Scholarly article on topic 'Spawning and rearing of Holothuria tubulosa
: A new candidate for aquaculture in the Mediterranean region'

Spawning and rearing of Holothuria tubulosa : A new candidate for aquaculture in the Mediterranean region Academic research paper on "Biological sciences"

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Academic research paper on topic "Spawning and rearing of Holothuria tubulosa : A new candidate for aquaculture in the Mediterranean region"

DOI: 10.1111/are.13487

ORIGINAL ARTICLE

Aquaculture Research

Spawning and rearing of Holothuria tubulosa: A new candidate for aquaculture in the Mediterranean region

Arnold Rakaj1 | Alessandra Fianchini1 | Paola Boncagni1 | Alessandro Lovatelli2 | Michele Scardi1 | Stefano Cataudella1'3

1Experimental Ecology and Aquaculture Laboratory, Biology Department, University of Rome "Tor Vergata", Rome, Italy

2Fisheries and Aquaculture Policy and Resources Division Food and Agriculture Organization of the United Nations (FAO), Rome,Italy

3General Fisheries Commission for the Mediterranean (GFCM) Palazzo Blumenstihl, Rome, Italy

Correspondence

Arnold Rakaj, Experimental Ecology and Aquaculture Laboratory, Biology Department, University of Rome "Tor Vergata", Rome, Italy. Email: arnoldrakaj@gmail.com

Abstract

Holothuria tubulosa (Gmelin, 1788) has recently shown an increased demand in Asian markets, becoming one of the intensively exploited holothurian species in the Mediterranean Sea. A risk is that over-harvesting is likely affecting both the species' natural stocks and the benthic communities. In this scenario, sea ranching and restocking through aquaculture could assist in mitigating its overexploitation. This study is the first to demonstrate the successful artificial breeding and rearing of H. tubulosa, and its consequent potential as a new species for the Mediterranean aquaculture industry. Here we describe the spawning induction, larval development and early juvenile growth in hatchery cultures, aimed at developing a spawning and rearing protocol for this species. The trials were conducted from July to October in both 2014 and 2015. Holothuria tubulosa was induced to spawn by testing four different methods. Thermal stimulation plus thermal shock emerged as the most efficient method to obtain active and healthy gametes. Larval development in H. tubulosa progressed through five stages, reaching the juvenile stage in 27 days. Two different microalgal feeding regimens were tested for larval breeding. Under the best feeding conditions, 7% of the larvae metamorphosed into settled juveniles, adhering to artificial substrates previously conditioned with benthic biofilm. Our results indicate that H. tubulosa shows good performance in hatchery rearing during the larval phases, indicating that this species could be a new candidate for aquaculture in the Mediterranean region, both for production and restocking proposes.

KEYWORDS

Holothuria tubulosa, larval feeding, larval rearing, mariculture, sea cucumber, spawning

1 | INTRODUCTION

Deposit-feeding sea cucumbers are common members of marine benthic communities and important ecosystem engineers that play a key role in sea floor dynamics by processing and bioturbating the sediment (MacTavish, Stenton-Dozey, Vopel & Savage, 2012; Purcell, Conand, Uthicke & Byrne, 2016).

Some species of sea cucumber are also edible and considered a delicacy in many Asian markets, where, in their dried form, they are traded as a luxury seafood called trepang or bêche-de-mer (Purcell, Hair & Mills, 2012; Toral-Granda, Lovatelli & Vasconcellos, 2008). Of the approximately 1,400 species of holothuroids worldwide (Han, Keesing & Liu, 2016; James, 2001; Pawson, 2007), nearly 60 species are commercially exploited (Kinch, Purcell, Uthicke & Friedman,

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2017 The Authors. Aquaculture Research Published by John Wiley & Sons Ltd

Aquaculture Research. 2017;1-12. wileyonlinelibrary.com/journal/are I 1

2008; Purcell, Samyn & Conand, 2012; Purcell, Hair et al., 2012). The high commercial value and the increasing demand has intensified exploitation in the wild, this remaining the only source available for many sea cucumber species (Conand, 2004; Purcell, Lovatelli, Vasconcellos & Ye, 2010; Purcell, Hair et al., 2012; Sicuro & Levine, 2011; Toral-Granda et al., 2008). The resulting massive and unregulated exploitation has led to the collapse of several natural stocks and is likely impacting significantly on vulnerable benthic communities (Purcell et al., 2013).

In this context, aquaculture could be a sustainable alternative to meet the current market demand (Anderson, Flemming, Watson & Lotze, 2011; Bartley & Bell, 2008) by reducing pressure on wild stocks and by providing restocking actions. Furthermore, several sea cucumber species have been shown to have a valuable application in Integrated Multi-Trophic Aquaculture (IMTA) (Paltzat, Pearce, Barnes & McKinley, 2008; Slater & Carton, 2007; Tolon, Emiroglu, Gunay & Ozgul, 2017; Yokoyama, 2013; Yu, Zhou, Yang, Ma & Hu, 2014). Their use in IMTA systems has been found to mitigate the negative effects of intensive aquaculture through waste particulate subtraction and processing (Ahlgren, 1998; Barrington, Chopin & Robinson, 2009; Slater & Carton, 2009; Tolon et al., 2017).

Recent years have witnessed a growing interest in the sea cucumber aquaculture industry on a global scale, as extensively noted and reported by FAO reports and numerous studies (Han et al., 2016; Lovatelli et al., 2004; Purcell, Hair et al., 2012; Toral-Granda et al., 2008). Aquaculture production has increased more than fourfold, rising from 46,000 tonnes in 2004 to 200,000 tonnes in 2014 (FAO, Global Production Statistics 1950-2014). To date, production of holothuroids by aquaculture has been developed mainly for six Indo-Pacific species (Dominguez-Godino, Slater, Han-non & Gonzalez-Wanguermert, 2015; Purcell, Hair et al., 2012), supplying a portion of the market demand, especially for the Japanese sea cucumber, Apostichopus japonicus (Chen, 2004; Han et al., 2016; Lovatelli et al., 2004; Purcell, Hair et al., 2012).

The increasing demand from Asian markets has led to the rapid exploitation of new fisheries elsewhere, such as the Mediterranean area (Antoniadou & Vafidis, 2011; Gonzalez-Wanguemert, Valente & Aydin, 2015) where the fishing effort for holothuroids is on the increase. In Turkish waters alone, there are approximately 120 active fishing vessels with a harvest that ranges from 720,000 to 1,080,000 sea cucumbers per day (Gonzalez-Wanguemert, Aydin & Conand, 2014; Gonzalez-Wanguemert, Valente, Henriques, Domi'n-guez-Godino & Serrâo, 2016).

In the Mediterranean Sea, the main target species displaying commercial interest are Holothuria polii, Holothuria tubulosa, Holothuria mammata and Parastichopus regalis (Gonzalez-Wanguemert et al., 2014, 2015, 2016; Purcell, Samyn et al., 2012). These sea cucumbers are considered a good source of nutrients and a seafood suitable for human consumption (Aydin, Huseyin, Bekir, Yilmaz & Sevim, 2011; Cakly, Cadun, Kisla & Dicer, 2004; Pereira, Valentao, Teixeira & Andrade, 2013; Roggatz et al., 2016; Sicuro et al., 2012).

Holothuria tubulosa, the object of this study, is a Mediterranean species (Gonzalez-Wanguemert et al., 2016; Valente, Serrao & Gonzalez-Wanguemert, 2015) generally associated with rich organic bottoms and seagrass beds where it thrives in a bathymetric range from 1 to 100 m (Bulteel, Jangoux & Coulon, 1992; Gustato & Vil-lari, 1979; Massin & Jangoux, 1976; Tortonese, 1965). This sea cucumber displays separate sexes without sexual dimorphism; it presents an external fertilization and has an annual reproductive pattern, spawning mainly during the summer (Despalatovic, Grubelic, Simunovic, Antolic & Zuljevic, 2004; Kazanidis, Lolas & Vafidis, 2014; Ocana & Tocino, 2005). Holothuria tubulosa, given its trophic behaviour, has been recently proposed as a candidate in the IMTA system by Tolon et al. (2017). The same authors have successfully reared wild specimens under finfish net cages, without supplementary food by exploiting aquaculture waste.

Holothuria tubulosa has become one of the target species actively harvested in Turkey, Greece, Italy, Spain and many other countries, prior to exportation to Eastern Asian markets (Aydin, 2008; Cakly et al., 2004; Gonzalez-Wanguemert et al., 2014, 2015). Unfortunately, to date, the management measures for a sustainable fishery of this target resource are still scarce or even completely absent along the Mediterranean coasts.

As yet, no artificial reproduction protocols have been developed for Mediterranean species, in Europe the only sea cucumber successfully reproduced in captivity is the Atlantic species Holothuria arguinensis (Dominguez-Godino et al., 2015).

In this study, we present details of the different methods for spawning induction, the development of embryos, larvae and juveniles of H. tubulosa, aiming at developing a hatchery-based larval-rearing protocol for this Mediterranean species.

2 | MATERIALS AND METHODS 2.1 | Broodstock collection and maintenance

Adult specimens of H. tubulosa (880 individuals; mean weight: 247.3 ± 15.7 g; mean ± SE) were collected by snorkelling (1-6 m depth) at Torre Astura (central Tyrrhenian Sea, Italy 41°24'29"N, 12°45'51"E) between July and October in 2014 and 2015. After collection, specimens were maintained in 30 L tanks equipped with aerators; a dry ice pack was placed inside the tanks to maintain the temperature below 28°C. No loss of broodstock occurred during transportation. The broodstock were transferred to the Laboratory of Experimental Ecology and Aquaculture (Tor Vergata University, Rome), and were acclimated and maintained indoors in 600 L tanks with filtered seawater at 23-24°C (in closed circulation systems with biological and mechanical depuration). The holding tanks had been previously filled with sediment collected from the sampling site. After 7 days, specimens were fed with additional dry extruded pelleted fish feed (EFICO YM 854 for Gilthead Seabream; Biomar SAS, Nersac, France) at a rate of 5 g per kg of weight broodstock per day. Broodstock were maintained in the holding tanks for 2 weeks prior to spawning induction at a density of approx. 40 specimens per tank.

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2.2 | Spawning induction and fertilization

Before stimulation, breeders were transferred and maintained for 48 hr in substrate-free aquaria to void their gut contents. The sea cucumbers were then cleaned and washed with filtered seawater to remove sediment and other organisms, and finally transferred to 600 L spawning tanks containing 5 im filtered and UV-sterilized seawater.

The following methods were performed in parallel on broodstock groups of 18-22 individuals to induce spawning. Each approach described below was replicated in total for 16 trials from July to October with six trials in 2014 and ten in 2015.

• Mechanical shock (dry shock): Broodstock were kept dry for approximately 30 min, subjected to a strong water jet, and were transferred back into the spawning tanks (Renbo & Yuan, 2004; Yanagisawa, 1998).

• Thermal shock: Water temperature was quickly raised by 3-5°C (from 23°C to 26-28°C), was maintained over 1.5 hr, and returned to the starting temperature (Battaglene, Seymour, Ramo-fafia & Lane, 2002; Dabbagh, Sedaghat, Rameshi & Kamrani, 2011; Dominguez-Godino et al., 2015).

• Thermal stimulation: Water temperature in spawning tanks was raised gradually by 2-3°C using aquarium heaters (from 23°C to 25-26°C) and maintained at the elevated temperature for at least 2 days (this method was drawn up on the basis of hatchery observations).

• Combined thermal stimulation and thermal shock: Water temperature in spawning tanks was gradually raised by 2-3°C (from 23°C to 25-26°C) and maintained. On day 2, a thermal shock was performed by quickly raising the water temperature by 3°C and returning it back to 25-26°C. The combination of these two methods was designed to ensure a better control of the spawning time.

When none of the methods induced spawning, the broodstock were maintained in the holding tanks and spawning induction was performed again 2 weeks later; instead, when spawning induction was successful, the broodstock showed signs of pre-spawning movements. Males were immediately identified, as they started spawning first, and were transferred into separate tanks. The remaining specimens manifesting pre-spawning behaviour were assumed to be female and isolated in 20 L spawning buckets until the release of eggs. Water in the buckets was stirred to uniformly distribute the eggs. Five samples (1 ml) were collected to estimate the number of released eggs/female; eggs were counted using a Sedgewick-Rafter cell chamber under a light stereomicroscope. At this stage, the spermatozoa solution (obtained from isolated spawning males) was added at low concentration (circa 1-5 x 104 spermatozoa/ml) in the female spawning buckets to minimize polyspermy (Hamel & Mercier, 1996). After the addition of the spermatozoa solution, eggs were continuously monitored. Thirty minutes after fertilization, eggs were removed from the tanks by using a 60 im bucket sieve. Collected

eggs were then washed with filtered and sterilized seawater to remove the residual sperm. Larval rearing was conducted from early August to September in 2014 and repeated in the same period in 2015.

2.3 | Larval and juvenile culture

The fertilized eggs were transferred and maintained in a 10 L round-bottom flask at a density of 1 egg/ml (Battaglene & Bell, 1999; Dominguez-Godino et al., 2015; Pitt, 2001). An air driven pipette was placed into each flask to maintain sufficient aeration and ensure gentle water circulation. During the first day, embryonic development was followed continuously by taking 10 ml water samples (five replicates) every hour, after which samples were taken every 6 hr until the early auricularia stage was reached. From the first occurrence of the early auricularia stage, the larvae were monitored daily to observe development. Larvae were reared indoors using 5 im filtered and UV-sterilized seawater. Water temperature was maintained at 24°C by conditioning the hatchery room environment. Salinity was monitored daily using a Sucrose Brix Refractometer and was maintained between 36& and 37&. The hatchery photoperiod was designed according to the natural circadian cycle using low brightness, and avoiding direct lighting on cultures. EDTA was dissolved in culture water at low concentrations (2 mg/L) to improve water quality by reducing heavy metal concentrations (Agudo, 2006). Water was partially exchanged (50%) on a daily basis using a 60 im mesh screened siphon. Dead larval deposits, when they were observed, were removed by gently siphoning the bottom of the flasks with a pipette.

When a fully developed gut was observed (third day following fertilization), larvae were fed daily using a mixture of microalgae Isochrysis galbana, Chaetoceros calcitrans and Tetraselmis suecica in equal proportions (cells/ml). The microalgae were cultured in 2 L plastic bag photo-bioreactors using Guillard's f/2 growth medium (Guillard & Ryther, 1962) at 24°C using a 12:12 hr light-dark pho-toperiod. Only algae under exponential growth were administered. Two different feeding regimes were tested for larval rearing:

• High microalgae regime of 20,000-40,000 cells/ml from early auricularia to late auricularia, as recommended in the literature for several sea cucumber species (Agudo, 2006).

• Low microalgae regime of 5,000-10,000 cells/ml from early auric-ularia to late auricularia.

The experiment was repeated twice with three replicates for each condition. When the larvae reached the doliolaria stage, they were transferred from the 10 L round-bottom flask to 30 L aquaria, each containing 6 PMMA (Plexiglas®) settlement panels (0.20 m x 0.40 m stacked with a 30 mm gap), covered with a well-developed diatom-biofilm. The settlement panels were prepared in advance by immersing them for 2 weeks in naturally illuminated tanks inoculated with benthic diatoms (Navicula spp., Nitzschia spp. and Phaeodactylum tricornutum), and supplemented with f/2 beta

growth medium to promote biofilm development. Since not all larvae metamorphosed at the same time, microalgae mixture was administered in the 30 L aquaria during the first 3 days to feed the remaining auricularia larvae. To provide sufficient aeration and to ensure water circulation, two air stones were placed on the bottom of each aquarium. Approximately 50% of the water was changed daily using 60 im mesh siphons. Juveniles were fed by adding ALGAMAC 3050 as supplementary feeding to the benthic diatoms. From the day of their appearance, 0.01 g of ALGAMAC was dissolved in each aquarium daily.

2.4 | Sampling and specimen preparation

Water samples (10 ml, five replicates) were taken daily after the first day to follow larval development, record developmental stages, and register the mean length and width for a minimum of 30 larvae during the planktonic phase. After settlement, the number of juveniles was estimated by counting the juveniles in five subareas, using prepared sampling plates. In the following results we present the mean ± SE of this sampling.

Eggs, embryos, larvae and juveniles were fixed in 2% formaldehyde in filtered seawater. The specimens were viewed using a Leica MZ12 stereomicroscope and a Leica DMLB 2000 light microscope (Leica, Wetzlar, Germany).

3 | RESULTS

3.1 | Spawning induction and fertilization

The following outcomes were observed during the trials performed to test various spawning induction methods: (i) Mechanical shock: no response was observed applying this method in all 16 trials; (ii) Thermal shock: spawning occurred sporadically (i.e. spawning successful in four trials); (iii) Thermal stimulation: spawning was successful in 8 out of 16 trials (pre-spawning behaviour was mainly observed after the first day of treatment); and (iv) Combined thermal stimulation and thermal shock: spawning was successful in 11 out of 16 trials. Thermal shock, performed on the second day of the thermal stimulation, permitted better control of the spawning event.

Among the different trials, thermal stimulation + thermal shock was selected as the best method to induce spawning in H. tubulosa. Pre-spawning behaviour was identified in broodstock. It was observed that individuals sought the standing position, many of them being deployed on tank walls close to the water surface. Breeders clung to the substrate with the posterior part of the body, and extended the anterior end of the body to facilitate the dispersal of gametes from the apical gonopore (Figure 1).

Males spawned before females by releasing a thin and steady stream of sperm from gonopore. Males spawned continuously in the erect position for about 2-2.5 hr with short interruptions. Females began spawning approximately 1-1.5 hr after males. They often raised the anterior body, moving and twisting upward until a short

(a) t. (b)

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FIGURE 1 Spawning of Holothuria tubulosa. (a) Male: gonopore (G), sperm (S). (b) Female: gonopore (G), eggs (E)

and powerful (4-8 s) squirt of eggs was released from the gonopore. Each female completed spawning with 4-6 distinct squirts/jets with a 5-10 min interval between each squirt. This powerful ejection dispersed the eggs much more widely in respect to the streams of sperm.

Mature eggs of H. tubulosa were spherical, visible to the naked eye, with a mean diameter of 151.2 ± 2.1 im (Figure 2a). The quantity of eggs varied widely among individuals, ranging from 0.4 to 12 million per spawning female (mean 3.48 ± 1.41 million; n = 12). During spawning induction, low aeration was provided using air pipettes inside the spawning tanks. Since the eggs were heavier than seawater, they quickly sank to the bottom of the bucket.

3.2 | Embryonic and larval development

The fertilization membrane of mature eggs (mean length 151.2 ± 2.1 im) was inflated within 1-5 min after the addition of sperm, increasing their dimension to a mean length of 172.1 ± 1.7 im (n = 30 larvae). At this stage, the fertilization envelope became clearly visible (Figure 2b). The first cleavage appeared 110 min after fertilization. Cell division was holoblastic, generating two blastomeres of similar size (Figure 2c). The second and third cleavages were observed at 150 and 195 min, respectively, after fertilization (Figure 2d, e). The morula stage was observed at approximately 6 hr after fertilization and the blastula stage was reached 12 hr after fertilization, exhibiting a typical central blastocoele surrounded by a small and single layer of cells (Figure 2f). At this stage, the embryo started rooting and moving (Figure 2g). At 20 hr, the invagination of the blastula wall began and the first gastrula was observed (mean length 177.1 ± 1.6 im) (Table 1). In the following

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FIGURE 2 Embryonic development of Holothuria tubulosa (a) Unfertilized egg, (b) Fertilized egg with a fertilization envelope (FE) and two polar bodies (PB), (c) First cleavage, (d) Second cleavage, (e) Third cleavage, (f) Morula, (g) Rotary blastula, (h) Early gastrula with blastopore (Bl), (i) Mid gastrula;mesenchyme cells (MC), Scale bars = 100

24-30 hr, the gastrula continued to elongate until they reached the early auricularia stage (Figure 3a).

Under the rearing conditions in this study, H. tubulosa progressed through five stages of larval development: early auricularia, mid auricularia, late auricularia, doliolaria and pentactula. Early auricularia larvae were completely developed by day 3 post fertilization. The mean length and width of early auricularia larvae were 434.2 ± 5.0 im and 260.9 ± 3.5 im, respectively, and the larvae appeared translucent with a functional digestive tract, including a

buccal cavity, oesophagus and stomach (Figure 3b). The first feeding instance was observed at this stage. Larvae increased in size, reaching the mid auricularia stage on day 7 post fertilization (Figure 3c). The mean length and width at this stage were 693.2 ± 7.2 im and 464.1 ± 4.9 im respectively. Larvae reached the late auricularia stage on day 20 post fertilization (Figure 3d), and had a mean length of 786.7 ± 12.3 im and width of 532.4 ± 7.2 im, respectively, these being the maximum dimensions registered during any of the planktonic stages. Larval intestines increased in size throughout the

TABLE 1 Embryonic development and size (mean ± SE; n = 30) of Holothuria tubulosa at 24°C

Stage Time (min, hr, d) Size (im)

Unfertilized egg - 151.2 i 2.1

Elevation of the fertilization envelope G-5 min 172.1 i 1.7

2-cell division 11G min -

4-cell division 15G min -

8-cell division 195 min -

16-cell division 255 min -

Morula 36G min -

Blastula 12 hr 173.G i 1.7

Early gastrula 2G hr 177.1 i 1.6

Late gastrula 2d 3G2.7 i 4.3

Early auricularia 3d 434.2 i 5.G

Mid auricularia 7 d 693.2 i 7.2

Late auricularia 2G d 7B6.7 i 12.3

Doliolaria 24 d 423.6 i 6.6

Pentactula 26 d 431.7 i B.G

Juvenile 27 d 512.3 i 1G.1

Juvenile 37 d 7B3.2 i 2G.7

Juvenile 45 d 1479.9 i 7B.1

Juvenile 9G d B7GG i 14GG

hr, hours; min, minutes; d, day.

development of auricularia larvae. The axohydrocoel made its first appearance during the mid-auricularia stage (Figure 3c), widely expanding during the late auricularia stage (Figure 3d). Late auricu-laria larvae became non-feeding doliolaria after 24 days post fertilization. At this stage, a rapid metamorphosis from the late auricularia to the doliolaria stage took place in 82% of the larvae. Metamorphosis did not take place in the remaining (18%) of the larvae.

Doliolaria larvae of H. tubulosa displayed the characteristic barrel shape with five ciliary bands (Figure 4a). At this stage, the larvae became smaller than in the previous stages, with a mean length and width of 423.6 ± 6.6 im and 267.3 ± 3.9 im respectively. During this stage, the digestive tract disappeared, and in the late doliolaria stage, tentacles became visible (Figure 4b) and larvae started swimming close to the substrate. Hyaline spheres appeared in late auricularia (Figure 3d) and remained visible during the doliolaria stage. At 26 days post fertilization, the first pentactula larvae were observed and became translucent with five anterior tentacles (Figure 4c) and podia. The mean length and width of pentactula larvae were 431.7 ± 8.0 im and 298.6 ± 4.9 im respectively. Juveniles appeared at 27-30 days post fertilization. At 37 days post fertilization, juveniles measured a mean size of 783.2 ± 20.7 im, at 45 days juveniles reached the size of 1479.9 ± 78.1 im, and their bodies were covered with ossicles of varying shapes (Figure 4d). At 90 days post fertilization, juveniles reached the mean size of 8.7 ± 1.4 mm (Figure 5). Juvenile growth of H. tubulosa was variable and some individuals become considerably larger than others.

3.3 | Feeding protocols

Varying the feed rate yielded differences in larval growth, development and survival. Food administered at a high concentration (20,00040,000 cells/ml) prevented the metamorphosis of larvae, which never progressed past the mid auricularia stage. At the mid-auricularia stage, larvae degenerated and displayed: (i) a spherical shape; (ii) a drastic reduction in the digestive tract; (iii) the disappearance of cilia and ecto-dermic protrusions; and (iv) larvae sunk to the bottom of the flask, exhibited benthic behaviours, and maintained this form for over 20 days (Figure 6). Food administered at a low concentration (5,00010,000 cells/ml) resulted in high numbers of metamorphosed larvae and a high number of embryos reaching the juvenile stages.

In this study, 98% of deposited eggs of H. tubulosa were mature. We observed a high proportion (96% on average) of successfully fertilized embryos that reached first cleavage, whereas 72% developed into the early auricularia stage. Under the low feeding regime, 51% of larvae reached the late auricularia stage, and 42% metamorphosed into doliolaria. We estimate that, finally, 7% of fertilized eggs metamorphosed into juveniles at 30 days post fertilization and 4% reached the 45th day becoming visible to the naked eye (Figure 7). The survival rate at 90 days post fertilization was estimated to be 1.4%, at this stage. Finally juveniles were transferred into larger aquaria. Considering that some juveniles may have escaped the count by adhering to the internal surface of plate supports instead of to the panels, the survival rate of juveniles reported here may be a slight underestimate.

4 | DISCUSSION

Research involving spawning induction is essential for controlling sea cucumber reproductive processes. Many authors have suggested that physical and mechanical treatments are effective methods for inducing spawning in both tropical and temperate sea cucumbers (Battaglene et al., 2002; Costelloe, 1985; Ramofafia, Byrne & Batta-glene, 2003). However, results vary and the responses are species-specific (Abdel Razek, Abdel Rahman, Moussa, Mena & El-Gamal, 2012; Agudo, 2006; Dominguez-Godino et al., 2015; Hu et al., 2010; Kumara, Jayanatha, Pushpakumara, Bandara & Dissanayake, 2013; Zacari'as-Soto, Olvera-Novoa, Pensamiento-Villarauz & Sánchez-Tapia, 2013). On the basis of the literature data, we tested out, on H. tubulosa, two common methods utilized to induce spawning in sea cucumbers. The mechanical shock treatment, successfully employed in species such as A. japonicus, Bohadschia marmorata, Holothuria scabra and Stichopus horrens (Al Rashdi, Eeckhaut & Claereboudt, 2012; Hu et al., 2013; Laxminarayana, 2005; Renbo & Yuan, 2004) proved unsuccessful in H. tubulosa broodstock, this remaining unresponsive during the trials. The thermal shock treatment, another effective method widely used to induce spawning in species such as Actinopyga mauritiana, Australostichopus mollis, H. ar-guinensis, Holothuria atra, Holothuria fuscogilva, S. horrens and H. scabra (Battaglene et al., 2002; Dominguez-Godino et al., 2015;

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FIGURE 3 Larval development of Holothuria tubulosa. (a) Late gastrula: enterocoeles are forming (En). (b) Early auricularia with buccal cavity (BC), oesophagus (O), stomach (S). (c) Mid-auricularia: cilia (C), axohydrocoel (A) are forming. (d) Late auricularia with an axohydrocoel (A) and hyaline spheres (HS). Scale bars = 100 im

Hu et al., 2013; Laxminarayana, 2005; Morgan, 2009; Ramofafia et al., 2003) proved to be only occasionally effective for H. tubulosa.

In wild reproduction, temperature increase is probably an indispensable condition for the induction of gonadal maturation in sea cucumbers (Guzman, Guevara & Hernandez, 2003; Muthiga, Kawaka & Ndirangu, 2009; Tehranifard & Uryan, 2006). In artificial reproduction, a slow and progressive increase in temperature is successfully applied to induce gonadal maturation in captivity maintained brood-stock of A. japonicus. This method allows Chinese farmers to obtain healthy gametes even outside the reproductive season (Xilin, 2004; Xiyin, Guanghui, Qiang, Lian & Benxue, 2004).

The two methods below both include a slow increase in temperature and were applied to induce spawning in H. tubulosa brood-stock. Thermal stimulation treatment successfully induced gamete release 1-4 days after stimulation began. Hence, we hypothesize

that this treatment may induce the gonadal maturation, which culminates in a spawning event. Lastly, the combined thermal stimulation and thermal shock method resulted in the best spawning performance for H. tubulosa, offering more control of the spawning time respect to thermal stimulation method alone.

During spawning, H. tubulosa, as well as many other holothuroid species, raise the anterior end of the body off the substrate to ensure the effective dispersion of gametes (McEuen, 1988). Males spawn first, releasing sperm that potentially stimulates and synchronizes the females to release eggs in short and powerful squirts to facilitate dispersion as observed by Battaglene et al. (2002).

The larval cycle H. tubulosa was consistent with that of holothurians reared previously; however, the duration of the larval stages differs from other species (Agudo, 2006; Asha & Muthiah, 2002; Battaglene et al., 2002; Dominguez-Godino et al., 2015). In

FIGURE 4 Metamorphosis of Holothuria tubulosa from doliolaria to juvenile. (a) Early doliolaria, cilia bends (CB) and hyaline spheres (HS). (b) Late doliolaria with tentacles (T). (c) Pentactula with tentacles (T) and podia (P). (d) 45 days juvenile: podia (P), ossicle (Os) and tentacles (T). Scale bars = 100 im

H. tubulosa, while development from the first divisions to the early auricularia stage took nearly the same length of time as observed in other holothurians, the time period for H. tubulosa to reach the doliolaria stage (24 days) was approximately 10 days longer than that observed in many other reared sea cucumbers, including S. hor-rens (Hu et al., 2013), B. marmorata and H. atra (Laxminarayana, 2005), H. scabra (Agudo, 2006), H. Arguinensis (Dominguez-Godino et al., 2015) and A. japonicus (Renbo & Yuan, 2004). In this regard Hoegh-Guldberg and Pearse (1995) stated that temperature and

other factors such as food availability might influence developmental timing in marine invertebrate larvae. Changes in temperature have been observed to result in differences in the larval timing of sea cucumber species such as Cucumaria frondosa and Holothuria spini-fera (Asha, 2004; Hamel & Mercier, 1996).

The larval-rearing protocol developed in this study was compared with those published for other species of sea cucumber. Many authors suggest that water quality management and disease control are key factors for successful breeding (Agudo, 2006; Asha &

Muthiah, 2005; Battaglene & Bell, 1999; Battaglene et al., 2002; James, Gandhi, Palaniswamy & Rodrigo, 1994; Pitt & Duy, 2005; Stenton-Dozey & Heath, 2009). According to our observations, the feeding regime is also a relevant factor supporting the success of culturing during the larval stage (Archer, 1996; Asha, 2004; Morgan,

2001). While previous reports involving other species recommend a microalgal concentration of 20,000-40,000 cells/ml (Agudo, 2006; Asha & Muthiah, 2005; Ramofafia et al., 2003), we observed optimal growth and development in H. tubulosa larvae with a lower algal concentration (5,000-10,000 cells/ml). Our results indicate that high algal concentration leads to larval degeneration and loss, highlighting overfeeding as a bottleneck for juvenile production in this species. Low rations in microalgae feeding have been reported to be optimal for another species the temperate sea cucumber A. mollis, which like H. tubulosa, show a similarly slow timing in larval development (Heath, Stenton-Dozey & Jeffs, 2015; Morgan, 2008, 2009; Stenton-Dozey & Heath, 2009). Thorson (1950) suggested that poor food conditions during the larval stages could cause slow growth and prolong larval life in marine invertebrates. However, little is known about the life history strategies and the influence of feeding behaviour on the duration of development in sea cucumbers. Larval duration in H. tubulosa could be related to its feeding behaviour, which seems to be adapted for low feeding concentrations.

This study, to the best of our knowledge, is the first to successfully induce H. tubulosa to spawn and to rear larvae until the juvenile stage using an established hatchery protocol. Considering the species' important ecological role, its high commercial value and its potential use in IMTA systems, we assume that the development of effective breeding and rearing techniques for H. tubulosa would be extremely valuable to the Mediterranean aquaculture industry.

5 | CONCLUSION

We have successfully established a method for the hatchery production of H. tubulosa juveniles. This method could constitute the basis for future research aimed at evaluating juvenile rearing for direct consumption or for use in stock enhancement programmes. Our preliminary trials regarding H. tubulosa reproduction show that this species should be considered as a potential candidate for future marine aquaculture assays. The ability to induce spawning in adults, and the successful rearing of larvae to juvenile stages under hatchery

Time (days)

FIGURE 7 Survival of larval stages and juveniles of Holothuria tubulosa during 90 days of monitoring in two feeding conditions. High concentration (triangles) 20,000-40,000 cells/ml; Low concentration (circles) 5,000-10,000 cells/ ml (mean ± SE; n = 6) and dotted line representing the degenerative larval stage

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conditions, could both support the development of a diversified aquaculture sector in the Mediterranean region.

To build upon the results of this study, further research on optimum larval feeding regimes will be crucial both for effective hatchery production and for clarifying differences in larval ecology between sea cucumber species. In addition, the rearing response in multi-trophic integrated aquaculture should be investigated to assess this species potential for large-scale production.

ACKNOWLEDGMENTS

This research was supported with the contribution of "Piscicoltura del Golfo di Gaeta (P2G)". Special thanks to "Ufficio Tecnico Territoriale Armamenti Terrestri Nettuno" for their help during broodstock collection.

Arnold Rakaj C http://orcid.org/0000-0003-2213-7131 Alessandra Fianchini © http://orcid.org/0000-0002-1229-7134 Paola Boncagni http://orcid.org/0000-0002-2222-9557 Michele Scardi http://orcid.org/0000-0003-0684-3941 Stefano Cataudella http://orcid.org/0000-0001-5196-0499

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How to cite this article: RakajA, Fianchini A, Boncagni P, Lovatelli A, Scardi M, Cataudella S. Spawning and rearing of Holothuria tubulosa: A new candidate for aquaculture in the Mediterranean region. Aquac Res. 2017;00:1-12. https://doi.org/10.1111/are.13487