2016 and December 2017 (except in July at Skhirat due to logistic issues). At Skhirat, 455 individuals (28–40 individuals/month) were randomly sampled by scuba diving between a depth interval of 5 and 15 m. At Souiria K'dima, 209 individuals (6–26 individuals/month) were handpicked from the intertidal zone. Each individual was kept separate in plastic bags and transported live in a cooler filled with seawater (de Jesus-Navarrete et al., 2018) to the coastal resources prospecting laboratory at INRH. Once they arrived at the laboratory, they were stored in the refrigerator for relaxation at 4°C for 12 h before taking measurements and dissection (Navarro et al., 2012; Benítez-Villalobos et al., 2013).

Since temperatures and chlorophyll-a do not differ significantly from the sea surface to a depth greater than 20 m (Bessa et al., 2019), we used the MODIS-aqua satellite database available online at NASA's Ocean Color (2022). The sea temperature (ST) (1.5 m below sea level) and chlorophyll-a concentration were extracted for both study sites, with a spatial resolution of 5 km and 8 days of temporal resolution, spanning an equivalent of four measurements/month for a period of 13 months from December 2016 to December 2017. Salinity data were acquired from the NASA Open Data Portal (2024) and sourced as a level four product with a spatial resolution of 0.25° and a monthly temporal grid. This product represents the monthly mean of the level four Ocean Salinity Interpolation System Sea Surface Salinity (OISSS) dataset, amalgamated from three satellite missions: Aquarius/SAC-D, Soil Moisture Active Passive (SMAP), and Soil Moisture, and Ocean Salinity (SMOS), utilizing Optimal Interpolation (OI) with a 7-day decorrelation time scale. Subsequently, the NASA SeaDAS software (8.3.0) was employed for visualization and data extraction purposes.

Biometric parameters

After the relaxation of individuals, total body length (TL) was measured using an ichthyometer to the nearest 0.01 cm (Ramón et al., 2010). Total body weight (TBW) before dissection and gutted body weight (GBW) (following removal of all viscera, gonads, and respiratory tree) (Conand, 1981) were measured using an electronic scale to the nearest 0.01 g. After dissection, the gonads were removed from each individual, dried on blotting paper, and weighed (GW ± 0.01 g). For further detailed measurements and their analysis of H. arguinensis see materials and methods in Haddi et al. (2022). The sex of each individual was recognized visually by the appearance and colour of the gonads; creamy or white for the males and pink or orange for the females (Siddique and Ayub, 2015; Leite-Castro et al., 2016), this was confirmed with histological images. The macroscopic characteristics of the gonadal tubules involved in this study were the colour, weight, and branching of gonads.

Histological analysis

Histological analysis was performed at the histology laboratory of the INRH. For this purpose, small sections (0.5–1 cm in length) from the mid-region of randomly selected tubules were removed from each specimen (Marquet et al., 2017) and fixed in Davidson's solution for 24–48 h. Tubule dehydration through graded ethanol solution of increasing concentrations until 100%, clearing in xylene, and infiltration in paraffin wax were performed through an automated tissue processor (Leica ASP300). Paraffin-embedded tissues were cut with a rotary microtome (Leica RM2255) to obtain 2 μm-thick tissue sections. The ribbon of sections was mounted on glass slides, dried in an oven for 12 h at 60°C, and stained with haematoxylin-eosin (Martoja and Martoja-Pierson, 1967). The slides were subsequently observed under a light microscope (Olympus BX53) to identify the sex and assign a gonadal developmental stage (GDS). Gonads development was classified into five gonadal development stages according to the scale adopted by Ramofafia et al. (2000), I. Recovery, II. Growing, III. Mature, IV. Partially spawned, and V. Spent stages. This GDS allocation followed a microscopic examination across each transverse-section of the gonads, and it draws into account the gonadal wall thickness, tubule appearance, filling of the lumen with gametes, and the gamete types.

Gonadsosomatic index (GSI)

The gonadosomatic index (GSI) (which refers to the intensity of the reproduction regardless of each GDS) shows a good picture of the reproductive season (Abadia-Chanona et al., 2018) and was calculated according to the formula GSI = (GW/GBW) × 100 (Conand, 1981, 1993a, 1993b; Ramofafia and Byrne 2001; Abdel Razek et al., 2005; Benítez-Villalobos et al., 2013). Here, the highest GSI values obtained show that most specimens have gonads full of gametes (III – mature stage), whereas the lowest values show the prevalence of V – spent, I – recovery, and/or II – growing stages.

Sex ratio and reproductive cycle (GDS frequencies)

The sex ratio (male: female) was calculated monthly and annually. A χ ² test was performed to check if the sex ratio differed from 1:1 (Zar, 2010), where P < 0.05 was recognized as statistically significant. Reproductive cycles of H. arguinensis (temporal differences in the occurrence of animals in each gonadal maturity stage) for males, females, and combined sexes were assessed. A stacked bar graph is used to represent combined sex data, as H. arguinensis does not show external sexual dimorphism, and for practical management and comparison of fishery resources data usually do not distinguish between the sexes (Abadia-Chanona et al., 2018).

Size at sexual maturity

Size at sexual maturity was defined as the size (TL₅₀) or total body weight (TBW₅₀) and or gutted body weight (GBW₅₀) at which gonads of 50% of the individuals were mature (Conand, 1993a, 1993b). This was determined by grouping the sampled individuals by sex and size class. Then, the ratio of mature individuals in each size class was calculated (stage II is taken as the beginning of the gonadal development phase).

To estimate the TL₅₀, the proportion of mature individuals at different size intervals are fitted to the symmetrical sigmoid logistic curve (Pope et al., 1983) whose mathematical expression is as follows:

$$P = 1/( {1 + {\rm e}( {a + b \times {\rm TL}} ) } ) $$

where P = percentage of sexually mature individuals; TL = total length (cm); a: original order and b: slope

The representation of the maturity ogive is carried out by considering all pairs of values except those with a proportion: P = 0 and P = 1.

$${\rm T}{\rm L}_{50} = {-}a/b$$

Oocyte diameter

Simultaneously, every female gonad slide was analysed monthly using the Image J 1.41 software to determine oocyte diameter. Following Costelloe (1985), only the oocytes with apparent nuclei on the cross-section were counted. Each section/slide was divided into 3–4 sub-samples and the widest part of the oocytes was registered for diameter determination (Walton, 1948). Absolute frequencies of all oocyte diameters were also calculated by grouping into 20 μm size classes.

Statistical analysis

The normality of data was evaluated by the Kolmogorov–Smirnov test. Likewise, data (which followed a normal distribution) were tested for differences by month using Student's t test (TBW, GBW at Skhirat, and TL at Souiria K'dima). Data that were not normally distributed, were tested using a Mann–Whitney test (TBW, GBW at Souiria K'dima, TL at Skhirat, GSI, and oocyte diameters in both locations). Multiple comparisons were carried out using either the Kruskal–Wallis or Games–Howell post-hoc tests to detect significant monthly variations in GSI and oocyte diameters. Sea temperature, salinity, and chlorophyll-a were compared between sites by Student's t test. To explore the relationship between the reproduction of H. arguinensis demonstrated by GSI and environmental parameters such as sea temperature, salinity, and chlorophyll-a, Spearman's rank correlation test was applied. The statistical analyses were carried out with IBM SPSS Statistics 25 software.

Results

Population characteristics and sex ratio

The TL of all the sampled H. arguinensis ranged from 6.00 to 22.00 cm and 6.50 to 23.00 cm at Skhirat and Souiria K'dima, respectively. The TBW ranged from 17.32–341.38 g and 38.86–425.20 g, while the GBW ranged between 10.82–205.04 and 13.49–214.44 g at Skhirat and Souiria K'dima, respectively (see Table 1 in Haddi et al., 2022). Mean (±SD) values of TL, TBW, and GBW of males (M), females (F), and combined (M + F) collected at both study sites are shown in Table 1. There were no significant differences in TL, TBW, and GBW among sexes and locations (Mann–Whitney's test, P > 0.05, P values are in Table 1).

Table 1. Biometric characteristics of male and female Holothuria arguinensis

Mean (±SD) total length (TL) (cm), total body weight (TBW) (g) gutted body weight (GBW) (g), and gonadsosomatic index (GSI) of males (M), females (F), and combine sex (M + F) sampled at Skhirat (SKH) and Souiria K'dima (SK).

The relationships among TBW and GW, TBW and GSI, GBW and GW, GBW and GSI, TL and GW, 204 and TL and GSI, were found to be non-correlated in both sexes at Skhirat and Souiria K'dima (r ² < 0.2, Table 2).

Table 2. The relationships among gonad index (GSI), gonad weight (GW), and different biometrics parameters total length (TL), total body weight (TBW), and gutted body weight (GBW) of Holothuria arguinensis sampled at Skhirat (SKH) and Souiria K'dima (SK)

Although H. arguinensis was commonly considered a gonochoristic species, a few hermaphroditic specimens were encountered during the present study at both localities (Figure 2a, b). Out of the 455 individuals collected at Skhirat, 6 were hermaphrodites (1.3%), 105 females (23.0%) and 93 males (20.4%), 3 were of undetermined sex (0.6%), whereas 248 did not have gonads (54.5%) (Figure 3A). The absence of gonads during particular months has been linked with the resorption of tubules after spawning (Conand, 1993b). At Souiria K'dima, 209 individuals were collected: 1 was hermaphroditic (0.5%), 66 females (31.6%), 67 males (32.0%), 1 undetermined (0.48%), and 74 without gonads (35.4%) (Figure 3B). The overall sex ratio (M: F) did not differ significantly from a 1:1 relation (χ² = 0.727, P = 0.393 and χ² = 0.008, df = 1, P = 0.930 at Skhirat and Souiria K'dima, respectively).

Figure 2. Gametogenesis in the hermaphrodite specimens of Holothuria arguinensis on the Moroccan coast. a: Macroscopic features; b: microscopic features; male gonad (MG); female gonad (FG); mature oocyte (MO); gonads wall (GW); lumen (L); spermatozoa (SZ); spermatocytes (SC).

Figure 3. Temporal variability of sex frequency of Holothuria arguinensis population at Skhirat (A) and Souiria K'dima (B).

Macroscopic and microscopic observation of gonadal developmental stages (GDS)

Based on the macroscopic and histological characteristics, the five maturation stages are:

Stage I: recovery

The macroscopic analysis could not determine the sex at this stage, only histological analysis could. The tubules were thin, translucent, slightly branched, and short for males and females (Figures 4a, b). Histologically, the tubules were characterized by an empty lumen and a thick gonadal wall where three basic layers were easily distinguished (Figures 5a–d). In male gonads, the spermatogonia were seen along the germinal epithelium, which has multiple infolds that enhance the surface area (Figures 5a, b). In female gonads, oogonia, previtellogenic oocytes, as well as some early vitellogenic oocytes, are present along the germinal epithelium. In the lumen, some phagocytes were also observed (Figures 5c, d).

Figure 4. Macroscopic features of male (left) and female (right) Holothuria arguinensis gonads at different maturity stages recovery (a, b); growth (c, d); mature (e, f); partially spawned (g, h); gonads showing unspawned and spawned tubules (arrows); spent (i, j).

Figure 5. Microscopic characteristics of different maturity stages in the ovary and testis of Holothuria arguinensis.

Stage II: growing

In both sexes, the tubule proportions, numbers, and branching increased as gametogenesis proceeded. The sex could be determined easily macroscopically through the colour of the gonad. The colour of male tubules progressed from clear cream to semi-opaque whitish (Figure 4c) and that of females from light pink to light orange (Figure 4d). At this stage, the thickness of the gonad wall is gradually reduced (Figure 5e). Vitellogenesis and spermatogenesis intensified as gonadal development progressed. In male tubules, numerous infolds and columns of germinal epithelium characterized the early growing stage. They were lined with a thick layer of spermatogonia and spermatocytes, which stretched for some expanse into the lumen (Figures 5e, f). In the late growing stage, the germinal infolds were reduced as the production of the gametes progressed. The spermatids were becoming more abundant, and a few spermatozoa were observed in the lumen. In female tubules, previtellogenic and early vitellogenic oocytes were visible along the germinal epithelium. The lumen was mostly occupied by mid and late vitellogenic oocytes surrounded by a single layer of follicular cells (Figures 5g, h). In the late growing stage, some mature oocytes, which contained prominent germinal vesicles and distinct nucleoli, were also present in the lumen (Figure 5h).

Stage III: mature

Mature tubules were bulgy, longer, and branched at their maximum levels in both sexes (Figures 4e, f). The female gonads were dark orange (Figure 4f). While the male gonads progressed from semi-opaque to opaque white (Figure 4e) gonadal wall had minimal thickness in both sexes at this stage, and the connective tissue could not be discerned (Figures 5i–l). In mature male tubules, the lumen was filled with spermatozoa. Few spermatocytes and spermatids were present throughout the germinal epithelium. The infolds of the latter were decreased or absent (Figures 5i, j). Histological analysis of the mature ovary showed densely packed, fully developed mature oocytes. They had a follicle, a polygonal shape with a well-defined nucleus, and several of these oocytes showed a germinal vesicle.

Stage IV: partially spawned

In this stage, the gonads of both sexes were composed of spawned and unspawned tubules. Spawning activity is indicated through their colour variation and density. Male gonads tubules were beige to translucent drab cream (Figure 4g). Female tubules were dull orange to translucent orange (Figure 4h). The gonads wall remained thin in partially spawned male and female tubules (Figures 5m–p). As for male tubules, the spermatozoa were less dense in the lumen and the distinct gaps in the spermatozoa mass were related to the partial release of mature gametes (Figures 5m, n). The reappearance of infolds was also observed in the male germinal layer (Figure 5m). The abundance of oocytes within female tubules was remarkably reduced, and empty spaces were seen in the lumen, while some tubules were still filled with mature gametes (Figure 5o, p). Spermatogenesis and oogenesis were re-initiated in partially spawned tubules, as demonstrated through, the occurrence of developing spermatocytes (Figure 5n) and previtellogenic oocytes (Figure 5p) in male and female tubules. Revived gametogenesis resulted in the existence of overlapping generations of germinal cells. Phagocytes were generally seen in both spawned and unspawned tubules of both sexes (Figure 5p).

Stage V: spent

The male gonads were light creamy to transparent, and the female gonads were clear pinkish to translucent (Figures 4i, j). Spent tubules of both sexes were thin, short, wrinkled, and narrowed. In females, the gonads wall became very thick, and a large area of connective tissue was observed in both male and female tubules (Figures 5q–t). The spent testes were an empty lumen except for some residual spermatozoa and numerous phagocytes (Figures 5q, r). Scattered spermatogonia lined the germinal epithelium of some tubules (Figure 5r). Spent ovaries showed high phagocytic activity and signs of degradation of unspawned relict oocytes, whereas some tubules were devoid of contents (Figures 5s, t).

Population-level gonadsosomatic index (GSI) across GDS

The Kruskal–Wallis test showed a significant difference in males' and females' GSI over the months at both locations (P < 0.001), except for Souiria k'dima no significant difference was found over the months (P = 0.256) at the population level (Figures 6a, 7a). However, there were no significant differences in the mature stages (III) between females and males at Skhirat (P = 0.344 and P = 0.121, respectively) and Souiria K'dima (P = 0.622 and P = 0.195, respectively), nor in the spawning stages (IV + V) between females and males at Souiria K'dima (P = 0.089 and P = 0.768, respectively), except for the spawning stage at Skhirat, which displayed a significant difference between females and males (P < 0.001, and P = 0.011, respectively) (Figures 6c, d & 7c, d).

Figure 6. Gonadosomatic index (GSI) of H. arguinensis from December 2016 to December 2017 at Skhirat. a: GSI for males and females; b: range for each GDS for males and females; c: GSI for mature stage males and females (III, n = 26); d: GSI for spawning stage males and females (IV&V, n = 80).

Figure 7. Gonadosomatic index (GSI) of H. arguinensis from December 2016 to December 2017 at Souiria K'dima. a: GSI for GSI for males and females; b: range for each GDS for males and females; c: GSI for mature stage males and females (III, n = 18); d: GSI for spawning stage males and females (IV&V, n = 61).

The monthly population means for GSI exhibited a similar pattern of variation for both sexes at both sites, with one peak observed throughout the sampled period (Figures 6a, 7a). The minimal values were recorded from December 2016 to February 2017 and from July through December 2017 at both locations (Figures 6a, 7a). In February, we noted a significant increase in GSI (P < 0.001), reaching a maximum value in April for females and in May for males at both sites. However, the effect of sex on GSI was not significant (Mann–Whitney test, P = 0.112, P = 0.982) at Skhirat and Souiria k'dima, respectively.

Males displayed monthly mean GSI values between 0.27 and 4.77 and 0.46 and 5.09 and females between 0.33 and 7.47 and 0.73 and 8.38 at Skhirat, and Souiria K'dima, respectively (Figures 6a, 7a) (Table 1). Both sexes combined mean GSI values showed 2.25 ± 1.89 (±SD) and 2.69 ± 2.64 (±SD) at Skhirat, and Souiria K'dima, respectively (Table 1). The median values for GSI across the GDS showed highly significant differences for both sexes at both sites (Kruskal–Wallis test, P < 0.001) (Figures 6b, 7b), with the mature (III) and spawning (IV, V) stages having the highest GSI values in both sexes and sites. However, in both sexes and sites mature (III) stages had higher GSI than spawning stages, possibly on account of depletion due to gamete release. There was no significant effect of sex on GSI at the mature stage at Skhirat (III; Mann–Whitney test, P = 0.231), while there was a significant effect at Souiria k'dima (III; t test, P = 0.021). However, no significant effect of sex on GSI at the spawning stage either at Skhirat (IV, V; P = 0.252) or at Souira k'dima (IV, V; P = 0.879). So overall, gonad expenditure seems to be equal for each sex at any given stage of gonad advancement.

For the mature stage (III), there were no significant differences in the median values of GSI among months (Figures 6c, 7c) for both sexes and sites; at Skhirat, females (F _{(2, 16)} = 1.142, P = 0.342) and males (Kruskal–Wallis test, P = 0.121) and at Souiria k'dima, females (F _{(2, 6)} = 0.514, P = 0.622) and males (F _{(2, 7)} = 2.087, P = 0.195). At Skhirat, for the two population peaks (April and May), there was no significant effect of sex on mature stage during April (III-stage, P = 0.264) or during May (III-stage; P = 0.345) (Figure 6c). At Souiria K'dima, same results during April (III-stage, P = 0.936) and during May (III-stage; P = 0.057; extended to include June, (P < 0.001) (Figure 7c).

Similarly, for the spawning stage (IV, V) (Figures 6d, 7d), there were significant differences in the median values of GSI among months for both females (P = 0.000) and males (P = 0.011) at Skhirat. While there was no significant effect of sex on spawning stages peaks in June, (IV, V stages; P = 0.186) and in August–September (IV, V stages; P = 1). At Souiria k'dima, there were no significant differences in the median values of GSI among months for females (P = 0.089) and males (P = 0.768), as well as no effect of sex on spawning stage during June, (IV, V stages; P = 0.385) and in August–September (IV, V stages; P = 0.879).

Reproductive cycle (GDS frequencies)

The reproductive cycle of H. arguinensis showed a clear seasonal pattern with synchronous development among the sexes and in both study sites (Figures 8 & 9). In general, the five GDSs showed a similar frequency pattern between sexes throughout the sampled months, with two stages (mature and spawning) displaying a predominance of high frequency. GDS frequencies for both sexes combined are also shown in Figures 8 & 9.

Figure 8. Monthly variation of the gonad developmental stages (GDS) of the Holothuria arguinensis population from December 2016 to December 2017 at Skhirat. (a) Males and females combined, (b) females, and (c) males.

Figure 9. Monthly variation of the gonad developmental stages (GDS) of the Holothuria arguinensis population from December 2016 to December 2017 at Souiria K'dima. (a) Males and females combined, (b) females, and (c) males.

At Skhirat, the Recovery stage (I) (sexes combined) was prevalent from November to January (with frequencies between 50.0 and 80.8%). This stage was also found in March (19.4%) due to the late spawning of some individuals supporting the hypothesis that H. arguinensis can spawn multiple times during the spawning season (Ramofafia et al., 2000; Foglietta et al., 2004). The percentages of both sexes were similar and varied between 47 and 100%, except for December 2016, where no males in this stage were detected in this stage. Growing stage (II) extended from January to April, with a high frequency of females in March (71.4%). The mature stage (III) was prevalent from April to May and peaked in April (51.4%). The percentage of mature females was higher than mature males (65.0 and 33.3%, respectively). Female mature sea cucumbers remained present in June but at the lowest frequency (9.0%). The spawning activity in females, including sea cucumbers in partially spawned (IV) and spent stages (V), started in April with a low frequency of 25.0% to reached higher frequencies in June with 54.55% in spent, whereas male spawning individuals achieved their highest frequency in June at 95.65% and extended until September when 50.0% of males were observed in partially spent stage (Figure 8).

At Souiria K'dima, the Recovery stage (I) was observed first in August (14.3%) and then from October to April (with frequencies of 36.8–75.0%) (sexes combined). The percentages of both sexes were similar and varied between 33.3 and 100%. The growing stage (II) prolonged from December to April, with a peak of females in March (83.3%). The mature sea cucumbers (III) were dominant from April to June, with the highest percentage in May (75.0%) for both sexes. They continued existing in June with the lowest percentage (10.0%) (combined sex) but females more than males 14.29 and 7.69%, respectively. The spawning phase, including partially spawned (IV) and spent stages (V), was predominant from June-October at Souiria K'dima occurred the high frequencies in October (63.6%) for both sexes (Figure 9).

Environmental parameters and gonadsosomatic index (GSI)

Seawater temperature showed a similar seasonal trend at both locations, peaking in August (Skhirat: 22.7 ± 0.2°C (±SD); Souiria K'dima: 20.5 ± 0.1°C (±SD), and the minimum monthly mean temperature was found in February at both sites (Skhirat: 16.5 ± 0.1°C (±SD); Souiria K'dima: 16.3 ± 0.1°C (±SD)) (Figures 10A, B). The salinity ranged between 36.4 and 36.5 psu at both study sites with average of 36.5 ± 0.0 (±SD). (Figures 10A, B). Chlorophyll-a concentration showed a seasonal pattern at Souiria K'dima, while it did not at Skhirat. Its annual mean value was higher at Skhirat (1.9 ± 0.5 (±SD) μg l⁻¹) than at Souiria K'dima (1.2 ± 0.2 (±SD) μg l⁻¹) (Figures 10A, B).

Figure 10. Seasonal variations of environmental parameters: ST: sea temperature (°C), salinity (psu), and chlorophyll-a (μg l⁻¹) Gonadosomatic index (GSI) at A: Skhirat and B: Souiria K'dima.

At Skhirat, there was a significant negative correlation of GSI with salinity and chlorophyll-a, while no significant relationship was found with sea temperature (Table 3). At Souiria K'dima, correlation analysis revealed no significant correlation between GSI and all environmental parameters (temperature, salinity, and chlorophyll-a) (Table 3). However, at Souiria K'dima the GSI and chlorophyll-a increased on and off together from January to May 2017, although, this trend changed dramatically from June to July 2017 as, the chlorophyll-a continued to rise but the GSI decreased gradually. Thus, the recovery period occurred at higher temperatures at both study sites.

Table 3. Results of the Spearman's correlation between the environmental parameters and GSI of H. arguinensis sampled at Skhirat and Souiria K'dima

Statistical significance level: P < 0.01**; P < 0.05*.

Size/weight at first sexual maturity

L₅₀ estimates at first sexual maturity were estimated to 15.9 cm in total body length (TL₅₀), 189.1 g in total body weight (TBW₅₀), and 92.5 g in gutted body weight (GBW₅₀).

Oocyte diameter

Monthly oocyte diameter provided a more precise representation of the seasonal gonadal development in female sea cucumbers. During the growing stage, from February to April, the diameter of the most representative oocytes ranged from 10 to 70 μm at Skhirat and from 10 to 90 μm at Souiria K'dima. It gradually increased as the gonads were maturing from 90 to 130 μm at both sites, reaching the highest values (130–150 μm) in the partial spawning and spent stages at Skhirat in June–August and in September at Souiria K'dima. The maximum measured diameter of mature oocytes was 190 μm. The oocyte diameter decreased progressively, reaching the lowest values during the recovery stage from November to January (10–50 μm) at both sites (Figure 11). The statistical analyses showed a significant difference in oocyte diameter between both locations (Mann–Whitney test, p < 0.05).

Figure 11. Frequency distribution of the oocyte diameter (μm) of Holothuria arguinensis at Skhirat (SKH) ■ and Souiria K'dima (SK) .

Discussion

Sex ratio

In this study, our observations agree with previous studies that reported a sex ratio of 1:1 for Holothuria species (Despalatovic et al., 2004; Kazanidis et al., 2010; Benítez-Villalobos et al., 2013; Siddique and Ayub, 2015; Marquet et al., 2017; Ramos-Miranda et al., 2017; Rogers et al., 2018). This balanced sex ratio is not evident in all Holothuria species. Higher proportions of females have been reported in H. leucospilota and H. arenacava, respectively (Muthiga, 2006; Gaudron et al., 2008). Sex ratio in favour of males was reported such as 125:73 in H. whitmae in Queensland (Shiell and Uthicke, 2006), 1.7:1 in H. fuscogilva in the Maldives (Reichenbach, 1999), 4:1 in H. nobilis in Micronesia (Amesbury et al., 1996), and 31:1 in S. chloronotus in La Reunion (Conand et al., 2002). The deviation in sex ratio was linked characteristically with species that experience asexual reproduction (Harriott, 1982; Uthicke et al., 1998). However, differential growth and mortality could also be associated with the dominance of one sex over the other (McPherson, 1965) and may be related to gonad maturation in echinoderms (Brookbank, 1967). On the other hand, some former studies pointed to fishing pressure as a possible root cause for the unbalanced sex ratio (Shiell and Uthicke, 2006; Muthiga et al., 2009; Santos et al., 2017). However, this study was carried out in an area where the population is unexploited, suggesting this ratio is a natural attribute for these detritivorous species. Furthermore, we did not notice any fission during our collection, but the presence of hermaphroditism in this species was reported for the first time. Marquet et al. (2017) did not report any case of hermaphroditism in the samples population from southern Portugal. A low proportion of hermaphroditic individuals in populations of sea cucumbers has also been reported in several other species, including, Stichopus mollis (Sewell, 1990), Isostichopus fuscus (Herrero-Pérezrul et al., 1998; Pañola-Madrigal et al., 2017), Holothuria mexicana (Rogers et al., 2018), Patinata ooplax (Kubota, 2000), and Pentactella perrieri (Martinez et al., 2020). It has been assumed that a low population density of a species, may intensify hermaphroditism and self-fertilization (Rogers et al., 2018). On the contrary, the existence of hermaphrodites will decrease if the population density increases (Ghiselin, 1969).

Reproductive cycle

In Morocco, H. arguinensis followed an annual reproductive cycle with spawning from summer (June-August) through autumn (September–October). This pattern, based on evidence from GSI, histology, and spawning observations, is analogous to that described for several other temperate holothurians (e.g. Hamel and Mercier, 1996; Despalatovic et al., 2004; Navarro et al., 2012; Kazanidis et al., 2014; Mezali et al., 2014), with maximum reproductive activity observed during the warmer months and the lowest activity (resting) in the colder months.

GSI showed a synchronous reproduction pattern in both males and females at both study sites, indicating a close relationship with the local oceanic dynamics (Benítez-Villalobos et al., 2013). Early gametogenesis started in January–February, and a distinct spawning season was recognized in April–October. In contrast, Marquet et al. (2017) in H. arguinensis populations in southern Portugal observed different periods of early gametogenesis (October, November, and December at three different locations, respectively), but similar spawning periods (July–October). Other species of the genus Holothuria, show similar patterns of reproduction to the present study, such as e.g. H. arenicola from Pakistan (Siddique and Ayub, 2015), H. mexicana from Caribbean Panama (Guzmán et al., 2003), and Belize (Rogers et al., 2018).

We did not find any relationship between GSI and any of the biometric parameters registered (Table 2), which shows that the gonad growth of this species is widely variable. Sea cucumbers have unique body structures, including a soft, leathery body wall, and internal organs that can vary significantly in size and density. This can make it challenging to establish a consistent relationship between body size and gonad weight.

The absence of gonads (resting period) was observed in October–January and has in some species been associated with the complete resorption of the gonads after spawning (Conand, 1993b). Because they possessed all of their internal organs except gonads, the possibility of auto-evisceration can be excluded (García-Arrarás and Greenberg, 2001). An increased number of individuals without gonads was found after the spawning months, which has also been observed in species, belonging to the order Aspidochirotida by Herrero-Pérezrul et al. (1999) in the southern Gulf of Mexico in Holothuria sanctori by Navarro et al. (2012) in Gran Canaria, H. arenicola by Siddique and Ayub (2015) in Pakistan, and Isostichopus badionotus by Acosta et al. (2021) in Colombia. The absence of gonads during the resting stage has been related to their resorption and suggests that the tubule recruitment model (TRM) that has been suggested to describe ovarian development in sea cucumbers (Smiley and Cloney, 1985; Smiley, 1988, 1994; Smiley et al., 1991) may not apply to H. arguinensis, although tubule growth was not uniform throughout the gonads in some individuals; and simultaneously displayed different degrees of maturation. Exceptions to the TRM have been also observed for I. badionotus (Foglietta et al., 2004) and H. glaberrima (Gómez, 2011). Gonad development in some species of the Holothuria genus do seem to follow the TRM model, notably H. arguinensis from southern Portugal (Marquet et al., 2017), H. fuscogilva (Ramofafia et al., 2000), H. scabra (Ramofafia et al., 2003), H. leucospilota (Purwati and Luong-van, 2003), H. whitmaei (Shiell and Uthicke, 2006), and H. floridana (Ramos-Miranda et al., 2017). Other Holothuria species with gonad development that does not seem to conform to the TRM, include H. atra (Chao et al., 1995), H. tubulosa (Despalatovic et al., 2004), H. sanctori (Navarro et al., 2012), and H. arenicola (Siddique and Ayub, 2015). According to Frick et al. (1996) and Sewell et al. (1997), the TRM model only describes gonad development in a few specimens of a few species in the genus Holothuria at different geographical positions. Hamel and Mercier (1996) have proposed that local environmental conditions possibly can affect tubular growth, but this aspect needs more systematic research.

Environmental parameters and gonadsosomatic index

Generally, the ubiquity of the reproductive traits of sea cucumbers is correlated with environmental factors such as seawater temperature, food availability, photoperiod, and chlorophyll-a (Smiley et al., 1991; Hamel et al., 1993; Muthiga, 2006; Toral-Granda and Priscilla, 2007; Omar et al., 2013; Leite-Castro et al., 2016). Our study reported a significant negative correlation of GSI with salinity and chlorophyll-a, but no significant correlation was observed with sea temperature. Water temperature is the environmental key factor mostly cited as influencing holothuroid reproduction (Hopper et al., 1998; Muthiga et al., 2009; Omar et al., 2013). A positive correlation between GSI and sea temperature was found in H. arguinensis from southern Portugal (Marquet et al., 2017), as well as in H. leucospilota from the western Indian Ocean (Gaudron et al., 2008) and H. mexicana from Belize (Rogers et al., 2018). On the other hand, Siddique and Ayub (2015) reported no correlation between GSI and temperature for H. arenicola from Pakistan, North Arabian Sea and Ramón et al. (2022) observed no influence of temperature on the gonadal cycle on Parastichopus regalis from the Mediterranean Sea. The reason may be that these species have less thermal sensitivity to the production of reproductive hormones (Miller et al., 2015). Further studies would be necessary to define the proximate environmental factors triggering the seasonality of gametogenesis and spawning of H. arguinensis on the Moroccan coast. The negative correlation of GSI with salinity at Skhirat may indicate that specific salinity ranges are conducive to favouring reproductive conditions, possibly by affecting physiological processes associated with gonad development. However, at Souiria K'dima, no significant correlation between GSI and salinity was found. This may suggest that other factors, such as food availability, may have a stronger influence on the reproductive cycle of H. arguinensis on this site At Skhirat, a negative correlation was observed between GSI and chlorophyll-a levels, where chlorophyll-a served as an indicator of phytoplankton biomass, which increased following the GSI peak. This suggests that the rise in phytoplankton biomass during the summer months (June–August), coinciding with the spawning of H. arguinensis. Indeed, a synchronization of spawning according to phytoplankton availability is considered a common advantageous adaptation in marine invertebrates (Starr et al., 1990; Watson et al., 2003; Navarro et al., 2012; Santos et al., 2017). In contrast, no correlation was found between GSI and chlorophyll-a at Souiria K'dima, suggesting that the larvae were able to develop in a low-food environment. It can be concluded that the cues to spawning and gonad maturation seems to be determined by different attributes depending on the site. This is consistent with the findings of Marquet et al. (2017), who reported a significant positive correlation of GSI with sea water temperature and photoperiod (with a time lag) but no correlation with chlorophyll-a, for the same species, H. arguinensis from southern Portugal. Venâncio et al. (2022) for H. mammata and Rogers et al. (2018) for H. mexicana reported positive correlations of GSI with all environmental parameters as significant. However, the satellite measurements used in the present study may not reveal local conditions, and therefore further studies would be necessary on the reproductive strategy of H. arguinensis to define the proximate environmental factors triggering gametogenesis and spawning in H. arguinensis on the Moroccan coast.

Size/weight at first sexual maturity

This study estimates L₅₀ at first sexual maturity in the H. arguinensis population; it was estimated to be 15.9 cm in total body length (TL₅₀); similar to that of other species of Holothuria, sharing a similar size range (5.0–21.0 cm) such as H. floridana from Campeche, Mexico (Ramos-Miranda et al., 2017) and H. mexicana from the Caribbean Sea of Southern Belize (Rogers et al., 2018). However, the total body weight at first maturity (TBW₅₀) was estimated at 189.13 g, which is smaller than that of H. leucospilota from the Western Indian Ocean, sharing a similar range of gutted body weight (26–203 g) (Gaudron et al., 2008). Though, there is a remarkable difference with Marquet et al. (2017) observation for the same species H. arguinensis, which reported total length at first sexual maturity was between 21.0 and 23.0 cm, while gutted body weight at first maturity was between 110 and 130 g and total body weight at first sexual maturity was between 220 and 260 g. Biologically, smaller sizes at maturity could be a sign that H. arguinensis had been overfished by local fishermen on the Moroccan coast compared to southern Portugal. Besides this, data represents the first estimation of L₅₀ for H. arguinensis from Morocco and may be considered as a preliminary evaluation, requiring additional data for lower-size classes. Because size and weight at first maturity are considered necessary to improve fisheries management and conserve exploited holothurians. Such small immature sea cucumbers have less commercial importance, and their exploitation reduces the sustainability of this population.

Oocyte diameter

In the present study, H. arguinensis oocyte diameter ranges from <10 to >190 μm. Marquet et al. (2017) also reported almost a similar interval of oocyte diameter for H. arguinensis (<20 to 180 μm) from southern Portugal. According to Sewell et al. (1997), Strathmann et al. (1992), and Ramirez-Llodra (2002), holothuroids with oocytes ranging from 50 to 300 μm indicate planktotrophic larval stages. During planktotrophic development, the larvae depend on food available in the ambient water because their eggs lack sufficient energy reserves. Thus, species with such types of larvae usually coincide spawning with rainfall periods because the primary production is in optimal conditions. It allows the larvae to feed and survive (McEdward and Miner, 2003; Reitzel et al., 2004), as seemingly occurs with the reproductive cycle of H. arguinensis off the coast of Morocco, where rainfall is reported from November to March (climates to travel, n.d.). Our result is in good agreement with those reported earlier by Chao et al. (1995) for three orders of intertidal holothurians from southern Taiwan and H. spinifera by Asha and Muthiah (2008) from Tuticorin (India). The oocyte diameters were represented in size classes, showing that at both sites females had a smaller oocyte in higher frequency (<30 μm) between November and January, when they were in recovery or post-spawning stages. The growing stages had oocytes with a diameter of up to 100 μm (February–April), mature females had oocytes with 100–130 μm, and spawning occurred from May to August and September, when the oocytes reached a diameter greater than 130 μm at both sites. These results support the study of Santos et al. (2017) and Venâncio et al. (2022), who documented mature oocytes had a diameter between 108–122 and 100–120 μm, respectively, for H. mammata. Additionally, Tuwo and Conand (1992) demonstrated that mature individuals of H. forskali had oocytes with a diameter between 90 and 120 μm. Some previous studies reported that mature and non-mature oocytes are present at all gonad maturation stages, like in Holothuria tubulosa (Despalatovic et al., 2004), Holothuria sanctori (Navarro et al., 2012) and Holothuria fuscogilva (Ramofafia et al., 2000); conversely, in this study, no mature oocytes were witnessed in the recovery stage, and no immature oocytes were found in the spawning stage.

Conclusion

Our study has shown for the first time that Holothuria arguinensis on the coast of Morocco displays hermaphroditic characteristics, an equal sex ratio and an annual reproductive cycle, with gonad maturation happening during spring (April–May), spawning occurring in summer to autumn (June–October), and with a resting phase in autumn–winter (October–January). For the Moroccan coast, it would help to establish a harvest season with closure during the spawning season. The sea cucumber fishery in Morocco is currently not regulated. The biological data obtained for H. arguinensis in this study, ought to serve as a preliminary reference for the implementation of management protection and establishing regularity measures of this important resource (e.g., high reproductive activity (April–October), legal catch size (greater than 15.9 cm), and allow fishing on foot only (as a preventive method). However, more studies must be conducted to manage sea cucumber populations in Morocco, and collect more data on population density, fecundity per size, distribution, and recruitment per size studies to facilitate stock assessments with high accuracy.