Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e60485, enero-diciembre 2025 (Publicado Jun. 10, 2025)

Reproduction of the sea cucumber Holothuria (Halodeima) grisea

(Holothuriida: Holothuriidae) from Santa Catarina coast, southern Brazil

Yara Aparecida Garcia-Tavares1; https://orcid.org/000-0002-4190-8029

Yara Nantes-Vasconcelos1; https://orcid.org/0009-0001-6038-2288

Guilherme Sabino-Rupp2; https://orcid.org/0000-0002-5476-9689

Pablo Damian Borges-Guilherme1; https://orcid.org/0000-0001-7471-6907

Adriano Weidner Cacciatori-Marenzi3; https://orcid.org/0000-0002-8154-5867

1. Programa de Pós-Graduação em Ambientes Costeiros e Insulares (PALI), Universidade Estadual do Paraná

(UNESPAR) campus de Paranaguá, Paranaguá, Paraná, Brasil; yara.tavares@unespar.edu.br (*Correspondence), yara.

nantesv@gmail.com, pablo.borges@unespar.edu.br

2. Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina (EPAGRI), Centro de Desenvolvimento em

Aquicultura e Pesca, Florianópolis, Santa Catarina, Brasil; rupp@epagri.sc.gov.br

3. Universidade do Vale do Itajaí, Itajaí, Santa Catarina, Brasil; marenzi@univali.br

Received 05-VII-2024. Corrected 25-II-2025. Accepted 21-V-2025.

ABSTRACT

Introduction: The sea cucumber, Holothuria (Halodeima) grisea, is a common species in intertidal rocky shores

along the Brazilian coast. Due to its abundance, it is considered a potential resource for aquaculture development.

Objective: To characterize some reproductive features of H. (H.) grisea in southern Brazil, including sex ratio,

gonadosomatic and maturity indices, and macro and microscopical observations of gonad tubules.

Methods: Adult individuals were randomly collected in August and October 2019; February, March, May, and

October 2020 at the intertidal region during low tides on the coast of Santa Catarina, Brazil. The individuals were

transferred to the laboratory, where they were subject to biometry, dissection, and preservation to further macro

and microscopic examinations.

Results: The sex ratio was 1 : 1 and the gonad indexes were similar in both sexes and between months. Two

main cohorts (25.3 and 85.3 μm) and three cytometric intervals (CI) were established. Based on histology for

males, and mainly cohorts and CI for females, the individuals were categorized into five stages of the gonadal

development scale (GDS) in tubules: growing, premature, mature, spawning and post spawning. Maturation and

spawning events were frequent in all months, mainly February, March, and May 2020. Seven colors of the tubules

were presented (transparent, transparent-pink, white, transparent-white, creamy-white, cream, and pink) with a

large overlap between sex and GDS.

Conclusions: This study is the first to describe the reproductive behavior of H. (H.) grisea on the coast of Santa

Catarina and this population exhibits a continuous reproductive pattern with reduction in winter season. Our

results can offer a baseline for future research, providing various qualitative and quantitative description tools

for greater effectiveness in understanding the maturity cycle of populations and managing the sea cucumber

fishery in Brazil.

Key words: Holothuroidea; reproduction; gametogenesis; gonad index; maturity index; oocyte size.

https://doi.org/10.15517/rev.biol.trop..v73i1.60485

INVERTEBRATE BIOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e60485, enero-diciembre 2025 (Publicado Jun. 10, 2025)

INTRODUCTION

Sea cucumbers (Holothuroidea) are echi-

noderms found in all regions of the oceans,

from intertidal regions to the deep-sea and

from polar regions to the tropics. With approxi-

mately two thousand species they represent

important components of marine ecosystems

(Belbachir & Mezali, 2018; Marquet et al., 2017;

Purcell et al., 2016; Tolon & Engin, 2019). In

addition to their ecological significance, the

body walls of sea cucumbers are considered

highly nutritional and valuable seafood (bêche-

de-mer or trepang) that is particularly demand-

ed by Asian consumers (Hamel et al., 2001;

Purcell et al., 2013; Ramofafia et al., 2003; Rob-

inson & Lovatelli, 2015), reaching high prices

and considered a commodity in the interna-

tional seafood market (Benítez-Villalobos et

al., 2013; Purcell & Eriksson, 2015). Moreover,

holothurians possess a wide range of bioactive

compounds that can be used in the production

of pharmaceutical, nutraceutical and cosmetics

products (Egloso & Delantar, 2018; Marrugo-

Negrete et al., 2021; Mezali et al., 2014; Purcell,

2014). The strong demand for these organisms

has depleted stocks worldwide, and has led to

the collapse of natural populations, classifying

some species as threatened (Carleton et al.,

2013; Diupotex-Chong et al., 2022; Laguerre et

al., 2020; Lewerissa et al., 2021; Purcell et al.,

2012; Santos et al., 2015; Shedrawi et al., 2019;

Tolon & Engin, 2019).

Holothuria (Halodeima) grisea Selenka,

1867 is one of approximately 120 large and

conspicuous sea cucumbers species of the gen-

era that occur in shallow tropical or subtropical

waters (Hamel et al., 2001). It has been rec-

ognized as amphi-Atlantic species, from the

Gulf of Mexico to Brazil and found in the

Eastern Atlantic off West Africa (Pawson et al.,

2010). Along the Brazilian coast H. (H.) grisea

RESUMEN

Reproducción del pepino de mar Holothuria (Halodeima) grisea (Holothuriida: Holothuriidae)

de la costa de Santa Catarina, sur de Brasil

Introducción: El pepino de mar, Holothuria (Halodeima) grisea, es una especie común en las costas rocosas

intermareales de la costa brasileña. Debido a su abundancia, se considera un recurso potencial para el desarrollo

de la acuicultura.

Objetivo: Caracterizar algunos rasgos reproductivos en el sur de Brasil, incluyendo proporción de sexos, índices

gonadosomáticos y de madurez y observaciones macro y microscópicas de los túbulos gonadales.

Métodos: Los individuos adultos fueron recolectados al azar en agosto y octubre de 2019; febrero, marzo, mayo y

octubre de 2020 en la región intermareal durante las mareas bajas de la costa de Santa Catarina, Brazil. Los indi-

viduos se transfirieron al laboratorio, donde fueron objeto de biometría, disección y preservación para posteriores

exámenes macro y microscópicos.

Resultados: La proporción de sexos fue de 1 : 1 y el índice gonadal resultó similar en cada sexo y entre los meses.

Se establecieron principalmente dos cohortes (25.3 y 85.3 μm) y tres intervalos citométricos (IC). Basándose en la

histología de los machos, y principalmente en las cohortes y los IC en hembras, se categorizaron cinco estadios de

desarrollo gonadal (GDS) de los túbulos: en crecimiento, prematuro, maduro, desove y postdesove. Los eventos de

maduración y desove fueron frecuentes en todos los meses, principalmente en febrero, marzo y mayo de 2020. Se

presentaron siete colores de los túbulos (transparente, transparente-rosado, blanco, transparente-blanco, blanco-

crema, crema y rosa) con un gran solapamiento entre sexo y GDS.

Conclusiones: Este estudio es el primero en describir el comportamiento reproductivo de H. (H.) grisea en la

costa de Santa Catarina, cuya población exhibe un patrón reproductivo continuo con reducción en la estación

invernal. Nuestros resultados pueden ofrecer una línea base para futuras investigaciones, proporcionando diver-

sas herramientas de descripción cualitativa y cuantitativa para una mayor eficacia en la comprensión del ciclo de

madurez de las poblaciones y el manejo de la pesquería del pepino de mar en Brasil.

Palabras clave: Holothuroidea; reproducción; gametogénesis; índice gonadosomático; índice de madurez; tamaño

del ovocito.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e60485, enero-diciembre 2025 (Publicado Jun. 10, 2025)

represents a very common sea cucumber usu-

ally, living under rocks or burrowing into sandy

substrata, inhabiting the intertidal and subtidal

habitats protected from wave action (de Lima-

Bueno et al., 2018; Sabino-Rupp et al., 2024).

A fundamental barrier to improvements

in the management of sea cucumber fisheries

is a lack of data regarding the basic biologi-

cal parameters of the most exploited species

(Laguerre et al., 2020; Ramos-Miranda et al.,

2017; Tolon & Engin, 2019; Venâncio et al.,

2022). Studies on the reproduction of marine

invertebrates are an important source of infor-

mation for the potential sustainable exploita-

tion of these resources for those species already

exploited, not only for proposing manage-

ment plans and closed seasons for commercial

catches but also to analyze the potential use of

this species in an aquaculture production (de

Lima-Bueno et al., 2015; Muthiga et al., 2009;

Sabino-Rupp et al., 2021). The knowledge of

the reproductive characteristics of H. (H.) gri-

sea is essential because this species is already

subject of indiscriminate catch and commer-

cialization in Brazil (Zacagnini-Amaral et al.,

2008; Rodrigues-Ponte & Vieira-Feitosa, 2019;

Sabino-Rupp & Cacciatori-Marenzi, 2021).

Southern Brazil is the high latitude limit

of geographic distribution of H. (H.) grisea

along with two other holothuroids: Isosticho-

pus badionotus and Parathyone braziliensis (de

Lima-Bueno et al., 2018; Sabino-Rupp et al.,

2023; Slivak et al., 2022; Ramos-Xavier, 2010).

Due to its great abundance (Mendes et al.,

2006; Sabino-Rupp et al., 2023; Sabino-Rupp et

al., 2024) and potential for aquaculture devel-

opment (Sabino-Rupp et al., 2021) this work

aimed to study for the first time the reproduc-

tive biology H. (H.) grisea in Santa Catarina

coast. Aiming to generate information about its

reproductive characteristics and to contribute

to aquaculture development, this study focuses

on important aspects such as sex ratio, gonad

(tubule development) and maturity indices,

macro and microscopical analysis of the tubules

in different periods of the year.

MATERIALS AND METHODS

Study site: South Brazil is in a subtropi-

cal region, which is considered a transition

area between the tropical marine biogeographic

province and the warm-temperate province

(Briggs & Bowen, 2013). Santa Catarina coast-

line comprises around 550 km, which includes

hundreds of beaches, several coastal islands,

mangroves, coastal lagoons, bays and estuaries

(Sabino-Rupp et al., 2023). Large cities are also

located in the coastal zone holding a population

of more than 1 million people, which is increas-

ing pressure on marine ecosystems (Ventura-de

Souza et al., 2022).

The regional climate is dominated by the

Atlantic Tropical air mass, with average month-

ly temperatures between 15 °C and 18 °C in

winter and between 24 °C and 26 °C in sum-

mer. Average monthly rainfall varies between

100 and 350 mm, with a tendency for rain to

be concentrated in the spring and summer

months, and relative humidity can reach up to

85 % (Rodrigues-Filho et al., 2016). The pre-

vailing winds are Northeasterly throughout the

year and Southwesterly in winter (de Araújo

et al., 2006). The water masses that occur near

the continental shelf are the Tropical Water

(summer and fall) influenced by the Brazil

Current and, eventually, the Central Water of

the South Atlantic (ACAS), in summer, in the

lower layers of the water column in coastal

areas (Resgalla-Jr & França-Schettini, 2006;

Sabino-Rupp et al., 2005)

Armação do Itapocoroy Bay is placed

on the North-central coast of Santa Catarina

State (Penha municipality) in Southern Brazil

(26º47’S & 48º 36’W). It is a sheltered bay with

low-hydrodynamic wave action, gently slop-

ing bedrock, and a sandy substrate consisting

of coarse grain-sized sediment (Mendes et al.,

2006). Along the coast H. (H.) grisea was com-

mon in lower intertidal rocky shores in the

Northern and central portions of the littoral

(Sabino-Rupp et al., 2023).

Sampling: Twenty adult individuals per

month (the largest sizes observed in the field)

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e60485, enero-diciembre 2025 (Publicado Jun. 10, 2025)

were randomly collected by hand from a rocky-

sandy shore where the intertidal region was

enlarged and had low slope. Specimens were

sampled for over one year (August and Octo-

ber 2019; February, March, May and Octo-

ber 2020). This study was carried out under

a license (number 68215-1) to collect native

fauna from the Brazilian Biodiversity Authori-

zation (Instituto Chico Mendes de Conserva-

ção da Biodiversidade, 2007) and Information

System and registration (number A61927E) of

the National System for the Management of

Genetic Heritage and Associated Traditional

Knowledge (Alves et al., 2018).

All individuals were transported to the

Centro de Maricultura of CTTMar/UNIVALI

laboratory where they were placed in buckets

with 7 % magnesium chloride in seawater

to relax until processing. Gonads were sexed

through visual observation of the color and

subsequent confirmation by histological exami-

nation. Gonads that could not be sexed macro-

scopically were recorded as indeterminate.

Measurements of total fresh weight (g)

and total length (cm) were recorded with Mark

M223 digital analytical balance (0.01 g) and a

manual vernier caliper (0.02 mm) After that

an incision was made along the ventral surface

to remove the organs, including gonads; the

gonad wet weight (g ± 0.01) was also recorded.

Gonad indexes (GI) were calculated using the

ratio between the gonad weight (GW), total

wet weight (TWW) and gutted body wet weight

(GBW) for comparisons of their effectiveness as

proposed by the literature (Arsad et al., 2017;

Gaudron et al., 2008; Tahri et al., 2019): GI1

(GW × 100) / TW) and GI2 (GW × 100) / GBW.

Histological analysis: The gonads were

immediately fixed in a buffered 4 % formalin

solution for 48 h and subsequently transferred

to Davidson solution or formalin solution until

the histological procedures. The samples were

then processed with sequential submersions

in graded ethanol for dehydration followed by

xylene for clarification and impregnation with

paraffin wax at 60 °C. After the gonad samples

were embedded in 100 % (v / v) paraffin,

they were cut with a thickness of 7 µm using

a manual rotary microtome (Minot type) and

stained with Harris’ Haematoxylin solution and

alcoholic Eosin Y (yellowish) solution.

Only a section of a gonad tubule was

used to obtain definitive preparations. Digi-

tal images of the histological sections were

captured for sex diagnosis and the gonadal

development scale (GDS) under a light optical

microscope (Olympus CX43) coupled with a

camera (Olympus EP50). GDS was classified

for each sex were based on the most outstand-

ing histological characteristics specified for the

stages used in other following previous works

for the genera (Benítez-Villalobos et al., 2013;

de Lima-Bueno et al., 2015; Leite-Castro et

al., 2016; Ramofafia et al., 2003; Navarro et al.,

2012; Venâncio et al., 2022).

To quantify reproductive status the maturi-

ty index (MI) is also calculated by the formula:

MI = Σ (ni × si) / N

Where ni is the number of sea cucumbers

at each tubule development stage (i.e. GDS), si

is the numerical score attributed to that stage,

and N is the total number of sea cucumbers

collected monthly (Arsad et al., 2017; Benítez-

Villalobos et al., 2013; Venâncio et al., 2022).

Each MI value indicates its respective GDS

throughout the collecting period.

Oocyte size: Measurements of female gam-

etes (i.e. non-vitellogenic ‘primary/secondary’

and vitellogenic oocytes) were performed along

the major axis and only those whose nucleus

was visible. The diameter of all oocytes (per

female) was recorded at least five areas in each

histological preparation using Feret’s diameter

measurement (the longest distance between

any 2 points along the selection perimeter)

(Benítez-Villalobos et al., 2013) with the image

analysis package ImageJ 2 (Rueden et al., 2017).

The absolute frequencies of all oocyte diam-

eters were grouped into size ranges each month.

The intervals of the size classes of the

diameter average (Da) of the oocyte were deter-

mined by the Sturges’ rule (Sturges, 1926), the

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e60485, enero-diciembre 2025 (Publicado Jun. 10, 2025)

main cohorts of the oocytes were determined

using the Bhattacharya method (Bhattacharya,

1967). The development of oogenesis was ana-

lyzed through the evolution of oocyte size in

relation to time. Bhattacharya’s (1967) method

was utilized to separate oocyte cohorts (Benítez-

Villalobos et al., 2013; Stakowian et al., 2020).

Data analysis: A theoretical 1 : 1 sex ratio

(female : male) was tested using Chi-square

test (χ2) considering the total sample over the

study period. Differences in Gonad Index (GI)

and mature oocyte diameter by months were

analyzed using Kruskal–Wallis non-parametric

test. All differences were considered statisti-

cally significant at the significance level of 5 %

(p-value < 0.05). All statistical analyses were

performed in R (R Core Team, 2021). Addi-

tional packages used included MASS (Venables

& Ripley, 2002), vegan (Oksanen et al., 2020)

and tidyverse (Wickham et al., 2019).

RESULTS

Sex ratio: A total of 83 H. (H.) grisea were

sampled for histological analyses, with an aver-

age length (± standard deviation) of 13.7 ± 2.5

cm (from 8.8 to 20.2 cm) and an average weight

of 52.2 ± 22.1 g (from 28.9 to 200.9 g). In total,

45 males (54.2 %), 36 females (43.4 %) and 2

undetermined (2.4 %) were sampled. The sex

ratio of H. (H.) grisea not differs significantly

from the 1:1 ratio. Macroscopic sexing is based

on visual analysis of the gonads diagnosed

correctly 85.5 % of the total organisms sexed

by histological analysis and most of the misdi-

agnosed individuals were those visually clas-

sified as indeterminate, due to the absence of

gonad coloration.

Gonad indexes: GI1 values ranged from

2.76 ± 1.81 to 5.83 ± 5.12 and GI2 from 4.12 ±

2.46 to 10.77 ± 15.46 with no significant differ-

ences detected between sexes or months (GI1:

χ² = 0.09, d.f. = 5, p = 0.999; GI2: χ² = 0.23, d.f.

= 5, p = 0.998) (SMF1A, SMF1B). The maxi-

mum values were recorded in October 2019

and 2020 (spring) and the lowest GI mean were

pointed in February and May 2020 (summer

and autumn months).

Gametogenic events: Histological diag-

nosis of gametogenic events were described

and were made by some features as tubules

and lumen aspects, degree of development

of gametes and presence of nutritive phago-

cytes (SMT1, SMF2). Qualitative and/or quan-

titative analysis allowed us to characterize five

different stages of gametogenic development:

growing, premature, mature, spawning and

post-spawning).

Biometric oocyte analysis: A total of 5

860 oocytes with diameter average (Da) rang-

ing from 5.3 to 135.3 μm were measured. The

Sturges’ method distributed the population into

14 size classes of 10 μm mm o μm / class. Bhat-

tacharya’s method identified 2 main cohorts

and then established three cytometric intervals

for oocyte population: (1) growing: Da < 2 5.3

μm, (2) premature: 25.3 < Da < 85.3 μm and

(3), mature: Da > 85.3 μm. Vitellogenic oocytes

(completely mature), were observed with aver-

age diameters from 65.0 to 85.3 µm.

Throughout the study period, most oocyte

population consisted of premature interval

(65 %) followed completely mature (19 %)

and growing (16 %) (SMF3). The latter were

observed in August 2019 (35 %), an important

gonial trigger, and in October 2019 (16 %) and

October 2020 (23 %). Premature gametes were

observed in all months with most cells mea-

sured in February/March and all in May 2020.

Complete cell maturation made up 25 to 30 %

of the cells in March and October 2020.

The females were categorized in four stag-

es based on size ranges: growing, when an

remarkable amount of primary oocytes was

observed (1 / 3 of cells were above to 25.3 μm)

and 2 / 3 are premature (in the process of vitel-

logenesis); mature, when 1 / 3 of oocytes were

up to 85.3 μm and 2 / 3 are premature; spawn-

ing with proliferation when primary, premature

and mature cells are in 1 : 2 : 1 ratio; and deple-

tion or post spawning when all cells (the lowest

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e60485, enero-diciembre 2025 (Publicado Jun. 10, 2025)

quantities observed over the period) were only

in intermediated diameter (SMF4).

Kruskal-Walli’s test comparisons of mature

(vitelogenic) oocytes (Da > 65 mm) showed sig-

nificant differences (H = 266.73, d.f.= 5 / 1677,

p = 0.0001) between October 2019 (Da = 85.3

μm; n = 757) and February, March and May

2020 that were grouped with a mean oocyte

diameter ranging from 71.0 to 75.8 μm (17 >

n > 447) (SMF5). Averages values close to 80

μm were recorded in August 2019 (n = 67) and

October 2020 (n = 310).

Tubule development stages (GDS) fre-

quency and Maturity Index (MI): Growing

and premature stages were usually observed

in August 2019 and October 2019 (winter

and spring months) (SMF6). Maturation and

spawning occurred in February, May and Octo-

ber 2020 (summer, fall and spring months).

Post spawning stage was observed mainly in

February 2020. The maximum MI values were

associated with maturation and spawning stag-

es from March to May 2020.

In males, the growing stage was not

observed. Premature stage was observed in

August 2019, October 2019 and 2020 (winter

and spring) but growing cells could be seen in

the same individual. Maturation and spawning

events were frequent in all months mainly Feb-

ruary, March and May 2020 (summer, autumn

and spring). The maximum MI values were

associated with spawning stage in March and

May 2020.

A summary of the macro-microscop-

ic (sex, GDS and tubules colors) diagnosis

monthly records was presented with seven col-

ors categories: transparent, transparent-pink,

white, transparent-white, creamy-white, cream

and pink with a large overlap between sex and

GDS (SMT2).

DISCUSSION

The reproductive pattern of H. (H.) grisea

from Santa Catarina population was character-

ized by a constancy of mature and releasing

gametes for most part of the year. The females

of this population showed a period of great

investment in cell growth (winter and spring

months) which allowed the development of

multiple cohorts of oocytes observed in sub-

sequent seasons and thus supported the great

reproductive period in practically three seasons

of the year (spring, summer and autumn). Post

spawning stage observed mainly in summer

end probably reflects a greater effort to release

gametes. It is speculated that favorable condi-

tions for the supply of energy resources from

feeding during the winter months were respon-

sible for this scenario.

The highest intensity of the reproductive

events occurring between the spring and sum-

mer months was also observed by de Lima-Bue-

no et al. (2015) in a population from the coast

of Paraná located around 100 km North from

Armação do Itapocoroy, in both locations the

months with lowest temperatures were charac-

terized by a reduction in reproductive activity:

growing (autumn) and post-spawning (winter).

The presence of underdeveloped gonads or

almost non-existent gonads described by de

Lima-Bueno et al. (2015) probably refers to a

large reduction in the size of the organ. This

process of resorption and disappearance of

gonads is considered a common phenomenon

in species of the order Aspidochirotida dur-

ing post-spawning stage (Hamel et al., 1993;

Hoareau & Conand, 2001; Ramofafia et al.,

2000; Rasolofonirina et al., 2005).

In comparison to Paraná population, it is

agreed that winter may be a recovering period

after the last reproductive effort during autumn.

Although both locations are geographically

close and within the same Spalding´s ecoregion

(Spalding et al., 2007). The identification of a

longer period of reproductive activity in the

present study may be due to interpopulation

variations (Arsad et al., 2017; Marquet et al.,

2017; Pasquini et al., 2022; Tahri et al., 2019)

where a series of environmental parameters

that are acting successively or in combination

to orchestrate spawning in the sea cucumbers

(Marquet et al., 2017; Tolon & Engin, 2019)

or even inter-annual variation (Marquet et al.,

2017; Venâncio et al., 2022) as observed in this

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e60485, enero-diciembre 2025 (Publicado Jun. 10, 2025)

study with the difference frequency of stages

between October 2019 and 2020.

Another factor to be considered is the low

sampling frequency (de Lima-Bueno et al.,

2015), as pointed by Leite-Castro et al. (2016)

which can compromise the diagnosis of game-

togenic stages and possibly explain why only

three stages of tubules development were found

in Paraná. We also believe that a major point to

be discussed is how to quantify gonad maturity,

since tubule recruitment growth model can

occur (see Smiley, 1988). If no synchroniza-

tion was observed in tubules development in

each gonad, we could expect a high variability

among the same individual (Hamel et al., 1993;

Marquet et al., 2017; Mercier & Hamel, 2009;

Rasolofonirina et al., 2005).

The present study reveals that the spring

and summer seasons are the mainly reproduc-

tive period over Brazilian coast. Besides small

differences between Southern (present study)

and Northeast populations (Leite-Castro et al.,

2016) of H. (H.) grisea along to Brazilian coast,

individuals have a long reproductive period

(mainly spring-summer) which suggests the

existence of a similar pattern between these

tropical and subtropical environments. Holo-

turia species have a wide geographical range

and when exposed to different environmental

factors follow a biannual or continuous repro-

ductive pattern with variations in the onset

of gametogenesis and spawning (Abdel-Razek

et al., 2005; Dissanayake & Stefansson, 2010;

Mercier et al., 2007; Muthiga & Kawaka, 2008;

Muthiga et al., 2009; Rogers et al., 2018).

A review of publications on reproduction

in sea cucumbers indicates that several spe-

cies of Holothuria both near the equator and

at higher latitudes showed reproductive peri-

odicity at least a half of the year with patterns

which reflect the seasonal differences in the

environment they inhabit (Benítez-Villalobos

et al., 2013) (SMT3). Generally, the size of

the organism, in particular body length and

weight, can be associated with gonad growth

and in Holothuria, the body size is highly vari-

able (SMT3). In the present study the sizes of

H. (H.) grisea can be considered intermediate

to small, compared to the other species of the

same genus.

In the present study fluctuation of both

gonadal indices (GI1 and GI2) could be asso-

ciated with the longer period of highest vari-

ability of cells ratios in the females (immature

and mature in oocytes) and the continuous

reproductive activity of males during the year.

Although GI1 has been widely used as a good

indicator of gonad maturity of sea cucumber

(Arsad et al., 2017; Bahida et al., 2022; Gaud-

ron et al., 2008; Rogers et al., 2018; Tolon &

Engin, 2019), many times this index could be

influenced by gonad recovery period (Benítez-

Villalobos et al., 2013) or by resorption

process (Drumm & Loneragan, 2005; Rasolo-

fonirina et al., 2005).

We also agree with the use of GI rela-

tionship with the wet weight of the body wall

consider a more reliable parameter for sea

cucumber size for some authors (Muthiga et al.,

2009; Ramofafia et al., 2000; Rasolofonirina et

al., 2005). Furthermore, in reproductive season

large-sized individuals could have less devel-

oped gonads or, inversely, or even gonads not

developed at all or missing. This phenomenon

in holothuroids could be also influenced by

evisceration and subsequent viscera regenera-

tion (Ramos-Miranda et al., 2017).

For most benthic marine species with com-

plex life cycles, the larval stage is the dominant

dispersal stage, this being directly correlated

to the temporal extension of the larval phase

(Rakaj et al., 2018). Moran et al. (2013), Sta-

kowian et al. (2020) and Venâncio et al. (2022)

pointed out that development time is inversely

proportional to egg size. Larval period of H.

(H.) grisea in laboratory conditions was about

20 days, but it may be influenced by temperature

and diet conditions (Sabino-Rupp et al., 2021).

This reproductive strategy matches what

we observed in Holothuria species that pres-

ents an intermediate egg size-less than 200 μm

(Benítez-Villalobos et al., 2013; Huang et al.,

2018; Navarro et al., 2012; Rasolofonirina et al.,

2005; Rogers et al., 2018) with H. (H.) grisea

turned out to be considerably smaller than that

reported for other congeneric species which

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e60485, enero-diciembre 2025 (Publicado Jun. 10, 2025)

might also reflect its rapid larval development

as a life history strategy (Rakaj et al., 2018).

In laboratory condition carried out by Sabino-

Rupp et al. (2021) at Armação do Itapocoroy

the vitellogenic oocytes had an average diam-

eter of 135.8 μm (± 6.7 μm) for this species and

it may reach 160 μm (Rupp pers. obs.). Thus,

it is important to bear in mind that in studies

where histological techniques are used, we are

referring to a gamete that has undergone retrac-

tion after the fixation techniques and is not

considered a cell under fresh conditions.

The allocation of reproductive energy into

a large versus a small number of offspring based

on egg size is an important life-history strategy

for any species. When comparing the three Bra-

zilian populations of sea cucumbers of H. (H.)

grisea we can hypothesize that the smaller size

ranges of the oocytes in southern Brazil would

be associated with differences in the metabolic

demands of each population under local envi-

ronmental conditions.

The present study integrated qualitative

(macroscopical diagnosis and microscopic

assessment) and quantitative tools (oocyte size

scale and gonad and maturity indexes) to evalu-

ate the reproductive activity and the gamete

(oocyte) investment of H. (H.) grisea from

Santa Catarina population. Integrative use of

quantitative and qualitative analyses for gonad-

al developmental stage determination proved to

be insightful, since qualitative methods alone

impair an adequate comparison due to the

lack of standardization. Quantitative tools such

as oocyte diameter distribution, tubule devel-

opment and maturity index provided strong

evidence to indicate that the species exhibits a

continuous reproductive pattern with a reduc-

tion in winter season.

Most studies on the reproduction of holo-

thuroids use the coloration of the gonads for

sexing and the degree of development of the

gonad (tubules). It is common to indicate that

creamy-colored gonads are testes and that pink-

colored gonads are ovaries (Arsad et al., 2017;

de Lima-Bueno et al., 2015; Muthiga et al.,

2009; Navarro et al., 2012; Pasquini et al., 2022;

Rogers et al., 2018; Santos et al., 2015; Venâncio

et al., 2022). In our study, the visual assessment

of sexual identification was mostly correct but

inconsistent for diagnosing the animal matu-

rity. Although no photographic records of the

colors were made in this study, recommended

that these are taken for further studies.

Aquaculture and fisheries remarks: As

sea cucumber fisheries along the Brazilian

coast is an unreported and unregulated activ-

ity (Rodrigues-Ponte & Vieira-Feitosa, 2019;

Sabino-Rupp & Cacciatori-Marenzi, 2021), it

is urgent to carry out biological and ecological

studies to develop effective management mea-

sures before the overexploitation of sea cucum-

ber populations. In Brazil, aquaculture efforts

have been attempted in the Northeast region

(Souza-Junior et al., 2017) and more recently

in the South (Santa Catarina). The availability

of H. (H.) grisea natural stocks is one of the

main reasons for the selection of this species

for aquaculture studies (Sabino-Rupp et al.,

2021; Sabino-Rupp et al., 2023; Sabino-Rupp

et al., 2024). The information from this study

may support spawning induction by knowing

when to capture broodstock but it does not

support larval culture or juvenile production.

Therefore, it is suggested that the activities to

induce spawning of H. (H.) grisea should not

be attempted during late autumn and winter

months. It is also recommended to carry out

further studies on growth, recruitment and

survivorship on longer time-scales, on determi-

nation of the first sexual maturity and on fecun-

dity per size distribution. Such features would

allow a better understanding of the population

ecology and the development of aquaculture

and stock management tools, as well as fishing

regulations based on precise information for

conservation of the Santa Catarina ecosystems.

In fact, this study is the first to describe

the reproductive behavior of H. (H.) grisea

on the coast of Santa Catarina, and specifi-

cally in Armação de Itapocoroy. This can offer

a baseline for future research, providing various

qualitative and quantitative description tools

for greater effectiveness in understanding the

maturity cycle of populations and managing

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e60485, enero-diciembre 2025 (Publicado Jun. 10, 2025)

the sea cucumber fishery in Brazil. Finally,

it is suggested that additional reproductive

studies be carried out over a longer period

of years, as inter-annual variation is likely to

occur, and ongoing climate change, along with

an above historical average increase in rainfall

and heat in Southern Brazil make this recom-

mendation imperative.

Ethical statement: The authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict 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 acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

See supplementary material

A34v73n1-suppl1

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