Timing of the reproductive cycle of Holothuria (Mertensiothuria) leucospilota (Brandt, 1835) from the Persian Gulf based on evaluation of gonad histomorphology and sex hormones

doi:10.21203/rs.3.rs-1958320/v1

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Timing of the reproductive cycle of Holothuria (Mertensiothuria) leucospilota (Brandt, 1835) from the Persian Gulf based on evaluation of gonad histomorphology and sex hormones

https://doi.org/10.21203/rs.3.rs-1958320/v1

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published 27 Aug, 2023

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Sea cucumber Holothuria (Mertensiothuria) leucospilota is a native export species in the Persian Gulf, and despite its high economic value, no study has been conducted on its reproductive biology in this region. The aim of the present study was to describe the reproductive biology of H. leucospilota using gonad tissue indices and steroid sex hormones (for the first time in sea cucumbers). Spawning was observed only once in this species. The annual reproductive cycle of H. leucospilota can be divided into six stages including resting (recovery), growth, advanced growth, mature, spawning and spent. These stages were simultaneous among the population. The beginning of gametogenesis coincided with the end of January and continued until the end of July. From May to July, the majority of individuals in the population were mature and ready to spawn. Spawning finally took place in August when the water temperature reaches its maximum value. However, spawning continued until the beginning of October. In general, according to the present study, the reproductive season of H. leucospilota is the summer of the Persian Gulf, when the temperature reaches its maximum. Most individuals were in the resting phase between late October and early January, during which it was impossible to distinguish the sex of them.

Sea cucumber

Holothuria (Mertensiothuria) leucospilota

Reproduction

Persian Gulf.

Sea cucumbers belong to Echinodermata phylum and Holothuroidea class. So far, about 1400 sea cucumber (Holothuroid) species have been reported worldwide, most of which belong to the Asia Pacific region (Pawson 2007). These benthic animals mainly live among coral reefs, although they are also found in sandy and muddy bed (Castro and Huber 2000). Most species of holothuroids are found in the intertidal zones; however, the deep oceans are the habitat of a few of them (Smirnov et al. 2000). Sea cucumbers play a beneficial role in the marine ecosystem as they help recycle nutrients, break down detritus and other organic matter, after which the bacteria can continue the decomposition process. Sea cucumbers play a beneficial role in the marine ecosystem as they help recycle nutrients, break down detritus and other organic matter, after which the bacteria can continue the decomposition process (Brukner et al. 2003).

Holothuroids are typically dioecious, with separate male and female individuals, but some species are protandric. They are mostly gonochoric and have a single gonad consisting of one or two elongated tubular tufts attached to the dorsal mesentery (Ramon et al. 2022). These organisms are mostly broadcast spawners, so that the mature male and females spawners release their sperms and eggs widely in the water column, and fertilization takes place in the water (Ramon et al. 2022). It is very difficult and mostly impossible to distinguish males and females using the morphological features of the body or gonads in sea cucumbers. However, in some species, mature male and female gonads can be distinguished by their different colors (Navarro et al. 2012).

Holothuria (Mertensiothuria) leucospilota (Brandt, 1835) (black sea cucumber or black tarzan) belongs to the family Holothuriidae. It is an opportunistic species with a wide distribution in most tropical areas of the Indian Ocean, western central Pacific, Africa and Asia. H. leucospilota is the dominant species in Persian Gulf too (Afkhami et al. 2012). Its habitat includes calm and slightly deep areas on the sandy bottom or on coral rubble (Yang et al. 2019). It has been shown that this species is relatively resistant to changes in salinity and temperature and continued to grow by changing these parameters in the laboratory; while changes in temperature and salinity led to shrinking and evisceration in Japanese sea cucumber (Apostichopus japonicus) and finally its death within three days (Yang et al. 2019). H. leucospilota is an edible sea cucumber with a high content of bioactive compounds that considered as a valuable fishery resource in many regions, especially in East Asia (Han et al. 2010). Despite the ecological and economic importance of H. leucospilota, little information is available on the reproductive biology of this species, and the available information is mainly related to its basic biology, food selection, and genetic.

Knowledge of sea cucumbers gonadal histomorphology is very important to understand their reproductive cycle. However, life history information obtained using advanced techniques is a prerequisite for efficient natural resource management. Therefore, the present study was carried out to elucidate the reproductive cycle of H. leucospilota in the Persian Gulf using the steroid hormones and gonads histological analysis, focusing on timing of the spawning period, as well as providing documents on the environmental conditions involved in the release of gametes. So far, parameters such as temperature (e.g., Chao et al. 1995), photoperiod (e.g., Muthiga 2006), or the salinity (e.g., Li et al. 2011) have been proposed as potential spawning inducers for temperate and tropical holothurian species. Finally, determining which of these environmental factors is the main driver of this phenomenon can be a difficult task (Olive 1995).

2.1. Study areas and sampling

About 262 individuals of Holothuria leucospilota (125 female, 105 male and 32 undifferentiated individuals based on the histological study of gonads) were randomly hand-picked monthly from Bushehr coasts, situated in the north of the Persian Gulf, (28^o 59′ N, 50^o 50′ E) (Fig. 1, 2) along the intertidal zone during the lowest low tide between April 2020 to August 2021. Sea cucumbers were anesthetized for 12 hours before being dissected by releasing them in seawater at 3°C. After measuring the total body weight and length, sea cucumbers were dissected and whole gonadal tubules were removed from the body and weighed. The macroscopic properties of each gonad including color, number, length, diameter and branching of the tubules were observed. The gonads (10 samples/month) were then fixed in 10% formalin buffer solution for 48 h and preserved in 50% ethanol until histological analysis. In addition, the gonads from 10 other samples were stored monthly at -80^o C for further analysis of sex steroids. Environmental parameters including salinity and temperature were also measured monthly (at the time of sampling). The care and use of experimental animals complied with the Iran Fisheries Organization. Animal welfare laws, guidelines and policies were approved by Iran Fisheries Organization and Iranian Department of Environment (IFO, program number 32886-1).

2–2. Microscopic examination

First, sex determination was performed based on the gonad color (The gonadal tubules are usually yellow or orange in the females and whitish in the males). Gonad samples were dehydrated in a graded series of ethanol, cleared in xylene, infiltrated and embedded in paraffin wax using tissue processing apparatus (Rotary model RX 11B, Tissue- Tek, Tokyo, Japan). Sections were cut at 5 to 6 µm thickness using a semi digital rotary microtome (RM2245; Leica, Wetzlar, Germany) and stained with haematoxylin and eosin (H&E). The stained samples were then analyzed microscopically to evaluate the sex and developmental stages of gonads using a light microscope (Olympus BX50) and digital images were taken using Dino Capture software (FDP2, New Taipei City, Taiwan). The different gonad developmental stages for each sex were determined according to the most histological characteristics specified for the stages used in other holothurians (Toral-Granda and Martínez 2007; Navarro et al. 2012; Benitez-Villalobos et al. 2013): inactive or resting (Stage I), growth (Stage II), advanced growth (Stage III), mature stage (Stage IV), spawning (Stage V), spent (Stage VI). Ten tissue sections from each sea cucumber (at least 6 calm/ each month) were studied to determine the degree of gonad maturity each month. The next three principals were used to define different gonad developmental stages for each sex: 1. A series of biological development trends of gametes, 2. Changes in gonadal tubules, 3. Mechanisms of gonadal regeneration or absorption after spawning (Benitez-Villalobos et al. 2013).

2–3. Quantitative Histology

2-3-1. Gonado-Somatic Index (GSI)

The Gonado-Somatic Index (GSI) was estimated using the following equation:

GSI = [Gonad weight (g)/Total body weight (g)]× 100

2-3-2. Mean gonadal index

Microscopic maturity index (MI) was calculated monthly for both sexes alone or in combination based on the microscopic gonad developmental stages using next formula (Machensen et al. 2011):

MI: (N_SI×0 + N_S1I×0.33 + N_SIII×0.66 + N_SIV×1 + N_SV×0.66 + N_SVI×0.33)/(N_{SI+S1I+SIII+SIV+SV+SVI})

Nx: Number of sea cucumbers at x-gonadal stage.

N_{SI+S1I+SIII+SIV+SV+SVI}: Total number of sea cucumbers evaluated per month.

The MI ranged between 0 (if all sea cucumbers sampled were in S0) and 1 (if all sea cucumbers sampled were in S5).

2-3-3. Oocyte Frequency

The percentage of the oocytes at each developmental stages were calculated in three regions of the ovary per female per month.

2-3-4. Gonad coverage area (GCA)

The area occupied by the gonad within an area of 7.9 mm² at ×4 from three different slices per each sample per month. The GCA was calculated as follows:

GCA= (gonad occupation area / total area) × 100

2-3-5. Oocyte Diameter

A diameter of at least 100 oocytes (with a visible nucleus) per female per month were measured (Galley et al. 2008).

2.4. Sex hormones analysis

The concentration of E2 and testosterone was measured in male and female sea cucumbers using enzyme-linked immunosorbent assay (ELISA) method according to Gauthier-Clerc et al. (2006). First, 0.5 g of gonad tissue from each sex (5 oyster/sex/month) were homogenized in distilled water (1:5 w:w). Then, 1200 µL of 25 mM HCl was added to 1500 µL of homogenate and incubated at 40 ^oC for 15 min. Before organic extraction, 3.75 mL of 0.07 M Na2HPO4 (pH 7.4) was added. The homogenates were then extracted twice using 42 ml of dichloromethane and the organic extracts were evaporated under nitrogen flow at 21–23 ^oC until dry. The resulting pellet was dissolved in 300 ml enzyme immuno-assay buffer and used for determination of sex hormones. The concentration of E2 and testosterone was determined using commercial ELISA kits (96 T MONOBIND company, Iran) based on the manufacturer’s instructions. The hormones concentration was then measured at 405 nm. Standard curves were obtained between 6.6 and 4,000 pg/mL for E2 and between 3.9 and 500 pg/mL for T and sample concentrations were assessed according to standard curves.

2–5. Statistical analysis

All data were presented as mean ± standard error (SE). The normality of data was assessed by shapiro-wilk test. Statistical differences in all studied parameters were tested using one way ANOVA followed by post hoc Duncan's multiple range test. Linear regression analysis was performed in order to determine the relationship between salinity and temperature (as independent variables), and the reproductive factors including GSI and MI (as dependent variables. A significance level of P < 0.05 was accepted.

3.1. Environmental changes

According to the Fig. 3, water salinity did not change significantly during the year. However, 40.2 and 39.52 PSU were the highest and lowest salinity recorded for the Iranian waters of the Persian Gulf that did not show significant difference (P > 0.05; Fig. 3). On the other hand, significant changes in the temperature of the Iranian waters of the Persian Gulf were measured during the year (P < 0.05; Fig. 3). The highest and lowest temperature were measured in August (33.9^oC) and February (19^oC), respectively. Temperature above 30^oC is common in this locality.

3.2. Gonad histomorphology

Figures 4, 5 and 6 show the gonad histomorphologic changes in the male and female H. leucospilota through the annual reproductive cycle. The gonad of H. leucospilota collected from the north of the Persian Gulf (Bushehr) was divided into numerous gonadal tubules that approximately all had the same diameter (ranged from less than 0.9 mm in inactive or sexually resting gonads to 2 mm in mature gonads) and length (ranged from 2.5 ± 0.5 cm in inactive or sexually resting gonads to 15 ± 1.2 cm in mature gonads). Approximately all the tubules in each gonad were in the same stage of development and simultaneously attained maturity (Fig. 4). Gametogenesis occurred uniformly along the germinal epithelium (the innermost surface of the gonadal tubules). The color of the tubules varied from yellowish cream in the inactive or sexually resting gonads to brown in the mature gonads (Fig. 4). All gonadal tubules were joined to a single gonoduct leading to a gonopore located between the tentacles.ning stage (SV); F. spent stage (SVI); GT: gonadal tubule.

Small hollow tubules with a thin wall consisting mainly of connective tissue were characteristic of gonads in inactive sea cucumbers (Stage I) (Figs. 5A and 6A). In growing gonads (Stage II), small oogonia and primary oocytes (contain nucleus (N) without obvious nucleolus) in female H. leucospilota and spermatogonia and primary spermatocytes in male H. leucospilota were visible in the developing tubules (Figs. 5B,C,D and 6B,C). Tubules were considerably expanded in advanced growth gonads (Stage III). In this stage, tubules contained many larger secondary oocytes (vitellogenic oocytes) with some oogonia and primary oocytes in females and spermatogonia, primary and secondary spermatocytes, spermatids and some spermatozoa in males (Figs. 5E,F and 6D,E,F). In the mature gonads (Stage IV), the expanded tubules were completely filled with vitellogenic oocytes (contain nucleus with obvious nucleolus and vacuolated cytoplasm) in females and the spermatozoa in males (Figs. 5G,H,I and 6G,H,I). In spawning stage (Stage V), gonad tubules contained some free vitellogenic oocytes in females and scattered spermatozoa in males (Figs. 5J,K and 6J,K). The germ cells were completely released or absorbed in spent H. leucospilota, and the gonadal tubules were mostly empty in males or mainly containing tissue debris and some atretic oocytes in females. (Table ????; Figs. 5L and 6L).

3.3. Annual reproductive cycle and frequency of gonad developmental stages

Based on gonadal histological study, only one reproductive cycle was identified for H. leucospilota in the Persian Gulf during the year, which included a relatively long period of gametogenic activity, a period of several months of spawning followed by a relatively short period of sexual rest. Figure 7 Shows the frequency distribution of the different reproductive stages observed in the present study.

As the water temperature dropped during November, 80–90% of the samples taken during November to mid-January were in the inactive or sexual rest stages (Fig. 7A,B). From mid-January, the inactive H. leucospilota gradually entered the growth stage, and 60% of females and 50% of males were in the growth stage, by mid-March (20.3 ^oC)(Fig. 7A,B). On the other hand, 20% of male and female H. leucospilota entered the advanced growth stage, by the end of March (Fig. 7A,B). From then by the end of May (30 ^oC), 75% of female H. leucospilota and 70% of male H. leucospilota were in the advanced growth stage (Fig. 7A,B). The mature H. leucospilota mostly were detected from the end of May to July. The highest amount of mature sea cucumbers was observed in July when the seawater temperature was more than 30°C (32.9°C)(Fig. 7A,B). 80% of the females and 70% of the males were mature by the end of July (Fig. 7A,B). When the water temperature reached its peak (33.9°C), many mature H. leucospilota simultaneously entered the spawning stage (80% of the females and 90% of the males), although few number of matures (5% of the females and 10% of the males) had already started spawning during July (Fig. 7A,B). Spawning continued until mid-October, although the spawning peak of H. leucospilota from the Persian Gulf was in August (Fig. 7A,B).

3.4. Biometric data and gonado-somatic index

Based on the gonad histological study, 125 samples of collected H. leucospilota were female (62.15 ± 8 gr mean body weight and 9.1 ± 1.54 cm mean body length), and 104 samples were male (58.41 ± 7.4 gr mean body weight and 8.5 ± 1.5 cm mean body length). Of these, 32 were sexually indistinguishable (43.5 ± 5 gr mean body weight and 5.36 ± 1 cm mean body length). In general, female H. leucospilota were larger than male H. leucospilota. Female H. leucospilota collected during mid-June to mid-August were significantly larger than the others (P < 0.05; Fig. 8A). Females collected in July were the largest (77.3 ± 10 gr mean body weight and 12.2 ± 2.3 cm mean body length) (P < 0.05; Fig. 8A). Sexually indistinguishable H. leucospilota collected during late-October to late-December were the smallest (43.5 ± 5 gr mean body weight and 5.36 ± 1 cm mean body length) (P < 0.05; Fig. 8A). Totally, the weight and length of H. leucospilota collected during the year followed the next pattern:

Mature H. leucospilota > Spawning H. leucospilota > Advanced growth H. leucospilota ≥ Spent H. leucospilota > Inactive or sexual resting H. leucospilota.

The weight of the gonads also followed a same pattern as total body weight and length. Mature male and female H. leucospilota collected in July had the largest gonads (17.05 ± 1.2 gr mean gonad weight) (P < 0.05; Fig. 8A). The smallest gonads were observed in inactive or sexual resting H. leucospilota (1.47 ± 0.08 gr mean gonad weight) (P < 0.05; Fig. 8A).

The GSI amounts in H. leucospilota collected during a year followed a similar trend. The highest and lowest amounts of GSI was recorded in both sexes collected in July (22.05 ± 3.4) and November (2.01 ± 0.05), respectively (P < 0.05; Fig. 8A). The pattern of GSI changes was as follows (Fig. 8A):

Mature H. leucospilota > Spawning H. leucospilota > Advanced growth H. leucospilota ≥ Spent H. leucospilota > Inactive or sexual resting H. leucospilota.

3.5. Mean gonadal index

The monthly changes of MGI had a similar pattern in male and female H. leucospilota. Moreover, a significant positive correlation (Rs = 0.7; P = 0.001) was observed between total MGI and temperature (Fig. 8B). The highest amounts of MGI (Total, male and female MGI) were recorded from mid-May to July with a peak in July in both males and females (P < 0.05; Fig. 8B). On the other hand, MGI (Total, male and female MGI) showed the lowest amount during November to mid-January (P < 0.05; Fig. 8B).

3–6. Gonad coverage area

Significant differences were observed in GCA between different gonadal developmental stage (P < 0.05). The most GCA was recorded in mature male and female H. leucospilota (SIV) in July (90 ± 8% in females and 87 ± 5% in males)(P < 0.05; Fig. 8C). In the other hand, male and female H. leucospilota in inactive or sexual rest stage (SI) showed the lowest GCA amount (4.3 ± .05% in both males and females) during November and December (P < 0.05; Fig. 8C). The amount of GCA in different male and female gonad developmental stages followed the next pattern:

SIV > SV > SIII > SII > SIV ≥ SI

3.7. Monthly changes in oocyte maturation stages and oocyte sizes

The gonadal tubules in inactive or sexually resting female H. leucospilota were mostly empty of germ cells from late October to mid-January. With the onset of gonadal growth in female H. leucospilota from mid-January, a large number of oogonia (22 ± 4.2 µm) and primary oocytes (45.2 ± 6.7 µm) were gradually observed on the inner surface of the gonadal tubes (Fig. 9A, 10A). From mid-March, the number of oocytes in the female gonad tubules increased and a significant number of secondary oocytes (68.42 ± 11.2 µm) were observed inside the tubules from April (Fig. 9A, 10A). During May and July, the number of oocytes and their size increased significantly, so that the oocytes, which were mainly vitellogenic (93.71 ± 16.2 µm), completely filled the developed gonadal tubules in mature females (Fig. 9A, 10A). During August and September, the female H. leucospilota was spawning and the oocytes observed in their gonadal tubes were mainly vitellogenic. From mid-September to mid-October, only tissue debris and a number of atretic oocytes (50 ± 7.12 µm) were observed in the gonadal tubules of spawned female H. leucospilota. From late October to mid-January, mainly no germ cells were found in the gonad tubules of inactive or resting sexual females (Fig. 9A, 10A).

Overall, the size of oocytes increased with the maturation of gonad (Fig. 9B). The mean size of oocytes gradually increased during winter and early spring and then peaked in July (Fig. 9B). The largest oocytes were in late vitellogenic stage and observed in mature females in July (P < 0.05; Fig. 9B, 10B). Female H. leucospilota in the early growth stage in January and February had the smallest oocytes (P < 0.05; Fig. 9B, 10B).

3.8. The concentrations of E2 and T in male and female gonads

The annual alteration pattern of E2 and T concentrations were consistent with the gonad development, so that their maximum and minimum levels were measured in mature male and female H. leucospilota during mid-June to late-July (E2: 471.68 ± 32.8 pg/g wet weight in females and 95.06 ± 12.5 pg/g wet weight in males; T: 28.14 ± 5.2 pg/g wet weight in females and 112.56 ± 16 pg/g wet weight in males) and the inactive or sexual resting H. leucospilota during November and December (E2: 68.9 ± 6.8 pg/g wet weight in females and 42.9 ± 3.4 pg/g wet weight in males; T: 18.5 ± 1.32 pg/g wet weight in females and 25.5 ± 2.8 pg/g wet weight in males), respectively (P < 0.05; Fig. 11A,B).

Until now, some aspects of the reproductive biology of H. leucospilota from other regions have been studied; however, there are differences from one population to another, as well as differences in the environmental conditions of their habitats. Therefore, it was necessary to investigate the Persian Gulf population in order to properly manage fisheries. According to Sewell (1992), reproductive biology may even differ among specimens of the same species collected at different latitudes.

H. leucospilota is a common species in the Persian Gulf that is gonochoric (having the sexes separate) and show no sexual dimorphism which is observed in many aspidochirotida holothurians such as H. fuscocinerea (Benitez-Villalobos et al. 2013), H. poli (Slimane-Tamacha et al. 2019) H. spinifera (Asha and Muthiah 2007) and H. (Platyperona) sanctori (Mezali et al. 2014). Drumm and Loneragan (2005) also reported no external sexual dimorphism for H. leucospilota feom Rarotonga. In the present study, the sex was determined using the mature gonad color (testes are beige and the ripe ovaries are pink/red (Purwati and Luongvan 2003; Drumm and Loneragan 2005)) and gonad histology.

Holothurians often exhibit an annual reproductive cycle (Chao et al. 1995); however, semiannual or even continuous reproductive cycles throughout the year are also common, especially in tropical regions (Conand 1993). Similar to H. poli from Lesvos Island in Greece (Bardanis and Batjakas 2018), the lack of hermaphroditic individuals and existence of an annual reproductive cycle were noted in the present study. In the present study, the annual reproductive cycle of H. leucospilota was determined using the histological and sex hormone analysis and the effect of environmental variables (including the salinity and temperature) on it was also evaluated. Based on the research, the main environmental stimuli that directly or indirectly affect the sexual cycles of organisms include changes in temperature, salinity, photoperiod, availability of food and habitat, and recruitment and survival of juveniles (Costelloe 1985). Among these, two factors (salinity and temperature) were investigated in the present study. The relationship between the reproductive cycle and natural environmental conditions has been investigated for a number of holothurians (Costelloe 1985; Sewell and Bergquist 1990; Hamel et al. 1993; Hopper et al. 1998; Asha and Muthiga 2008; Benitez-Villalobos et al. 2013; Kazanidis et al. 2014; Panola-Madrigal et al. 2017). Like H. Tubulosa and H. fuscocinerea, H. leucospilota reproduces in the warmer months when the days are longer (Benitez-Villalobos et al. 2013; Kazanidis et al. 2014). In the Persian Gulf H. leucospilota population, gamete release coincided with a significant increase in temperature (when temperatures reach 33.9°C in July-August. Similarly, H. tubulosa spawns during the warm months (from July to September) in the Adriatic Sea, when surface water temperature is at its warmest (Despalatovic et al. 2004). The main reason is that the larvae have more food available during this time (warm months) due to the phytoplankton proliferation (Kazanidis et al. 2014; Panola-Madrigal et al. 2017). According to Conand (1993), the seasonal reproductive cycle in holothurians is characteristic of most temperate species, whose spawning season occurs with increasing temperature, which is associated with increasing phytoplankton biomass and, in turn, more food source for sea cucumber. Unlike these species, the spawning period of some holothurians such as Aslia lefevrei (Costelloe 1985), H. forskali (Ballesteros et al. 2021)d whitmaei (Shiell and Uthicke 2006), occurs in colder months. Anyway, considering the stimulation of spawning by thermal changes, it seems that temperature is probably the most important factor in the release of gametes in many holothurian species, and for this reason, temperature changes are used abundantly to stimulate spawning in holothurian cultivation (Rakaj et al. 2018; Ballesteros et al. 2021). On the other hand, Vargas-Yanez et al. (2017) stated that temperature might not affect the gonadal cycle of Parastichopus regalis collected from depths of 60 to 150 m in the northwestern Mediterranean Sea, because the water temperature at these depths is quite constant and about 13–14 ^◦C in winter, spring and summer when the reproductive cycle develops. In autumn, when the temperature decreases with depth (from 18°C at 50 m to 13°C at 150 m), no gonads were found in the studied P. regalis (Vargas-Yanez et al. 2017).

Salinity could be another factor affecting the reproductive cycle in holothurians. However, there was no correlation between gonadal development in H. leucospilota and salinity in the present study. Analysis of salinity fluctuations in the Persian Gulf based on 10-year reports shows that changes in salinity in the region have a constant trend. The average salinity is 39.12 PSU during the year; it decreases to a minimum of 38.68 PSU in summer and increases to a maximum of 39.34 PSU in winter (Omidi and Norinezhad 2009). Therefore, salinity does not seem to have a significant effect on the reproductive cycle of H. leucospilota.

In the present study, H. leucospilota did not show the characteristic pattern of a continuous reproductive cycle. This is to be expected given that it is a tropical species and considering that most cycles are timed so that larval or juvenile production coincides with periods favorable for feeding or survival (Giese and Pearse 1974). Slimane-Tamacha et al. (2019) reported the same result with H. poli that exhibits a single annual reproductive cycle. On the other hand, Gaudron et al. (2008) reported a biennial cycle consisting of a dominant warm-season spawning and a smaller and more variable secondary peak in H. leucospilota in the western Indian Ocean.

In the present study, the gonad morphology and gametogenesis stages were similar to those obtained with holothurians such as H. leucospilota in the western Indian Ocean (Gaudron et al. 2008). the gonad of H. leucospilota from the Persian Gulf was similar to H. fuscocinerea (Benitez-Villalobos et al. 2013)d poli (Slimane-Tamacha et al. 2019), consisting of two bundles of long tubules, one on each side of the dorsal mesentery. However, the results of the present study differed in some aspects from the results obtained by other researchers even on the same species in other regions. Such as H. fuscocinerea (Kazanidis et al. 2014), no sexual dimorphism was observed in the color of the gonads of H. leucospilota from the Persian Gulf. This was in contrast to Gaudron et al. (2008), who noted sexual dimorphism for gonad color in H. leucospilota in the western Indian Ocean, a feature usual in many Pacific holothurians. In addition, gonad tubules were absorbed after spawning in Stichopus californicus (Smiley 1988), while in H. leucospilota (such as H. leucospilota in the western Indian Ocean (Gaudron et al. 2008)d fuscocinerea (Kazanidis et al. 2014)) the tubules were not absorbed and progenitor cells were visible along the germinal epithelium of those at the end of next January and growth continued.

In all holothurian reproductive research, although researchers may have used different names to express the stages of gonad development (e.g. two stages in a stage 1), they all express the stages of rest, growth, maturation, spawning, and exhaustion. The annual reproductive cycle of H. leucospilota culminated in spawning in mid-summer. Such as Cucumaria frondosa (Hamel and Mercier 1996), gametogenesis in H. leucospilota started in late January, after a short period of recovery. Intense synthesis of gametes and their storage continued until spawning, when gametes were released from the mature gonad tubules, which had reached their maximum size in late July to mid-October. This growth period (on average 6–8 months) appears to be necessary for each tubule to reach maturity. Such results were quite different from those reported by Gaudron et al. (2008) for the same species in the western Indian Ocean. In the present study, male and female H. leucospilota were mainly in stages 4 and 5 from July to mid-October, while at the same time, the western Indian Ocean H. leucospilota were reported in stage 3 (Gaudron et al. 2008). A Higher percentage of H. leucospilota were in post-spawning stage by the end of October, while the western Indian Ocean H. leucospilota were reported post-spawning stage at the end of February (Gaudron et al. 2008). Some western Indian Ocean H. leucospilota remained at this stage until April followed by a second spawning event in May. Gaudron et al. (2008) suggested that the occurrence of the second spawning event could be because the females that failed to spawn in February spawned in suitable environmental conditions during the wet season of the Australian summer. In agreement with Gaudron et al (2008), in the present study, gametes were in the maturation stage and distribution of oocyte diameters was more homogeneous in mature male and female H. leucospilota. While, coexistence of different stages of developing gametes within one tubule was usual in male and female growing H. leucospilota from the Persian Gulf (The present study) and the western Indian Ocean (Gaudron et al. 2008). In the present study, about 75–90% of male and female H. leucospilota had gonads every month of the year. However, the gonads were very small and undistinguishable in lower than 25% samples during the year especially during December to mid-January. It is also common to find a high proportion of specimens with gonads for several months among other sea cucumber species. Christophersen et al. (2020) showed that 67–100% of Parastichopus tremulus samples had gonads every month of the year.

Based on the microscopic observations, the gonad development in H. leucospilota male and female conforms to a synchronous "tubule recruitment pattern" and all gonad tubules are at the same stages of development in this species. It seems to be due to changes in water temperature and photoperiod (Drumm and Loneragan 2005). This is not unusual and many other holothurian species do not follow the model of an asynchronous "tubule recruitment pattern", such as H. fuscocinerea (Benitez-Villalobos et al. 2013). The same result was reported by Gaudron et al. (2008) on H. leucospilota in the western Indian Ocean. On the other hand, the asynchronous model is usual in some other holothurians such as H. atra (Chao et al. 1994), H. fuscogilva and H. mauritiana (Ramofafia and Byrne 2001).

Body size is an important factor to explain changes in reproductive function and index, both interspecies and intraspecies. For most holothurian species, with the increase in body size and weight, the weight of the gonads and thus the reproductive index increases (Conand 1993). In the present study, female and male H. leucospilota with a body weight between 61 and 70 gr and GSI more than 12% had gonads at stage III (Growing stage) of gametogenic development indicating that they were approaching sexual maturity. In addition, in H. leucospilota with a body weight between 72 and 85 gr and GSI more than 19%, the gonads were fully mature in stage VI of sexual maturity. However, these are more than the mean body weight reported for growing and mature H. leucospilota in southern Vietnam (45 gr and 67 gr, respectively) (Nguyen and Britaev 1993). Drumm and Loneragan (2005) reported female and male H. leucospilota with body weights more than 37.5 g and 55 were in growing and mature gonad development stages, respectively. Furthermore, H. leucospilota had the lowest values of GSI (between 1.2 to 2.7%) in resting months (late-October to early-January), during which the ‘sexually undifferentiated’ individuals were observed. Benitez-Villalobos et al. (2013) also recorded the lowest values of GSI (< 2) for Holothuria fuscocinerea during October to January.

In many holothurian species, the mean gonad index (MGI) has been used to determine the gonad growth pattern (Conand et al. 2002; Foglietta et al. 2004; Christophersen et al. 2020). In the current study, the MGI was used to document the development pattern of gonads, and due to the relatively low dispersion of MGI between individuals in both males and female H. leucospilota, we recommend its use to obtain suitable information on the reproductive cycle. However, in some researches on holothurian reproduction, the use of this index has not been successful. For example, Ramon et al. (2022) did not suggest the use of this index due to the high dispersion of the results in P. regalis, especially in summer when the gonad reached its maximum size. During the reproductive cycle (except from August to the end of November), the mean gonadal index in female H. leucospilota was higher than that of males. Tuwo and Conand (1992) also stated that female H. forskali had higher gonadal indices than male. Inversely, the mean gonadal index was reported to be higher in male Cucumaria frondosa (Hamel and Mercier 1996) and Psolus fabricii (Hamel et al. 1993) than that of female, before the spawning event. On the other hand, there was no inequality between male and female gonadal indices in holothurians such as Parastichopus californicus (Cameron and Fankboner 1986) and Stichopus mollis (Sewell and Bergquist 1990). It seems that the female gonads had larger gonads and higher MGI due to the need for more energy to synthesize an equivalent amount of gametes, especially considering that no significant difference was observed in the body size of males and females during the reproductive cycle. Lawrence and Lane (1982) showed that ovaries are richer than testes even though they are of similar size. According to Hamel and Mercier (1996), during periods of low food availability, the energy content decreases in the female body wall, even though its mass does not differ significantly. This indicates that a metabolic activity that requires energy is taking place and probably at the same time, (the early stage of gametogenesis), the transfer of energy to the gonads occurs. Synthesis of female gamete probably initially needs more energy than males (Hamel and Mercier 1996).

The oocyte size is another important factor in the reproductive cycle. In the holothurians studied, the size of the mature oocytes varies between 65–150 µm (Conand 1993; Hamel and Mercier 1996). In the present study, the mean diameter of the mature oocytes (a parameter that is commonly used in the population reproduction study (Conand 1993)) in H. leucospilota reached its maximum (86.57 ± 18 µm) in stage VI of maturity (May to July). This result was comparable with the reports about mature oocyte diameter in H. atra (88 µm; Harriott 1982); H. parvula (93 µm; Emson and Mladenov 1987).

The present study showed the existence of estradiol and testosterone in the gonad tissues of H. leucospilota. This helps build evidence for steroids in invertebrates, including holothurians. Although the presence of steroids has been proven in a limited number of species of holothurians such as H. scabra (Thongbuakaew et al. 2021), the amount of these hormones in these species has not been determined. The results presented here are consistent with data reported on the presence of T and E2 in other echinoderms such as the sea urchins Paracentrotus lividus (Barbaglio et al. 2007)d lividus (Janer et al. 2005). Such as other species, a close relationship was observed between T or E2 levels and the degree of gonad growth and gametes maturation in H. leucospilota in the present study. A significant difference in the concentration of T and E2 was found among the months as well as between males and females in H. leucospilota. This result contrasted with the results reported by Barbaglio et al. (2007) who found no clear correlation between T and E2 levels and distribution of reproductive stages along the year in sea urchin P. lividus. On the other hand, as in the present study, Janer et al. (2005) reported that the T level was significantly lower in the testis of P. lividus in the developmental stage (during early and advanced spermatogenesis) than in the mature stage (the end of gametogenesis). This result shows the role of T in regulating late spermatogenesis and spawning. T levels also begin to increase immediately after the resting (recovery) phase (when the gonad is preparing to reorganize for the next reproductive cycle), indicating the involvement of T in the initiation of a new spermatogenesis, as has been reported for sea urchin Lytechinus variegatus (Wasson et al. 2000). Hines et al. (1992) stated that, T levels are also elevated during the developmental stage in ovaries, during vitellogenesis, as observed in sea star Asterias vulgaris, thus indicating involvement of T also in oogenesis. In the present study, mean E2 concentration was more than T levels in females than in males, possibly reflecting a more important role of this hormone in females. As also reported in L. variegatus (Wasson et al. 2000)d vulgaris (Hines et al. 1992), mean E2 levels increased in ovaries immediately after recovery stages and it was high in the growth phase, during villogenesis, which indicates the special role of this hormone in the regulation of oogenesis. According to the data, the significant role of T and E2 in the reproductive biology of echinoderms, especially sea cucumber, seems evident.

The present study provides important information on the reproduction of H. leucospilota in the Persian Gulf. For a long time, H. leucospilota has been hunted in this area for export to other countries in an uncontrolled manner, and on the other hand, the Iranian Fisheries Organization is trying to culture this species. In conclusion, indices such as MGI and GSI used in the present study were reliable tools for reproductive cycle timing because microscopic examination was consistent with these indices. The present study showed an annual and synchronogamic pattern characterized by a spawning period in the summer months. It seems that high values of sea water temperature play a role in the release of gametes (Kazanidis et al. 2014). In the general reproductive pattern of H. leucospilota in the Persian Gulf, the maturation period was characterized by an increase in the percentage of matures (stages III and IV) from March to July, followed by spawning from August to mid-October, while the resting period lasted from late October to early January.

6. Acknowledgments

This work was practically supported by the Khorramshahr University of Marine Science and Technology.

7. Data Availability Statement

All data generated or analyzed during this study are included in this article.

8. Conflict of Interest

The authors report no declarations of interest.

9. Ethics approval

The samples were collected with a permission obtained from the Iran Fisheries Organization and Iranian Department of Environment.

10. Authors’ contributions

Negin Salamat conceived the ideas and designed methodology; Fatemeh Azimiyan Faridani and Ali Fakhri collected the samples and data; all authors contribute in analyzing data. Negin Salamat led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.

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Timing of the reproductive cycle of Holothuria (Mertensiothuria) leucospilota (Brandt, 1835) from the Persian Gulf based on evaluation of gonad histomorphology and sex hormones

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Study areas and sampling

2.4. Sex hormones analysis

3. Results

3.1. Environmental changes

3.2. Gonad histomorphology

3.3. Annual reproductive cycle and frequency of gonad developmental stages

3.4. Biometric data and gonado-somatic index

3.5. Mean gonadal index

3.7. Monthly changes in oocyte maturation stages and oocyte sizes

3.8. The concentrations of E2 and T in male and female gonads

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

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