by Álvarez-Blázquez1, B., Rodríguez Villanueva2, J.L., Pérez Rial1, E., Peleteiro1, J.B., Mylonas3, C.C., Papadaki3, M., Papadakis3 I., Fakriadis3, I., Robles4, R., Linares5, F.

1 Instituto Español de Oceanografía-IEO, Centro Oceanográfico de Vigo, Spain.

2 Instituto Galego de Formación en Acuicultura-IGAFA, Illa de Arousa, Spain.

3 Hellenic Center for Marine Research-HCMR, Heraklion, Crete, Greece.

4 CTAQUA, Cadiz, Spain; actual affiliation TESTING BLUE S.L., Cádiz, Spain.

5 Xunta de Galicia, Centro de Investigaciones Mariñas, Vilanova de Arousa, Spain.

 

Introduction

Following the series of articles published on the fish species included in the EU-funded DIVERSIFY project (see April, May, June, July and October issues of International Aquafeed), which ran between 2013 and 2018, we present here the wreckfish (Polyprion americanus), which was the most challenging species in the project.

The wreckfish is one of the largest Polyprionidae species, reaching a size of 100kg. It is a deep-water fish (100-to-1000m) found almost throughout the world and is characterised by an extended pelagic juvenile phase (Ball et al., 2000; Sedberry et al., 1999). It is one of the most interesting new species for aquaculture, due to its fast growth (Suquet & La Pomélie, 2002; Rodriguez-Villanueva et al., 2011), late reproductive maturation, high market price and limited fisheries landings and easy handling in captivity with no mortality recorded during the DIVERSIFY trials (Papandroulakis et al., 2008). Its large size makes it appropriate for processing and development of value-added products, which could be of interest for the EU market.

Wreckfish accepts inert food easily, being a very voracious carnivore. Wild-caught individuals kept in captivity grew from one-to-five kilogrammes in a period of 10 months (Rodriguez-Villanueva et al., 2011). The slow reproductive maturation of wreckfish, which occurs at an age of 5-10 years in captivity, may be a problem for broodstock development and management. On the other hand, its long juvenile stage is a great advantage from the aquaculture viewpoint, allowing for commercialisation before sexual maturity, and thus avoiding problems linked to maturation, such as reduction in growth. It has been demonstrated that growth is strongly influenced by sex and that female wreckfish are significantly heavier than males, as observed in many other marine fish species (Rodríguez, 2017).

The world wreckfish population is composed of three genetically distinct stocks: 1) the North Atlantic and the Mediterranean Sea, 2) Brazil and 3) the South Pacific (Ball et al., 2000). Wreckfish is a gonochoristic species with no sexual dimorphism and spawning occurs at the continental slope at depths of 300-500m, with the formation of spawning aggregations (Peres and Klippel, 2003). Demersal wreckfish individuals inhabit rocky and muddy bottoms, at depths of 40-200m; however, individuals are frequently found in waters deeper than 300m, with a maximum recorded depth of 1000m (Fischer et al., 1987). For the first part of its life (from hatching to a body length about 60cm) wreckfish is pelagic and lives in association with floating debris.

The work of DIVERSIFY for this species has focused in the areas of reproduction and genetics, larviculture and nutrition, which have been the major bottlenecks preventing wreckfish aquaculture so far.

Reproduction and genetics

Three stocks  have been maintained at the facilities of three Galician institutions (Spain): Institute of Oceanography (IEO), Ayuntamiento of A Coruña, (MC2) and Conselleria do Mar from the Xunta de Galicia (CMRM). In addition, one stock was maintained at the Hellenic Centre for Marine Research (HCMR) in Crete, Greece. The reproductive development of these stocks was followed for two years. The reproductive period (oogenesis, maturation and spawning) was rather long in wreckfish, beginning in October and lasting all the way to July, especially in the Spanish broodstocks that were exposed to lower temperatures during the year. The highest oocyte diameter of wreckfish was found between March and July, suggesting that this is the expected spawning period. The egg size of wreckfish (~2mm in diameter) is markedly larger than any other marine fish cultured in the temperate waters of the Atlantic coast of Europe and the Mediterranean Sea (Papadaki et al., 2018). A large egg size and lower fecundity is considered essential for demersal fishes, as it is related to higher individual survival in a relatively constant environment, in contrast to pelagic small eggs that have to face a changing environment, where survival is more difficult and thus high fecundity is more advantageous. Embryonic development and early life stages have been described (Papandroulakis et al., 2008; Peleteiro et al., 2011), indicating that the large egg size of this species may offer significant advantages for its larval rearing.

It is a constant temperature of 16°C throughout the year (HCMR, Greece), although it is probably more representative of the environment to which wreckfish breeders are exposed to in the deep waters that they inhabit in the wild, and they did not seem to have any beneficial effects on the reproductive development of captive female wreckfish. Fish held under naturally fluctuating temperatures in the Spain stocks exhibited the same or better reproductive performance than fish under constant temperatures in Greece, i.e. they matured and spawned spontaneously. On the other hand, males, maintained under constant temperature in the HCMR stock, remained in full spermiation for almost the whole monitoring period compared to males exposed to annual cycling temperatures in Spain, suggesting that perhaps males responded differently than females to the two different temperature regimes of the study.

Spontaneous spawns have been obtained from the three Galician stocks, with increasing regularity of spawns and fertilisation rates as the project progressed and more experience was acquired. Relative batch fecundity was also variable among the four wreckfish broodstocks in the present study, varying between 2,000 and 30,000 eggs spawn-1 kg-1 body weight. Limited egg collection has been also achieved from captive spawners after hormonal induction and tank spawning or manual stripping of maturing individuals. During the last year of DIVERSIFY in 2018, spawning became more consistent with better fecundity and fertilization in the Spanish broodstocks. Based on the obtained results, we expect that full acclimatisation of the species to captivity can result in consistent natural spawning and production of good quality eggs.

Spawning takes place during the night or very early in the morning (between 05:00 and 08:00h, with some exceptions at noon). In 2017 and 2018, spontaneous spawning at the IEO, MC2 and CMRM stocks produced a large number of fertilised eggs. Spawning periodicity was 3-5 days in all stocks and fertilisation success ranged between 50 and 100 percent with improved egg quality towards the second half of the spawning season for each female. It has been found that a female is able to spawn up to ten times per breeding season, while the same male has fertilised at least 30 spawns in a total period of 150 days.

The response of the females to treatment with gonadotropin releasing hormone agonists (GnRHa) implants in order to induce spawning has been variable, with irregular results in terms of fecundity and egg quality. In some occasions tank spawns or stripped spawns yielded non-viable eggs, and in other occasions even though the fertilisation was a success, hatching was zero. Artificial spawning by stripping could be the method of choice with mature females that exhibit problems to spawn spontaneously after GnRHa induction. In the case of females that naturally undergo oocyte maturation (without exogenous hormones) it is not advisable to use stripping, since the stress caused by the manipulations could result in poor egg quality and lack of fertilisation success.

With respect to the male characteristics, sexual maturation takes place from March to July as in the case of females, reaching its peak during April and June. All studied sperm quality parameters varied significantly during the two years of the study, but without any clear correlation with the female breeding season (March-June). Overall, sperm quality was considered high throughout the year. Mean sperm density ranged between 4.5-11.5 x 109 spermatozoa ml-1, sperm motility was always higher than 60 percent, motility duration ranged between 1.5 and 6 min and survival of sperm at 4°C ranged between 3 and 10 days, although in some cases a survival of 18 days was achieved (Pérez Rial, 2019). Sperm density was very similar to other pelagic species, such as European sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata) or meagre (Argyrosomus regius). However, wreckfish sperm concentration is higher than the one found in sole (Solea solea) and turbot (Scophthalmus maximus).

There are three possible procedures to obtain wreckfish spawns in captivity:

  1. Expose breeders to natural photothermal conditions in large tanks (>40 m3) and obtain spontaneous spawns
  2. Induce spawning with hormonal implants loaded with GnRHa, if fish do not undergo ovulation and spawning spontaneously, and allow the fish to spawn in the tanks
  3. Induce maturation and ovulation by hormonal treatment, and strip-spawn the eggs followed by in vitro fertilisation.

Despite the easy handling of this species in captivity, its large size requires large tanks to guarantee its well-being and avoid stress-induced reproductive dysfunctions, that would affect gametogenesis and maturation. Although it is possible to strip-spawn wreckfish and fertilise the eggs in vitro after hormonal induction of ovulation the frequent handling of the females is problematic when dealing with such large fish. During the project, it has been shown that wreckfish are able to reproduce spontaneously with very good results in egg fecundity and fertilisation, and this is the recommended method of reproduction with a view to the industrial aquaculture production of this species.

Reproduction and larval rearing of a very close species, the hapuku (Polyprion oxygeneios) has been achieved only recently in New Zealand (Anderson et al., 2012; Symonds et al., 2014; Wylie et al., 2018). The scarcity of broodstock is a disadvantage for the culture of wreckfish, but the clear biological and economical potential of this species justified allocation of part of the effort of DIVERSIFY in bringing together almost all European partners involved so far in wreckfish domestication, to overcome its documented bottlenecks --i.e., reproduction and larval rearing-- in order to produce appropriate numbers of juveniles to launch commercial production.

Larviculture

During the first stages of egg development, vulnerability to external conditions is the highest. Optimal incubation temperature and larval culture conditions have been adjusted in the facilities of the IEO in order to have a correct embryonic development (Álvarez-Blázquez et al., 2016). Zootechnical aspects such as aeration, tank hydrodynamics and water flow have been optimised. These are essential parameters to diminish larval malformation, which is an issue that would need further investigation. During wreckfish larviculture trials in DIVERSIFY, malformations were found that may be similar to a syndrome that is related to the consumption of the yolk sac (SYSS) described by Gunakesera et al (1998) or also to the 'blue sac disease' (BSD) described for common trout. Further studies will be necessary to identify this problem.

In wreckfish, the ontogenesis of the digestive system is considered a slow procedure compared to other species. The development of the digestive system is controlled by endogenous factors and is generally genetically programmed, but the time of appearance of the structures of the digestive system may be influenced by a series of exogenous factors, with temperature being one of the most important (Kamler, 2002). In addition, it was found that the ontogeny of the retina is similar to the general pattern of most fish species. In hatching, the retina is an undifferentiated and non-functional tissue, as occurs in most marine fish with pelagic stages of early life (Pankhurst and Eagar, 1996; Pankhurst and Hilder, 1998; Pankhurst et al., 2002; Roo et al., 1999; Shand et al., 1999). Cone cells were the first photoreceptors to appear (6 dph). This fact indicates that after 5 dph fish can identify food such as rotifers and Artemia nauplii.

Within the DIVERSIFY trials, larval hatching ranged between 42 and 82 percent, which can be considered very acceptable. However, larval survival has been rather limited. Only during the last year of the project, it has been possible to obtain some larvae that have been successfully weaned and juveniles have been obtained and cultured at the facilities of the IGAFA (Galician Institute of Aquaculture, Spain), which is an CMRM collaborating organisation. This was the first time in the project that we succeeded in producing juveniles weaned to inert food, and it is a milestone in the efforts to produce wreckfish under aquaculture conditions. This trial has provided important data on wreckfish growth and has increased our knowledge about the feeding protocol and the specific behavior and metamorphosis of wreckfish larvae (Rodríguez et al, 2019).

During the last year of the project, a large number of experimental trials were carried out in larval culture, focused on adjusting different culture systems (flow through, mesocosm and recirculating aquaculture systems, RAS) and environmental parameters such as water temperature, culture volume, larval density, food sequence, air, light, shape and color of the tanks.

The best results were achieved with RAS at the IGAFA facilities with two batches of larvae from IEO and MC2 spawnings. The food was based on rotifers, Artemia nauplii and enriched metanauplii. Both batches of larvae reached the weaning period and are currently in the juvenile phase. This is the first time that a batch of wreckfish juveniles was produced under cultivation conditions and represents a milestone in our efforts to make this species a firm candidate for exploitation on an industrial scale.

The main results from the wreckfish larviculture experimental work in the DIVERSIFY project have been published (Pérez Rial et el., 2019) and can be summarised as follows:

  • The ontogenesis of the organs related to the digestive system and vision system was not completed until day 23 post-hatching. Most of the organs (except the maxillary teeth in the upper jaw that became visible at 19 dph), appeared by 8 dph
  • The optimal temperature for egg incubation is in the range of 16.5-19.5ºC. Lower temperatures (14±0.5ºC) promote higher larval deformities and low hatching rates, with higher mortalities during the first three days of incubation. Larval culture temperature between 16-18ºC is adequate for greater survival and growth
  • The newly hatched larvae are characterised by their large yolk sac, storing endogenous reserves until day 20 dph at 17±0.5ºC. The large yolk sac and the slow absorption of the lipid droplet indicate a long period of autotrophic nutrition
  • A larval feeding sequence was defined in the recirculation system that gave good results at a temperature of 17.5-18ºC, with a natural photoperiod from 7-9 dph. Feeding live food protocol: rotifer enriched with an arachidonic acid (ARA) supplement form 8-19 dph, Artemia nauplii from 15 to 23 dph and Artemia metanauplii from 18 to 48 dph. Inert food was administered from 40 dph onwards
  • The incubation and larval culture technologies were adjusted to the requirements of this species and important results were achieved with the larval culture in RAS (Rodríguez et el., 2019)

Nutrition

Nutritional requirements of wreckfish were unknown so far and there were only a few references related to feeding habitats from commercial caught (Brick Peres & Haimovi, 2003) and feeding rates in captivity (Papandroulakis et al., 2004). Recently some studies were done on the composition of wild wreckfish (Roncarati et al., 2014; Linares et al., 2015) and their morphometric characteristics (Álvarez-Blázquez et al., 2015). The optimum development of broodstock diets for wreckfish is essential for the future of its aquaculture. Dietary lipids and especially fatty acids play a critical role in the successful production of high-quality gametes and eggs of marine fish (Izquierdo et al., 2001; Sargent et al., 2002). Also, the development of enrichment products of live prey is very important for the success in the larval culture. The understanding of the poly unsaturated fatty acid (PUFA) requirements of marine fish larvae requires the definition of optimal dietary ratio of DHA, EPA and ARA .

Based on data of biochemical analyses of gonads from wild wreckfish females, eggs and larvae obtained from reared fish, some live feed enrichment products were developed for larval wreckfish. Some nutritional experiments with wreckfish larvae were performed in DIVERSIFY showing that larvae exhibit, in general, a good acceptance of the enriched live prey tested and no differences in fatty acid composition of wreckfish larvae fed with the prey enriched with the enrichment products tested were found at different days of live. The fatty acid profile of wreckfish larvae along the larval development shows big amounts of PUFA specially DHA, EPA and ARA. Furthermore, there is a clear relationship between fatty acid profile of broodstock feed supplied and fatty acid profile of oocytes and eggs (Linares et al., 2016, 2018)

The results obtained regarding the composition of tissues of wild specimens were very useful to advance in the knowledge of the nutritional requirements of this species, which shows a large amount of proteins and a low level of lipids in the muscle. In addition, the results suggest that the enriched live prey used as food for the larvae is well digested. Although so far, the larvae have a very low survival rate, the results in 2018, with the obtaining of the first juveniles, are very promising.

It is quite apparent that this species exhibits a fast growth rate and an easy adaptation to the captive environment and handling procedures. Low feeding rates were recorded during the spawning season (from March to July) and high feeding rates occurred during autumn. Ingestion rate varied between 0.2 and 0.5 percent for fish fed with semi-moist diet, and between 1 and 1.8 percent for those fed dry pellets (Pérez Rial, 2019). The results obtained during the DIVERSIFY project in terms of nutrition can be summarised in the following points:

Wreckfish have a big amount of proteins in their muscle (84% in wild fish and 78% in reared fish) and the level of lipids is lower in the muscle from wild fish (7% DW) than in reared fish (25%). Concerning fatty acid composition in muscle, values of PUFA and ∑n-3 are higher in wild wreckfish (39 and 34% of total fatty acids respectively) than in reared fish (30 and 24%). DHA values represent 12 percent in cultured fish and 26 percent in wild fish. EPA content represents seen percent in reared fish and four percent in wild fish and ARA 1.6 percent and 3.1 percent in reared and wild fish respectively. The EPA/ARA ratio has values of 4.6 in reared fish and 1.5 in wild fish (Linares et al, 2015).

Significant advances were made in the knowledge of this species in terms of its biology, nutrition, reproduction and physiology, as well as its adaptation to captivity, reproduction technology and larval culture and the first knowledge of its larval ontogeny was provided. The results obtained as well as the shortage of specimens in the natural environment, are two aspects of great importance to continue dedicating research efforts on the development of aquaculture for this species. It is considered a high potential species with a view to the diversification of industrial aquaculture. Having managed to obtain juveniles in captivity has generated a great impact in the aquaculture sector, interested in diversifying and innovating. The general interest in scientific advances in cultivation is high, and in particular of the companies that currently hold stocks of mature specimens of this species (mainly in Spain).

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