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Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(S1): 101-117, March 2021 (Published Mar. 30, 2021)

Reproductive cycle of the sea cucumber Holothuria forskali

(Holothuriida: Holothuriidae) in the Ría de Vigo (NW of Spain)

Tania Ballesteros

Ana Tubío

Rosana Rodríguez

Alba Hernández

Damián Costas

Jesús Troncoso

1. Centro de Investigación Mariña, Universidade de Vigo, Grupo de Ecoloxía Costeira, Edificio CC Experimentais,

Campus Lagoas Marcosende, Vigo, Spain; tballesteros@uvigo.es (*Correspondence), atubio@uvigo.es,

alba.hernandez@uvigo.es, troncoso@uvigo.es

2. Centro de Investigación Mariña, Universidade de Vigo, Estación de Ciencias Mariñas de Toralla, Illa de Toralla,

Coruxo, Spain; rrodriguez@uvigo.es, dcostas@ecimat.org

Received 10-V-2020. Corrected 08-IX-2020. Accepted 20-X-2020.

ABSTRACT

Introduction: The exploitation of the sea cucumber (Holothuria (Panningothuria) forskali) in Ría de Vigo (NW

Spain) is recent (2015) and it has been done until now with scarce or no information about some key biological

aspects as reproduction, recruitment or growth. Objective: To describe the reproductive cycle of H. forskali

in Ría de Vigo. Methods: We sampled fortnightly throughout 2018. We calculated gonadal condition indices

(GCI) and gametogenic stages by classic histological methods. Results: The reproductive cycle of H. forskali

in the Ría de Vigo is characterized by a sexual resting stage during spring, when temperature and daylight hours

are lower; the beginning of gametogenesis during summer, when temperature is higher, daylight hours longer

and the sea bottom is rich in nutrients; then, a period of spawns interspersed with a gonadal restoration during

autumn and winter, when temperature is lower and food is scarce. Sex ratio is 1:1, however, the studied popula-

tion is not synchronized, because females initiate maturation earlier. The comparison of the histological results

with GCI indices suggest that GCI is a good indicator for gonadal stage. Fishery management strategies, such as

a closure period, must be adapted to the reproductive stage. We recommend avoiding fishing between November

and February to increase spawning potential ratio and, consequently, recruitment.

Key words: gonad restoration; abiotic factors; sex ratio; gonadal condition index; fisheries.

Ballesteros, T., Tubío, A., Rodríguez, R., Hernández, A.,

Costas, D., & Troncoso, J. (2021). Reproductive

cycle of the sea cucumber Holothuria forskali

(Holothuriida: Holothuriidae) in the Ría de Vigo

(NW of Spain). Revista de Biología Tropical, 69(S1),

101-117. DOI 10.15517/rbt.v69iSuppl.1.46331

For centuries, sea cucumbers have been

consumed as a luxury food item in Asian coun-

tries (Conand & Byrne, 1993; Bordbar, Anwar

& Saari, 2011; Mehmet, Hüseyin, Bekir, Yilmaz

& Sevim, 2011; Purcell et al., 2013) due to its

high protein content, low fat content and amino

acid profile (Chen, 2003, 2004; Taboada,

Millán, Míguez & Fernández-Pulpeiro, 2008).

Due to its nutritional richness, they have also

been used in artificial feed (Bakus, 1973) and

as a fishing bait (Entrambasaguas, 2008). They

have been used in the traditional medicine sys-

tems, too because of their effectiveness against

hypertension, asthma, rheumatism, cuts and

DOI 10.15517/rbt.v69iSuppl.1.46331

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Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(S1): 101-117, March 2021 (Published Mar. 30, 2021)

burns, impotence and constipation (Wen, Hu &

Fan, 2010; Bordbar et al., 2011). Also, they are

part of balms, oils, toothpastes, body lotions

and soaps (Poh-Sze, 2004; Conand, 2005). In

addition, new studies have attracted interest

from nutritionists and pharmacologists (Zhong,

Khan & Shahidi, 2007; Bordbar et al., 2011;

Santos et al., 2016).

All these possible applications have mas-

sively increased their demand, causing serious

problems of overexploitation around the world.

The high demand has caused 70% of the fisher-

ies to be depleted or over/fully exploited, and

that has led to the search for new less valuable

species in other distribution areas (Guzmán,

Guevara & Hernández, 2003; Purcell et al.,

2013; Sonnenholzner, Searcy-Bernal & Pan-

chana-Orrala, 2017; González-Wangüemert,

Domínguez-Godino & Cánovas, 2018). 16 out

of the 66 exploited species have been classified

as vulnerable or endangered by IUCN (Conand

et al., 2014; IUCN, 2019).

One of these new target species is Holo-

thuria (Panningothuria) forskali (González-

Wangüemert et al., 2018). This species is

present in the NE Atlantic Ocean, in Portugal

several companies are selling it (González-

Wangüemert, Valente, Henriques, Domínguez-

Godino & Serrão, 2016) and in Galicia (NW

of Spain) it began being exploited in 2015.

Galicia´s fisheries are co-managed by the

Autonomous Administration (Xunta de Gali-

cia) and fisher’s guilds (called “cofradías de

pescadores”) which nowadays design harvest-

ing plans every three years. H. forskali was

included in the sea urchin exploitation plan

(Plan Xeral de Explotación, 2015), as both

are echinoderms. Until now, the management

of the exploitation of this marine resource has

been done with scarce or no information about

some key biological aspects as reproduction,

recruitment and growth in this area. Studies

about the reproduction of holothurians are usu-

ally implemented when a fishery exploitation

has already started, or even when populations

are already overexploited (Navarro, García-

Sanz & Tuya, 2012).

There are only two studies on the repro-

ductive biology of this species. The first one

was carried out in Brittany-France by Tuwo

and Conand (1992) and the second in Peniche-

Portugal (2016) by Santos et al. Both agree

that it has an annual cycle, but disagree on

other aspects such as the resting period. The

gametogenic cycle may differ between popula-

tions in response to interactions with external

factors (e.g. seawater temperature, salinity,

light, food availability, parasitic infestations)

and internal factors (e.g. genetics, hormonal

cycle, nutrient reserves). This may generate

variations even within a small spatial range

like a ria, especially in sedentary invertebrates

such as sea cucumbers. In a study of gonadal

development of the temperate species Sewell

(1992) found that one population in northern

New Zealand had complete resorption of the

gonad after spawning, with no gonad material

present during the winter months. The same

species in the south retained a large volume of

tubules containing developing oocytes in the

resting period. Later Sewell, Tyler, Young and

Conand (1997) showed that gonad develop-

ment varies in holothurians as a function of

taxonomic position, geographical location and

habitat, even within individuals in the same

location. The aim of this paper is to present the

main features of the reproductive biology of

H. forskali in a bed of the Ría de Vigo, based

on histological examination of the gonads and

gonad indices, as well as to investigate the

relationship of the reproductive cycle with the

main environmental variables. This knowledge

would allow the development of effective

fishery management strategies, taking steps

toward conservation.

MATERIALS AND METHODS

Sampling collection: Samples were taken

fortnightly (each comprising 30 individuals)

during 2018 from the Ría de Vigo (Galicia, NW

Spain) (42°14’65’’ N & 8°49’63.4’’ W) (Fig. 1),

by scuba-diving, at a depth of 10 m, with a total

of 630 individuals. Samples were put in plastic

recipients with seawater and were brought to

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laboratories at Estación de Ciencias Mariñas de

Toralla (ECIMAT, University of Vigo).

Histological analyses and gonadal con-

dition index (GCI): Individuals were relaxed

for 15 min with 5 % MgCl

before the follow-

ing measurements were taken: total length (Lt)

and total wet weight (Wt). For histological

analysis, a piece of gonad was dissected and

routinely processed for histology: i.e. fixed in

Davidson formaldehyde for 24 h, dehydrated

in an ethanol series, embedded in paraffin, sec-

tioned at 5 μm and stained with hematoxylin

and eosin (Howard & Smith, 1983).

A gonadal status was assigned to each

individual according to a scale modified from

Tuwo et al. (1992), Ramofafia, Battaglene,

Bell and Byrne (2000), Navarro et al. (2012)

and Santos et al. (2016), which describes a new

stage of gonadal restoration, not described until

now for this species far as authors know. Gonad

development was therefore scored in five stag-

es: 0 (sexual rest), 1 (start of gametogenesis),

2 (advanced gametogenesis), 3 (maturation),

4A (spawning), 4B (gonad restoration) and 5

(exhaustion). For all collected individuals, the

gonad was removed, weighed (Gw) and mea-

sured (Gl). The eviscerated body weight (Ew),

which corresponded to the body wall weight,

excluding internal organs was also recorded.

The eviscerated body and the gonad were dried

for 48 h at 80 ºC to obtain dry weight of the

gonad (Gd) and dry weight of the eviscerated

body (Ed).

Two gonadal condition indices (GCI) were

calculated using wet and dry weights of the

body wall and gonads (Tuwo et al., 1992;

Navarro et al., 2012): GCI

= Gd100/Ed and

GCI

= Gw100/Ew.

The GCI values were plotted using the

mean and the standard deviation as estimators

of the central trend of the sample. Progress

through the gametogenic stages was deter-

mined by the histological analyses and changes

in the GCI; it was compared to confirm the

correlation between both methods.

Environmental variables: The environ-

mental variables selected to study its effect

on the reproductive cycle of H. forskali were

sea water temperature and photoperiod. Water

temperatures were obtained from Torallamar

Fig. 1. Location of the sea cucumber studied population (star).

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(Oceanographic and Meteorological Informa-

tion Portal developed by the Physical Oceanog-

raphy Group and the staff of the Marine Research

Center of the University of Vigo) (https://www.

pangaea.de/?q=event%3Alabel%3ACIM-ECI-

MAT) (González, Herrera & Varela, 2020) and

the number of hours of light per day was pro-

vided by the reference meteorological station

(www.meteogalicia.es).

Chi-square analysis was performed on the

data of all samples analyzed by histological

methods to test possible deviations from the 1:1

ratio of the male:female ratio. The normality

of the size and weight distribution was evalu-

ated by the Shapiro-Wilk test. The relationship

between size/weight and sex was examined by

a non-parametric test, unpaired two-samples

Wilcoxon test. The homogeneity of variance

(Levene test) and normality (Shapiro test) in

the GCI between groups was not achieved.

Therefore, to study possible differences of the

GCI between variables (sex-month and sex-

season) it has been used a robust method of

a single factor Welch-ANOVA for each of the

settings and Games-Howell post-hoc test to sta-

blish pairwise comparisons of means. Two-way

ANOVA to explore single and combined effects

of sex, months and seasons over GI (Wilcox,

2012; Liu, 2015; Mair & Wilcox, 2020).

RESULTS

Population characteristics: Of the 630

individuals, 49.37 % were males, 45.24 %

females and 5.40 % were undetermined sex

(Fig. 2). The sex ratio for all sampled speci-

mens not differ significantly from 1:1 (c

1.1342

P > 0.05). All indeterminate individuals

correspond to the period of sexual rest, when

the sexes cannot be differentiated. The aver-

age weight (± SD) of the males was 262.47

± 83.47 g and of females 271.62 ± 83.46

g; average length (± SD) was 27.68 ± 5.04

and 27.60 ± 5.04 cm, respectively (Table 1).

Although females were bigger (in weight) than

males, no significant differences in weight

were found between the sexes (Wilcoxon, P

> 0.05). Significant differences were found

between gonad measurements and sex. Males

had longer gonads than females, but weighed

less (Wilcoxon, P < 0.05).

Fig. 2. Proportion of sex of H. forskali by monthly sample (N = 630).

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Histological analyses: seven stages were

defined for H. forskali based on histological

observations (Table 2). A gonadal status was

assigned to each individual according to fol-

lowing scale:

Stage 0 - Sexual rest: most of the gonad

is occupied by connective tissue. The tubules

are small with very thick walls. There may be

primary germ cells but it is not possible to dif-

ferentiate the sex (Fig. 3A). This stage remains

from March to May, predominating in April.

Stage 1- Start of gametogenesis: the wall

of the tubule is reduced, but the connective

tissue still abounds. In males the germinal

epithelium forms numerous folds extending to

the lumen. The tubules are full immature cells:

oogonias and previtelogenic oocytes adhered

to the walls of the tubule in females (Fig.

3B) and spermatogonia of spherical appear-

ance and primary spermatocytes in males (Fig.

3C). This stage predominates from the end of

April to July.

Stage 2 - Advanced gametogenesis: the

inside of the tubule is filling up of cells, the

wall goes away slimming down and the folds

begin to disappear as gametogenesis progress-

es. All maturity stages of the reproductive cells

are observed, with more germ cells than mature

ones (in number). The immature oocytes are

fully adhered to the wall of the follicle while

mature oocytes are attached by one peduncle

or free in the follicle (Fig. 3D). The imma-

ture sperm (spermatogonia, spermatocytes, and

spermatids) are close to the follicle wall and

mature sperm (with tail) occupies the center

(Fig. 3E). This stage predominates from July

to October.

Stage 3 - Maturation: the tubules are very

big; have a thin wall, the longitudinal folds in

them disappear and no germ cells are found.

In females, oocytes measure 162.68 ± 6.1

µm and may acquire polygonal forms due to

them under pressure (Fig. 3F). In males, sperm

occupy all the lumen, hold on strongly and the

longitudinal folds disappear (Fig. 3G). This

stage is more frequent between September

and February.

Stage 4A - Spawning: the tubule wall

remains thin, with strangulations, and as the

stage progresses this begins to thicken again.

As a result of the partial release of gametes,

follicular pressure decreases, empty spaces

are observed inside the follicles (Fig. 3H for

females and Fig. 3I for males). Disorganiza-

tion of the sperm is stands out in males. This

stage predominates from the end of October

to March.

Stage 4B - Gonad restoration: after the

spawn, a new line of germ cells appears on

the wall of the tubules. This stage is similar to

stage 2, but it differs in the big size of tubules,

in the absence of connective tissue and in that

the wall is thin (Fig. 3J for females and Fig.

3K for males). In males there is a separation

between the mature spermatocytes at the center

of the lumen and the immature spermatocytes

at the periphery of the follicles. This stage is

more frequent between December and April but

in a low number of individuals.

Stage 5 - Exhaustion: the tubules with

numerous folds begin to degenerate (phago-

cytic cells appear), so they decrease in number

and size, being replaced by connective tissue.

In females, residual oocytes can be observed

TABLE 1

Biometric data attending to sex

Wet weight (g) Length (cm)

Mean ± SD Min Max Mean ± SD Min Max

Males 311 262.47 ± 83.47 100.4 522 27.68 ± 5.04 15 45

Females 285 271.62 ± 83.46 109.35 622 27.60 ± 5.04 12 42

Undetermined 34 241.94 ± 70.07 122 451 27.06 ± 4.13 19.5 37

*N = Population size, Mean ± standard deviation, Min = minimum size, Max = maximum size.

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(Fig. 3L) and in males, the spermatozoa are dis-

organized (Fig. 3M). The gonad is practically

reabsorbed by phagocytic activity. This stage

predominates from March to May.

Gonadal condition index (GCI), repro-

ductive cycle and environmental factors:

The mean (± SD) obtained from GCI

was

12% (± 11%) while that of GCI

was 11%

TABLE 2

Description of each gonadal state for females and males

Stage Month Males Females

0: sexual rest March-May Abundance of connective tissue

Small tubules

Abundance of connective tissue

Small tubules

1: start of

gametogenesis

April- July Abundance of connective tissue

Small tubules

Germinal epithelium with numerous folds

Immature cells: spermatogonia and

primary spermatocytes

Abundance of connective tissue

Small tubules

Immature cells: oogonias and

previtellogenic oocytes adhered to the

walls

2: advanced

gametogenesis

July-October Inside of the tubule is filling up of cell

Wall goes away slimming down

Folds begin to disappear

Cells in all stages of development

Spermatogonia, spermatocytes, and

spermatids are close to the follicle wall

and mature sperm (with tail) occupy the

center

Inside of the tubule is filling up of cell

Wall goes away slimming down

Cells in all stages of development

Immature oocytes adhered to the wall

and mature oocytes attached by one

peduncle or free in the follicle

3: maturation September-

February

Big tubules

Thin wall

No folds

No germ cells

Sperm occupy all the lumen

Filled tubules

Big tubules

Thin wall

No germ cells

Polygonal oocytes

Filled tubules

4A: spawning October- March Big tubules

Thin wall with strangulations

No folds

No germ cells

Empty spaces

Disorganization of the sperm

Big tubules

Thin wall with strangulations

No germ cells

Follicular pressure decreases

Empty spaces

4B: gonad

restoration

December- April Big tubules

Thin wall with strangulations

Absence of connective tissue

New line of germ cells on the wall of the

tubules

Mature spermatocytes in the center

Immature spermatocytes in the periphery

of the follicles

Big tubules

Thin wall with strangulations

Absence of connective tissue

New line of germ cells on the wall of

the tubules

Mature oocytes in the center

Previtellogenic oocytes in the periphery

of the follicles

5: exhaustion March- May Numerous folds

Phagocytic cells

Small tubules

Connective tissue

Phagocytic activity

Phagocytic cells

Small tubules

Connective tissue

Residual oocytes

Phagocytic activity

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(± 11%). Despite the fact that the GCI

was higher except in the months of sexu-

al rest (March, April and May), the trend

throughout the year is similar in both

indices (Fig. 4A, 4B and 4C). Conse-

quently, only GCI

will be kept in mind

for using the dry weight and not the wet

weight, resulting in less error. The GCI

varies throughout the year, significant

differences were found between months

(Welch’s ANOVA, F

11,216.33

= 146.26, P

< 0.05) and seasons (Welch’s ANOVA,

3,360.46

= 267.16, P < 0.05). Consider-

ing two-to-two comparisons between

months, there would be no difference

between the winter months and the

spring months. June shows more similar-

ity with the spring months, August with

the winter ones and in the winter months,

September differs from November.

There were significant differences

between sexes on the yearly mean GCI

(Welch’s ANOVA, F

1,524.03

= 6.05, P <

0.05) obtaining an annual mean (± SD)

12.2 % (± 11.7 %) for females and 10.2

% (± 8.8 %) for males. There is interac-

tion of the GCI

between sex-season (sex

x season, two-way ANOVA, P < 0.05)

and sex-month (sex x month, two-way

ANOVA, P < 0.05), the difference in

the GIC

between sexes is not constant

throughout the year, that difference is

more important in winter and more con-

stant in the other seasons (Fig. 4D). The

maximum values of the gonad index

Fig. 3. Gametogenic stages of female (B, D, F, H,

J, L) and males (C, E, G, I, K, M) Holothuria

forskali. A. Stage 0-sexual rest in indeterminates.

B-C. Stage 1-start of gametogenesis. D-E.

Stage 2-advanced gametogenesis. F-G. Stage

3-maturation. H-I. Stage 4A-spawning. J-K. Stage

4B-gonad restoration. M-N. Stage 5-exhaustion.

CT: connective tissue, CTF: connective tissue

folds, GV: germinal vesicle, GW: gonadal wall, L:

lumen, N: nucleus, O: oocyte, P: phagocytes, PO:

previtellogenic oocytes, PS: primary spermatocytes,

RO: residual oocyte, S: spermatozoa (bar: 100 µm).

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were achieved in February during the winter

season, whereas in spring the lowest values

were found, corresponding to the sexual rest

stage (stage 0) when there is hardly any gonad.

The successive ups and downs from October to

January may be indicative of partial spawning,

and those in February, of the main spawning.

The study of the reproductive cycle (Fig.

5) began in winter, when the H. forskali species

was in the maturation (stage 3) and spawning

(stage 4A) stages, with the predominance of the

latest ones. Then, the gonadal remnants were

resorbed to a stage of sexual exhaustion (stage

5) and rest (stage 0) that occurs in spring, find-

ing a higher percentage of individuals in these

stages in April. After a period of sexual rest

(the total number of individuals sampled in this

stage is never observed), a new gonadal cycle

begins. Individuals in the beginning phase of

gametogenesis (stage 1) were observed from

April to summer, a period in which this stage

dominates together with that one of advanced

gametogenesis (stage 2). At the end of June, the

first mature individuals appeared, the stage of

maturation remained until the end of the year

and overlaps with the first spawning which

started in November. The stage of gonad resto-

ration (4B) was observed after the first spawn-

ing, in the month of December and continued

always in a low percentage of the population

until the rest stage (end of April). Something to

note, is that the stage of gonadal recovery was

Fig. 4. H. forskali gonad condition index (GCI) through the year 2018. A. Represent GCI1 vs. GCI2 in females. B. Represent

GCI

vs. GCI

in males. C. Represent GCI

vs. GCI

in all population. D. Represent the interaction of sex and seasons over

GCI. GCI

-dark grey line, GCI

-light grey line, females-empty circles and males-full circles.

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uncommon (Fig. 6), and it appeared in females

during the months of March (Fig. 6A) and

April. However, it appeared in males between

December and February (Fig. 6B).

The studied population showed some asyn-

chronous gametogenic development between

sexes (Fig. 6), females initiated maturing ear-

lier than males. It is worth noting that, in addi-

tion, the females remained in a longer stage of

maturation, finding mature females in a higher

percentage until March. Another observation to

highlight between the sexes was that in males

the first gonadal restoration was observed in

December and continued until the exhaustion

stage, whereas in the females it appeared coin-

ciding with the resting stage (March).

When representing the reproductive cycle

against environmental variables (Fig. 5), it can

be observed that there is a negative correlation

between gonadal development and photope-

riod, as the hours of light increase, the gonadal

condition index decreases, coinciding with the

stages of sexual exhaustion and rest. The sexual

rest coincides with the increase in temperature

and hours of light, and the beginning of matura-

tion, with the decrease.

DISCUSSION

Although some aspects of reproductive

biology of H. forskali have been studied in

other areas, there may be differences from one

population to another. Therefore, it was neces-

sary to study the population of the Ría de Vigo

in order to carry out proper fisheries manage-

ment. The first study that reported data on the

reproductive cycle of this species was that of

Tuwo et al. (1992) in France. As in the French

Brittany population, this population did not

show significant differences in size between

males and females as in many other species

(Despalatović, Grubelić, Ŝimunović, Antolić &

Zuljević, 2004; Fajardo-León, Suárez-Higuera,

del Valle-Manríquez & Hernández-López,

Fig. 5. Temporal distribution of gametogenic stages of H. forskali and variation in gonadal condition index (GCI; mean). 0:

sexual rest; 1: start of gametogenesis; 2: advanced gametogenesis; 3: maturity; 4A: spawning; 4B: gonad restoration and 5:

exhaustion. Also are represented monthly variations in water temperature and in the hour of light.

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2008), and the sex ratio did not differ from the

1:1. In contrast, the population of the Ría de

Vigo presents bigger individuals with an aver-

age weight of 265.42 ± 83.40 g compared to

152.5 g of the French population. This may be

due to the fact that the holothurian fishery in

Galicia is still very recent or that Galicia waters

are richer in nutrients.

Despite the fact that the sex ratio is 1:1

in most holothurians (Ramofafia, Byrne &

Battangle, 2001; Asha & Muthiga, 2008;

Fajardo-León et al., 2008; Kazanidis et al.,

Fig. 6. Temporal distribution of gametogenic stages of H. forskali. 0: sexual rest; 1: start of gametogenesis; 2: advanced

gametogenesis; 3: maturity; 4A: spawning; 4B: gonad restoration and 5: exhaustion. A. Females. B. Males.

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2010; Peters-Didier, Pardo, Garrido & Gal-

lardo, 2018; Cahuzac et al., 2019), as in the

Ría de Vigo, some species demonstrated an

unbalanced ratio (Costelloe, 1985; Navarro et

al., 2012; Omar, Abdel Razek, Abdel Rahman

& El Shimy, 2013; Pañola-Madrigal, Calde-

rón-Aguilera, Aguilar-Cruz, Reyes-Bonilla &

Herrero-Pérezrul, 2017). In fact, Santos et al.

(2016) found in H. forskali, more female than

males; these authors explain that the place of

sampling is subject to strong tides which is

considered to be another major factor affecting

the balanced sex ratio due to the fact that strong

sea waves often promote species´ evisceration

as a stress response. However, this result may

also be a consequence of the low sample num-

ber (N = 91), Tuwo et al. (1992) sampled a total

of 409 mature individuals, and in this study,

596 individuals. Shiell & Uthicke (2006) also

found that in Holothuria whitmaei the males

and females’ ratio did not differ from unity in

a population of Western Australia (N = 325),

but in a population of Pacific Australia more

males than females predominated significantly

(N = 206). Something similar happened also

happens with the species Isostichopus fuscus in

two populations of Mexico (Pañola-Madrigal et

al., 2017). Another species of the same genus

(Holothuria tubulosa) was studied in the Aege-

an Sea (N = 166) by Kazanidis et al. (2010) and

in the Adriatric sea (N = 130) by Despalatović

et al. (2004), both obtained the same result

(1:1) regarding the sex ratio.

The measurements taken from the gonads

differ between sexes, agreeing with the data

selected by Santos et al. (2016) and Tuwo et al.

(1992) to H. forskali, male gonads are longer

than female gonads but with less weight. This

is common in sea cucumbers, females with

shorter tubules have a greater quantity because

they invest more in reproduction (Shiell &

Uthicke, 2006). The size of the gonad with

respect to each sex can differ between species

of holothurian and may reflect differences in

spawning behavior (Morgan, 2000).

In all the papers reviewed, although they

can designate the stages with another name or

include 2 stages in 1, basically all of them state

a phase of rest, growth, maturation, spawn and

exhaustion. However, in this study a gonadal

restoration phase has been observed, without

going through a previous rest, probably thanks

to the high sampling periodicity and by the

sample number (N = 630). To understand this

stage of gonadal restoration, it is necessary to

keep in mind that unlike other species (Costel-

loe, 1985), H. forskali does not present germ

cells in the mature stage. They appear after the

first spawning. The gonadal restoration phase

is frequent in many bivalves (Suárez, Álvarez,

Molist & San Juan, 2005; Martínez-Castro &

Vázquez, 2012; Hernández-Otero, Martínez-

Castro, Vázquez & Macho, 2014) but not in

holothurians. The low percentage of individu-

als found in this stage suggests that those who

have gutted use this strategy (gonadal restora-

tion), individuals who have gutted part of the

gonad in the period of maturity could regener-

ate it without going through a period of rest.

These individuals begin a new gametogenic

cycle without going through a resting stage to

save time and not lose the reproductive period.

Also, if we keep in mind the GCI, before appre-

ciating the steepest peak (February) we can

observe other weaker prior peaks, and as Tuwo

et al. (1992) indicate, after spawning, almost

the entire content of the gonads is released,

causing the decrease in GCI. If this is compared

with what happens in two species of mollusks

(Darriba, San Juan & Guerra, 2005), one of

them with recovery status and the other without

it, the following can be observed: Ensis siliqua

is a species that does not exhibit gonadal resto-

ration during the spawning period and for this

reason there is only one maximum in the GCI

rather than consecutive maxima, which would

be indicative of consecutive spawns over the

course of several months, as it occurs in Ensis

arcuatus. Females start before maturation,

which may be because it was underestimating

the rate of mature males, since this status has

only been assigned to males in which the tubule

walls were free of immature cells. Although

the criteria were the same in both sexes, males

may release sperm before immature cells stop

proliferating. In addition, some females start

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a new cycle of gametogenesis without going

through a resting stage, which may be the

reason why some mature females appear in the

month of July.

The reproductive cycle of H. forskali was

determined based on the histological analysis

and the GCI. The influence of environmental

variables on it was also observed. Different

gonadal indices have been used (Pérez &

Marcos, 1985) from the beginning, with them

a first idea of the periodicity of the reproduc-

tive cycles can be obtained (Engstrom, 1980).

Two indices were used in this study, the GCI

considering the dry weight and the GCI

with

the wet weight, this is the reason why the GCI

is superior and why the differences between

indices decrease in the months of rest when the

gonad is reduced, thus accumulating less water

inside. The highest percentages of GCI

are

reached between October and February. The

gonadal cycle of H. forskali in Galicia shifts

one month when compared to the study carried

out in Brittany (France) (Tuwo et al., 1992), in

which they obtained the highest percentages

of GCI

between November and March, on

the other side it agrees with the data obtained

by Santos et al. (2006) in Peniche (Portugal),

who found the highest values in November

and February.

The GCI

trend of the was similar in both

sexes, but females have a higher ICG than

males, this difference being more evident in the

reproductive period just like in other species

(Sewell & Bergquist, 1990; Asha & Muthiga,

2008; Omar et al., 2013; Woong, Lee, Yoo &

Hang, 2017). These results coincide with those

obtained by Tuwo et al. (1992) in this species,

but they differ from those found by Santos et

al. (2006), also for H. forskali. No significant

differences were found either between sexes

or among months and in other species the

opposite occurs, where the male has a higher

GCI than that of the females except in the

maturation stage (Peters-Didier et al., 2018).

Other species of the same genus, such as Holo-

thuria sanctori, have the highest GCI values

in the summer months and the lowest between

November and March. Its reproductive cycle is

contrary to that of H. forskali, because despite

being an Atlantic species in this region there

is a subtropical climate (Navarro et al., 2012).

Comparison of the histological results with

GCI indices suggests that GCI is a good indi-

cator for the gonadal stage. A good homol-

ogy between the GCI and the stages of the

gonads with a histological analysis has been

reported for various aspidochirote holothuri-

ans (Conand, 1993; Shiell & Uthicke, 2006;

Kazanidis et al., 2010), thus GCI seems to be

reliable for the description of the reproductive

cycle in holothurians. Although a first idea of

the periodicity of the reproductive cycles can

be obtained through GCI (Engstrom, 1980)

such as Fajardo-León et al. (2008) indicate in

their studies, the GCI can vary with the physi-

ological stage of the organism, due to environ-

mental factors and diet, therefore it is necessary

to complement it with a histological study.

H. forskali showed an annual cycle, with

spawning in winter and rest in spring. These

results concur with that found for this species

by Tuwo et al. (1992), the maturation starts

in October. However, these authors conclude

that there is a short spawning in April, but in

this case, we have found partial spawns from

November and a large one in February. On

the other hand, Santos et al. (2006) did not

find a resting period in males and they found

resting females in January, May and June.

These differences can be attributed to the fact

that the frequency of gonad maturity stages

used by these authors was determined by the

physical characteristics of large tubules and

not by histology. In H. forskali there could be a

latitudinal gradient as with other species. It has

been noticed that the sexual rest of this species

in Galicia-Spain takes place one month earlier

than in Brittany-France (Tuwo et al., 1992) and

some females were found at rest in January

in Peniche-Portugal (Santos et al., 2016), two

months earlier than in Spain. A gradient lati-

tudinal from south to north has been described

(Fajardo-León et al., 2008) to Parastichopus

parvimensis, in the south the maturity-spawn-

ing period is in winter / spring and in the north

in spring / summer.

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Correlation of reproductive cycle with con-

ditions existing in the natural environment has

been examined for a number of sea cucumbers

(Costelloe, 1985; Sewell & Bergquist, 1990;

Hamel, Himmelman & Dufresne, 1993; Hop-

per, Hunter & Richmond, 1998; Asha & Muth-

iga, 2008; Pañola-Madrigal et al., 2017) since,

the reproductive activities of echinoderms are

regulated by endogenous factors, which, in

turn, are affected by exogenous environmental

factors (Costelloe, 1985). According to Costel-

loe (1985), the environmental stimuli that have

the most influence on sexual cycles directly

or indirectly are: changing temperature, salin-

ity, photoperiod, food and habitat availability

and juvenile recruitment and survival. In this

study, two of these factors have been studied;

temperature and photoperiod.

The reproduction pattern of this species is

contrary to that the other holothurians, which

reproduce during the warmer months when the

days are longer. In this way the larvae have

greater food availability due to the prolifera-

tion of phytoplankton (Cameron & Fankboner,

1986; Sewell & Bergquist, 1990; Conand, 1993;

Hamel et al., 1993; Morgan, 2000; Ramofafia

et al., 2001; Guzmán et al., 2003; Despalatović

et al., 2004; Kazanidis et al., 2010; Omar et

al., 2013; Pañola-Madrigal et al., 2017). The

spawning period of H. forskali takes place

when the temperatures are lower as well as

are the species Holothuria whitmaei (Shiell

& Uthicke, 2006), Aslia lefevrei (Costelloe,

1985) or Holothuria nobilis (Conand, 1993).

In fact, the induction of spawning by thermal

stimulus supports the suggestion that tempera-

ture may be a factor that cues gamete release.

This is a widely used spawning method in the

cultivation of holothurians (Rakaj et al., 2018;

Hartai & Pringgenies, 1998; Agudo, 2006)

and has also been successful for this species

(Laguerre et al., 2020).

It can be observed that there is a negative

correlation between gonadal development and

photoperiod, as the hours of light increase, the

gonadal condition index decreases. By contrast,

according to Cameron & Fankboner (1986),

most holothurians according spawn in longer

photoperiod; for example, Parastichopus cali-

fornicus initiates spawning with the increase

in daylight length and intensity which means

an increase in phytoplankton biomass. The

same happens with Actinopyga mauritiana,

the observations of Hopper et al. (1998) and

Ramofafia et al. (2001) support the suggestion

that spawning coincided with increased water

temperature and day length, and the beginning

of the wet season.

The strategy followed by H. forskali could

be to accumulate reserves in the hot months

with more hours of light, when the food is pre-

dominant, in order to use these reserves during

the maturation and spawning periods together

with the accessible food in this moment. In

this way, the larvae would be favored, they

would have greater availability of food, since

from February the hours of light and the tem-

perature increase. This idea was also proposed

by Costelloe (1985) and Costelloe & Keegan

(1984) for A. lefevrei, this species accumu-

lates most of the nutrients during the warmer

months and depends on these food reserves for

general metabolism and gametogenic activity

during the winter. Galicia has waters very rich

in nutrients and although the upwelling events

are a frequent phenomenon during the spring

and summer months, there is also in the pos-

sibility of observing some these events during

the maturation-spawning periods of this species

(November-February) (Álvarez, Prego, deCas-

tro & Varela, 2012). The results of this study

confirm that the abiotic factors are probably

important signals for the gonadal development

of this species.

Data obtained on the reproductive biol-

ogy of H. forskali can be useful to implement

a sustainable exploitation of this resource, as

it would help to stablish a harvest season and

a closure during the spawning period. The

sea cucumber fishery is recent in Galicia and

therefore, it is important to develop this type

of study and apply effective management mea-

sures that avoid overexploitation of this sea

cucumber species.

The tendency of the two GCI was the

same, the GCI

is a good tool to be used by

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fishermen’s guilds to check the maturity period

in which the population is, because it is faster

and does not require special equipment.

The closure period must be adapted to the

reproductive stage. Thus, it is recommended to

avoid harvesting between November and Feb-

ruary in order to increase the spawning poten-

tial ratio and consequently the recruitment.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

To all the personnel of the Toralla Marine

Science Station (ECIMAT) and to the fisher´s

guild of Cangas. This study was conducted

under the project “Study of the biology, popu-

lation status and genetic diversity of holoturia

(Holothuria forskali) in the Ría de Vigo”,

financially supported by the Consellería do

Mar, Xunta de Galicia.

RESUMEN

Ciclo reproductivo del pepino de mar Holothuria fors-

kali (Holothuriida: Holothuriidae) en la Ría de Vigo

(NW de España)

Introducción: La explotación del pepino de mar

(Holothuria (Panningothuria) forskali) en la Ría de Vigo

(NO España) es reciente (2015) y se ha llevado a cabo

con poca o ninguna información sobre algunos aspectos

biológicos claves como la reproducción, el reclutamiento

o el crecimiento. Objetivo: Describir el ciclo reproductivo

de H. forskali en un banco de la Ría de Vigo. Métodos: Se

tomaron muestras quincenales durante el 2018. Se calcu-

laron índices de condición gonadal (ICG) y se obtuvieron

los estados gonadales siguiendo métodos histológicos

clásicos. El estado gonadal de cada individuo se asignó de

acuerdo con una escala modificada por otros autores, y en

ella se describe un nuevo estado de recuperación gonadal,

no descrito hasta ahora para esta especie. Resultados:

El ciclo reproductivo de H. forskali se caracteriza por

una etapa de reposo sexual en primavera, un inicio de la

gametogénesis durante el verano y un período de pues-

tas sucesivas intercaladas con recuperaciones gonadales

durante el otoño e invierno. La proporción de sexos es 1:1

y la población estudiada no está sincronizada, las hembras

inician la maduración antes. La comparación de los resul-

tados histológicos con los índices ICG sugiere que este

índice es un buen indicador del estado gonadal. El período

de veda debe adaptarse a la etapa reproductiva, por lo que

recomendamos evitar la extracción del recurso al menos

entre noviembre y febrero para asegurar un mayor número

de puestas y, en consecuencia, el reclutamiento.

Palabras clave: recuperación gonadal; factores abió-

ticos; proporción sexual; índice de condición gonadal;

pesquerías.

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