Wreckfish (Polyprion Americanus)

Communication Wreckﬁsh (Polyprion americanus). New Knowledge About Reproduction, Larval Husbandry, and Nutrition. Promise as a New Species for Aquaculture

Evaristo Pérez 1,Fátima Linares 2, José Luis Rodríguez Villanueva 3, Antonio Vilar 4, Constantinos C. Mylonas 5, Ioannis Fakriadis 5 , Maria Papadaki 5 , Nikos Papandroulakis 5, Ioannis Papadakis 5, Rocío Robles 6 , Christian Fauvel 7, Javier Roo 8, José Benito Peleteiro 1, Nuria Lluch 1, Gema Pazos 2, Belén Méndez 2, Irini Sigelaki 5, Castora Gómez 1, Montse Pérez 1 and Blanca Álvarez-Blázquez 1,*

1 Instituto Español de Oceanografía, Centro Oceanográﬁco de Vigo, 36280 Vigo, Spain; [email protected] (E.P.); [email protected] (J.B.P.); [email protected] (N.L.); [email protected] (C.G.); [email protected] (M.P.) 2 Centro de Investigacións Mariñas, Xunta de Galicia, 36620 Vilanova de Arousa, Pontevedra, Spain; [email protected] (F.L.); [email protected] (G.P.); [email protected] (B.M.) 3 Instituto Galego de Formación en Acuicultura, Xunta de Galicia, 36626 Illa de Arousa, Spain; [email protected] 4 Aquarium Finisterrae, 15002 A Coruña, Spain; [email protected] 5 Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Center for Marine Research, P.O. Box 2214, 71500 Heraklion, Crete, Greece; [email protected] (C.C.M.); [email protected] (I.F.); [email protected] (M.P.); [email protected] (N.P.); [email protected] (I.P.); [email protected] (I.S.) 6 Centro Tecnológico de la Acuicultura, 11500 Puerto de Santa María, Cádiz, Spain; [email protected] 7 Institut Français de Recherche pour l’Exploitation de la Mer, Jean-Jacques Rousseau 155, 92138 Issy-les-Moulineaux, France; [email protected] 8 Universidad Las Palmas de Gran Canaria, Calle Juan de Quesada, 30, 35001 Las Palmas de Gran Canaria, Las Palmas, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-906-492-111

Received: 9 January 2019; Accepted: 18 February 2019; Published: 25 February 2019

Abstract: Four different wreckfish (Polyprion americanus) broodstock batches were maintained in research facilities under different photo and thermo-period conditions, one in Greece, the Helenic Center for Marine Research (HCMR, n = 3) and three in Spain: Instituto Español de Oceanografía (IEO, n = 13) in Vigo, Aquarium Finisterrae (MC2, n = 21) in A Coruña and Consellería do Mar (CMRM, n = 11). The CMRM includes two centers that work together: Instituto Galego de Formación en Acuicultura (IGAFA) and Centro de Investigacións Mariñas (CIMA), both in Pontevedra. During the five years of the project DIVERSIFY (Exploring the biological and socio-economic potential of new-emerging candidate fish species for the expansion of the European aquaculture industry, 2013–2018) works focused on the reproductive biology of the species, broodstock, and larvae nutrition and development of incubation and larval rearing protocols have been carried out. In terms of reproduction, catch methods of new wild animals, the reproductive cycle, sperm characteristics evaluation, and spontaneous and induced spawning methods have been described for wreckfish. Regarding nutrition, the positive effect of two types of enrichment on the fatty acid profiles of Artemia and rotifer has been verified. The relationship between the fatty acid profile of the diets supplied to the broodstock and the fatty acid profile obtained in the oocytes and eggs of the females fed with different diets, has also been demonstrated. Finally, early larval ontogeny has been described and incubation and larval rearing protocols have been proposed based on the results obtained in the different experiments of temperature, growth, survival, and larval feeding that were carried out.

Fishes 2019, 4, 14; doi:10.3390/ﬁshes4010014 www.mdpi.com/journal/ﬁshes Fishes 2019, 4, 14 2 of 14

Keywords: wreckﬁsh; broodstock; larval husbandry; nutrition; natural spawns; live prey; lipid proﬁle; ontogeny

1. Introduction The 5-years DIVERSIFY project [1] identified a number of new/emerging finfish species with a great potential for the expansion of the aquaculture industry. Six species were selected based both on their biological and economical potential: Meager (Argyrosomus regius), greater amberjack (Seriola dumerili), wreckfish (Polyprion americanus), Atlantic halibut (Hippoglossus hippoglossus), grey mullet (Mugil cephalus), and pikeperch (Sanders lucioperca). This new/emerging species are fast growing and/or a large species marketed at a large size and can be processed into a range of products to provide to the consumer. Wreckfish (Polyprion americanus) is one of the largest serranid species, reaching a size of 100 kg. It is a deep-water fish found almost throughout the world and is characterized by an extended pelagic juvenile phase [2–4].It is one interesting new species for aquaculture, due to its fast growth [5,6], late reproductive maturation [2], high market price and limited fisheries landings—quotas have been reduced by 90% in 2012 in the U.S.A. [7] and 80% in 2017 in Galicia, Spain [8]—and easy manipulation in captivity [6,9]. Its large size lends itself to processing and development of the value-added products. Wreckfish acclimatizes easily to captivity and, despite its large size, no mortalities have been reported due to handling. It accepts commercial feed easily, being a very voracious carnivore. In a study of wild-caught individuals it was shown that the fish grew from 1 kg to 5 kg in a period of 10 months [6]. The slow reproductive maturation, which occurs at an age of 5–10 years in captivity, may be a problem for broodstock development and management. On the contrary, its long juvenile stage is a great advantage from the aquaculture viewpoint, allowing for commercialization before sexual maturity, and thus avoiding problems linked to maturation, such as reduction in growth, or loss of flesh quality and organoleptic properties. It has been demonstrated that growth is strongly influenced by sex and that wreckfish females are significantly heavier than males, as observed in many other marine fish species [10]. There are three genetically different subpopulations [2]: North Atlantic and Mediterranean Sea, Brazil, and South Pacific. Wreckfish is a deep-water fish found almost throughout the world. The first part of their life phase (from hatching to a body length about 60 cm) is pelagic and juveniles live associate with floating debris near the coast. Demersal wreckfish individuals inhabit rocky and muddy bottoms at depths of 40–200 m, however, individuals are frequently found in waters deeper than 300 m with a maximum recorded depth of 1000 m [11]. It is a gonochoristic species with no sexual dimorphism and spawns at the continental slope at depths of 300–500 m, with the formation of spawning aggregations [12]. In Galicia, Spain, the cities of Vigo and A Coruña are the two main ports for wreckfish sales and most of the catches came from the Azores fishery. The price varied in last ten years between 13–22 €/Kg (Figure1). The establishment of methods for the control of spawning and the production of good quality eggs are essential for the culture of any animal species. The description of the reproductive cycle, along with allowing for the identification of the spawning period and spawning preferences of each species (temperature and photoperiod), enables the recognition of possible reproductive dysfunctions and leads to the development of protocols for spawning induction and production of viable eggs [13]. Fishes 2019, 4, 14 3 of 14 Fishes 2019, 4, x FOR PEER REVIEW 3 of 15

Kg Sales Price €/Kg €/Kg 800000 30

700000 25 600000 20 500000

400000 15

300000 10 200000 5 100000

0 0 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

FigureFigure 1. Sales 1. Sales and andprices prices evolution evolution of wreckfish of wreckfish in the in Galician the Galician market market from from 2008 2008 to 2018 to 2018 [8]. [8]. The lack of reproductive control and larval rearing protocols have been the major bottlenecks The lack of reproductive control and larval rearing protocols have been the major bottlenecks preventing wreckfish aquaculture thus far. Limited egg collection has been achieved from captive preventing wreckfish aquaculture thus far. Limited egg collection has been achieved from captive spawners using hormonal induction [9] or stripping [14]. Embryonic development and the early life spawners using hormonal induction [9] or stripping [14]. Embryonic development and the early life stages have been described [9,13], indicating that the large egg size of this fish (~2 mm in diameter) stages have been described [9,13], indicating that the large egg size of this fish (~2 mm in diameter) may offer significant advantages for its larval rearing. Reproduction and larval rearing of a very may offer significant advantages for its larval rearing. Reproduction and larval rearing of a very close close relative, the hapuku (Polyprion oxygeneios) has been achieved recently in New Zealand [15–18]. relative, the hapuku (Polyprion oxygeneios) has been achieved recently in New Zealand [15–18]. The The scarcity of broodstock is a disadvantage for this fish, but the clear biological and economical scarcity of broodstock is a disadvantage for this fish, but the clear biological and economical potential potential of this species justified the invested effort to overcome its documented bottlenecks (i.e., of this species justified the invested effort to overcome its documented bottlenecks (i.e., reproduction reproduction and larval rearing) in order to produce appropriate numbers of juveniles to launch and larval rearing) in order to produce appropriate numbers of juveniles to launch commercial commercial production. production. In the present work, we provide a global information of the studies carried out during the 5-years In the present work, we provide a global information of the studies carried out during the 5- of the DIVERSIFY project, related to the acquisition of new broodstock, control of reproduction, years of the DIVERSIFY project, related to the acquisition of new broodstock, control of reproduction, nutrition, and larval rearing. This information is targeted towards commercial and research nutrition, and larval rearing. This information is targeted towards commercial and research organizations interested in the potential of wreckfish for aquaculture. organizations interested in the potential of wreckfish for aquaculture. 2. Reproduction 2. Reproduction 2.1. Acquisition of Wild Fish and Establishment of Captive Broodstocks 2.1. Acquisition of Wild Fish and Establishment of Captive Broodstocks There are two main methods for catching wreckfish: By a net (similar to a purse seine) that surroundsThere are a floating two main object methods with fishfor underneathcatching wreckfish: for juveniles By a net (weight (similar <3 kg)to a directly,purse seine) and bythat a surroundslong-line for a floating adults (weight object with >3kg). fish The underneath fishing season for juveniles of wreckfish (weight takes <3 place kg) directly, between and April by and a long- July. line forThe adults decline (weight in wreckfish >3kg). catchesThe fishing in Galicia season it difficultof wreckfish to obtain takes individuals place between to increase April theand wreckfish July. broodstocks.The decline A small in wreckfish number ofcatches young in fish Galicia (<3kg) it weredifficult captured to obtain on the individuals surface with to increase a net in the wreckfishAtlantic coast broodstocks. (NW of Spain) A small by number a coastal of boat young and fish kept (<3kg) in quarantine. were captured For the on adults the surface (>3 kg), with the a lines net inwere the liftedAtlantic very coast slowly (NW from of Spain) the bottom by a coastal of the seaboat to and avoid kept the in overquarantine. expansion For andthe adults rupture (>3 of kg), the theswimming lines were bladder lifted very that wouldslowly producefrom the hemorrhage,bottom of the collapsesea to avoid of internal the over organs, expansion and and death rupture of the ofanimals the swimming in the following bladder hours. that would Once theproduce fish were hemorrhage, on the surface, collapse they of were internal placed organs, inside and a tank death with of theanesthetic animals (clove in the oil) following at a concentration hours. Once of the 0.03 fish mL were L−1 [on19 ]the and surface, the hooks they were were removed. placed inside Finally, a tank they −1 withwere anesthetic transferred (clove to the oil) facilities at a concentration in a truck and of kept0.03 mL in quarantine. L [19] and the hooks were removed. Finally, they were transferred to the facilities in a truck and kept in quarantine. 2.2. Description of the Reproductive Cycle in Captivity 2.2. Description of the Reproductive Cycle in Captivity From March 2015 to October 2016 the broodstocks of all the facilities were monitored and samples of oocytes,From March sperm, 2015 and blood to October were taken 2016 tothe describe broodstocks the reproductive of all the facilities cycle of wreckfish were monitored in captivity. and samplesThe results, of oocytes, published sperm, in reference and blood [20 ],were showed taken that to describe vitellogenesis the reproductive began several cycle months of wreckfish before the in captivity.spawning The period results, in wreckfish. published The in reference analysis of [20], the showed ovarian biopsiesthat vitellogenesis in females, began the sperm several samples months in beforemales, the and spawning the blood period samples in inwreckfish. both sexes The showed analysis a correlation of the ovarian between biopsies estradiol in females, and testosterone the sperm sampleslevels in in females males, as and vitellogenesis the blood samples progressed, in both as in sexes the casesshowed of testosterone a correlation and between 11- ketotestosterone estradiol and testosteronethat were higher levels in in males females with as high vitellogenesis rates of spermiation. progressed, as in the cases of testosterone and 11- ketotestosterone that were higher in males with high rates of spermiation.

FishesFishesFishes 2019 20192019,,, 4 44,,, 14 xx FORFOR PEERPEER REVIEWREVIEW 4 4 of of 14 15 15

2.3.2.3. SpontaneousSpontaneous andand InducedInduced SpawningSpawning 2.3. Spontaneous and Induced Spawning TheThe four four wreckfish wreckfish broodstocks broodstocks at at the the Hellenic Hellenic Center Center for for Marine Marine Research Research (HCMR), (HCMR), the the The four wreckfish broodstocks at the Hellenic Center for Marine Research (HCMR), the Instituto InstitutoInstituto EspañolEspañol dede OceanografíaOceanografía (IEO),(IEO), thethe AquariumAquarium FinisterraeFinisterrae (MC2)(MC2) andand ConselleríaConsellería dodo MarMar Español de Oceanografía (IEO), the Aquarium Finisterrae (MC2) and Consellería do Mar (CMRM) (CMRM)(CMRM) facilitiesfacilities werewere monitoredmonitored fromfrom JanuaryJanuary 20142014 toto JulyJuly 20182018 andand thethe bestbest conditionsconditions toto obtainobtain facilities were monitored from January 2014 to July 2018 and the best conditions to obtain spontaneous spontaneousspontaneous andand inductioninduction spawnsspawns forfor thethe speciesspecies werewere evaluated.evaluated. LowLow feedingfeeding ratesrates werewere recordedrecorded and induction spawns for the species were evaluated. Low feeding rates were recorded during the duringduring thethe spawningspawning seasonseason fromfrom MarchMarch 20152015 toto JulyJuly 2015,2015, whilewhile highhigh feedingfeeding ratesrates werewere observedobserved spawning season from March 2015 to July 2015, while high feeding rates were observed during autumn duringduring autumnautumn inin thethe twotwo tankstanks atat IEOIEO facilitiesfacilities (Figure(Figure 2).2). DuringDuring thisthis period,period, theythey werewere fedfed adad in the two tanks at IEO facilities (Figure2). During this period, they were fed ad libitum twice a week libitumlibitum twicetwice aa weekweek withwith twotwo differentdifferent diets:diets: TheThe wetwet feedfeed (semi-moist(semi-moist diet)diet) waswas aa mixturemixture ofof whitewhite with two different diets: The wet feed (semi-moist diet) was a mixture of white fish (15%), fish oil fishfish (15%),(15%), fishfish oiloil (15%),(15%), musselsmussels (18%),(18%), squidsquid (18%),(18%), andand fishmealfishmeal (34%),(34%), withwith 64.7%64.7% andand 17.3%17.3% ofof (15%), mussels (18%), squid (18%), and fishmeal (34%), with 64.7% and 17.3% of total proteins and totaltotal proteinsproteins andand lipidslipids respectively;respectively; andand aa specificspecific drydry feedfeed formulatedformulated forfor wreckfishwreckfish broodstockbroodstock lipids respectively; and a specific dry feed formulated for wreckfish broodstock (Sparos Lda, Portugal) (Sparos(Sparos Lda,Lda, Portugal)Portugal) composedcomposed byby 68.2%68.2% ofof proteinsproteins andand 12.5%12.5% ofof lipids.lipids. composed by 68.2% of proteins and 12.5% of lipids.

FeedFeed intake intake (gr/fish/month)(gr/fish/month) WetWet feedfeed DryDry feedfeed 30003000 25002500 20002000 15001500 10001000 500500 00

FigureFigure 2. 2 2.. MeanMean feed feed intake intake intake (gr/ﬁsh/mounth) (gr/fish/mounth) (gr/fish/mounth) of of of the the indoor indoor Instituto Instituto Español Español de de Oceanograf Oceanografía Oceanografíaía broodstocksbroodstocks duringduring 2015.2015.2015.

AA variety variety ofof of environmentalenvironmental environmental conditionsconditions conditions likelike like photothermalphotothermal photothermal regimeregime regime andand and tanktank tank sizesize sizeandand location andlocation location werewere wereused:used: used: IndoorIndoor Indoor (IEO)(IEO) (IEO) andand outdooroutdoor and outdoor (CMRM)(CMRM) (CMRM) tankstanks tanks withwith natural withnatural natural photothermalphotothermal photothermal conditions,conditions, conditions, indoorindoor indoor tankstanks tankswithwith simulatedsimulated with simulated naturalnatural natural photothermalphotothermal photothermal conditionsconditions conditions (MC2)(MC2) (MC2) andand constantconstant and constant temperaturetemperature temperature (HCMR).(HCMR). (HCMR). ThreeThree Threedifferentdifferent different strategiesstrategies strategies toto obtainobtain to obtain wreckfishwreckfish wreckﬁsh spawnsspawns spawns inin captivitycaptivity in captivity werewere tested weretested tested (Figure(Figure (Figure 3).3). 3).

FigureFigure 3.33.. MethodsMethods toto obtainobtain wreckfishwreckﬁshwreckfish spawnsspawns inin captivity.captivity.

3 1.1.1. NaturalNatural and and spontaneous spontaneous spawns spawns in in large large tankstanks (>40(>40 m m33),), collecting collectingcollecting the thethe eggs eggseggs as as theythey exitexit thethethe outflowoutﬂowoutflow of of the the tank tank (a). (a).

Fishes 2019, 4, x FOR PEER REVIEW 5 of 15 Fishes 2019, 4, 14 5 of 14 Fishes 2019, 4, x FOR PEER REVIEW 5 of 15 2. Spawning induction with exogenous gonadotropin-releasing hormone agonist (GnRHa) (b) 2. carriedSpawningSpawning out in inductionlarge induction tanks with with(>40 exogenous exogenousm3) under controlled gonadotropin-releasing gonadotropin-releasing photothermal conditions, hormone hormone agonist agonist allowing (GnRHa) (GnRHa) the fish (b) (b) to spawncarriedcarried spontaneously. out out in in large large tanks tanks (>40 (>40 m m33)) under under controlled controlled photothermal photothermal conditions, conditions, allowing allowing the the fish ﬁsh 3. Spawningtoto spawn spawn induction spontaneously. spontaneously. followed by in vitro fertilization by stripping of the mature females and 3. males,SpawningSpawning maintained induction induction in smaller followed followed tanks by by (<40 inin vitrom vitro3) (c). fertilization by by stripping stripping of of the mature females and 33 Naturalmales,males, vitellogenesis maintained maintained in inand smaller smaller oocyte tanks tanks maturation (<40 (<40 m m )took )(c). (c). place in females in captivity and the number of spontaneous spawns increased while the number of induced spawns was reduced during the last Natural vitellogenesis and oocyte maturation took place in females in captivity and the number year (2018), mainly in the three Spanish stocks (Figure 4). of spontaneous spawns increased while the number of induced spawns was reduced during the last year (2018), mainly in the three Spanish stocks (Figure4 4).). Natural spawns 50 (nº) Natural spawns45 50 (nº) 40 45 35 40 30 35 IEO 25 30 MC2IEO 20 25 CMRMMC2 15 20 CMRM 10 15 5 10 0 5 2015 2016 2017 2018 0 2015 2016 2017 2018 Figure 4. Total number of natural spawns from the three Galician broodstock (IEO, MC2 and CMRM) Figure 4. Total number of natural spawns from the three Galician broodstock (IEO, MC2 and CMRM) duringFigure 2015–2018. 4. Total number of natural spawns from the three Galician broodstock (IEO, MC2 and CMRM) during 2015–2018. during 2015–2018. The reason was probably a better adaptation of the females to the captive conditions and the The reason was probably a better adaptation of the females to the captive conditions and the promotionThe of reason a natural was maturation probably a cycle, better resulting adaptation in not of onlythe females vitellogenesis to the captivebut also, conditions spontaneously, and the promotion of a natural maturation cycle, resulting in not only vitellogenesis but also, spontaneously, oocytepromotion maturation. of a natural The natural maturation spawning cycle, resulting behavior in was not characterizedonly vitellogenesis by males but also, chasing spontaneously, females oocyte maturation. The natural spawning behavior was characterized by males chasing females followedoocyte by maturation. the release Theof the natural gametes. spawning Spawning behavior took place was in characterizedthe night or very by early males in chasing the morning. females followed by the release of the gametes. Spawning took place in the night or very early in the morning. In 2017followed and by2018, the spontaneous release of the spawns gametes. at SpawningIEO, MC2, took and placeCMRM in thefacilities night orproduced very early a large in the number morning. In 2017 and 2018, spontaneous spawns at IEO, MC2, and CMRM facilities produced a large number of of fertilizedIn 2017 and eggs 2018, (Figure spontaneous 5). spawns at IEO, MC2, and CMRM facilities produced a large number fertilized eggs (Figure5). of fertilized eggs (Figure 5). 2015 2016 2017 2018 Spontaneous (%) 2015 2016 2017 2018 12.5 9.6 Spontaneous (%) 25 18.5 12.5 9.6 Stripping (%) 25 18.5 Stripping (%) 13.5 59.4 81.5 Induction (%) 87.5 13.5 59.4 90.4 81.5 Induction (%) 87.5 90.4

Figure 5. Percentage of spontaneous, artiﬁcial (by stripping), and GnRHa-induced spawns during Figure2015–2018 5. Percentage in the three of spontaneous, Galician wreckﬁsh artificial broodstock. (by stripping), and GnRHa-induced spawns during 2015–2018Figure 5in. Percentagethe three Galician of spontaneous, wreckfish artificialbroodstock. (by stripping), and GnRHa-induced spawns during 2015–2018Sexual maturation in the three inGalician males wreckfish covered broodstock. the same period as females, with high values of sperm concentrationsSexual maturation between in Aprilmales andcovered June andthe maximumssame period of as 25–35 females,× 10 9withspermatozoa high values mL −of1. sperm concentrationsSexualFor hormonal maturationbetween trials, April in 11 malesand females June covered inand the maximums threethe same Spanish periodof 25–35 stocks as × werefemales,109 spermatozoa inducted with high with mL GnRHavalues−1. of implants sperm concentrationsbetweenFor hormonal June 2015between trials, and 11 2018April females with and in dosesJune the threeand between maximums Spanish 25 and stocks of 100 25–35 wereµg kg× inducted10−19 spermatozoaof female. with GnRHa The mL results−1 .implants of these betweentreatmentsFor June hormonal varied2015 and fromtrials, 2018 no 11 with response females doses in in between the 2015, three spontaneous 25 Spanish and 100 stocks orµg stripping kg were−1 of inducted female. spawns Thewith with results GnRHa non-viable of implants these eggs treatmentsbetweenin 2016 to variedJune good 2015 from fertilization and no 2018response resultswith in doses 2015, but notbetween spontaneous hatching 25 and in or 2017. 100stripping µg There kg −1spawns of was female. only with oneThe non-viable spawnresults that ofeggs these was in 2016treatmentssuccessfully to good varied reared fertilization from until no 25 results response dayspost but in hatchingnot 2015, hatching spontaneous (dph) in in 2017. 2016. or There stripping In 2018, was after spawnsonly 6 daysone with spawn from non-viable the that hormonal was eggs successfullyininduction, 2016 to reared angood implanted untilfertilization 25 female days results post from hatching MC2but not facilities (dph)hatching spawnedin 2016. in 2017. In periodically 2018, There after was every6 days only 5 from daysone thespawn with hormonal egg that fertility was induction,successfully an implanted reared until female 25 days from post MC2 hatching facilities (dph) spawned in 2016. Inperiodically 2018, after every6 days 5 from days the with hormonal egg induction, an implanted female from MC2 facilities spawned periodically every 5 days with egg

Fishes 2019, 4, 14 6 of 14 higher than 50%. Similar results in spawning frequency were achieved with the GnRHa injection test a year before at the HCMR facilities. Ovarian biopsies were performed before the implants to determine the size of the oocytes. Results showed that females with oocyte size <1200 µm did not respond to the implants doses used. In addition, it was found that GnRHa injections were as effective as GnRHa implants and, as observed with the implants, a response time of approximately six days after the injection was observed. On the other hand, the effect of recombinant sea bass gonadotropins hormones follicle-stimulating hormone (FSH), and luteinizing hormone (LH) produced by Rara Avis Biotec, Spain, has been evaluated in some wreckfish specimens (n = 4) presenting arrest of the oogenesis in captivity. The results showed that the use of FSH and LH stimulated gonadal development [21], but more trials with a greater number of individuals and different hormonal doses are required to verify the effects in wreckfish. It could be possible to apply the method of artificial spawning by stripping in wreckfish only with mature females that exhibit problems of spontaneous spawns after GnRHa induction but not for females that undergo oocyte maturation naturally, because the adult fishes are large and heavy and the stress caused by handling resulted in problems with egg quality and fertilization success.

2.4. Sperm Characteristics and Cryopreservation In DIVERSIFY, we established a Computer Assisted Sperm Analysis (CASA) for the evaluation of wreckfish sperm. This method is available as a movie describing the procedure of sperm activation and CASA analysis on the website of the project [1]. The best adapted CASA parameters for wreckfish sperm analyses were determined and reported to optimize their abilities to check fertility potential of the semen in the course of their future spawning induction experiments. The analyses demonstrated that sperm of captive wreckfish share a common pattern of motility with both marine and freshwater fish, based on a general activation of all the sperm at the same time of ejaculation in the activating environment, then a decrease with time down to zero in a rapid lapse of time from 30 s to more than 20 min due to exhaustion of energy stores, that are not replenished due to poor respiration. The mean concentration of wreckfish sperm was 2.41 × 1010 (standard deviation: 0.4 × 1010, n=9) spermatozoa mL−1 in Galicia in January, while it remained around 1 × 1010 from April to September, with no significant variation between sampling dates in Crete, Greece. In 2015, the concentration reached higher values of up to 2 × 1010 spermatozoa mL−1. These concentrations levels did not differ from earlier data (from 1.5 to 2.71 × 1010 spermatozoa mL−1) from the HCMR broodstock. Finally, the spermatozoa concentration in wreckfish stripped semen was of the same order of magnitude as that of pelagic fish such as European sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata) or meagre (Argyrosomus regius)[22–24] and it was higher than that of sole (Solea solea) and turbot (Scophthalmus maximus)[25,26].

3. Nutrition Nutrition studies performed in the DIVERSIFY project were focused mainly on the development of adequate live prey enrichments for wreckfish larvae and broodstock feeds for enhancing fecundity and spawn quality. These are very important for the improvement of wreckfish nutrition and culture development of this polyprionidae species. The quality of the first feeding regimes play an important role in the success of larval culture with dietary lipids being recognized as one of the most important nutritional factors that affect larval growth and survival [27], and an adequate broodstock nutrition is essential to get success in fish intensive culture. In marine fish, dietary lipids and, in particular, polyunsaturated fatty acids (PUFAs) play a critical role in the successful production of high-quality gametes and eggs of marine fish [28,29]. Marine fish have restricted ability or are unable to synthesize n-3 and n-6 LC-PUFAs from their precursors, alpha linolenic acid (18:3n-3) and linoleic acid (18:2n-6) respectively [30–32]. Therefore, 22:6n-3 (docosahexaenoic acid, DHA), 20:5n-3 (eicosapentaenoic acid, EPA) and 20:4n-6 (arachidonic acid, ARA) are considered essential fatty acids for marine fish and must be included in their diets. Fishes 2019, 4, 14 7 of 14

Due to the scarce information about wreckfish nutrition and with the objective of knowing the nutritional requirements, a study of the composition of different tissues (muscle, liver, and gonad) from wreckfish wild fish was done to get some basic information of this species. Furthermore, studies carried out in DIVERSIFY about the composition of wild wreckfish mature gonads and eggs from reared females were very useful as the basis to formulate the enrichment products for live prey used as feeding of wreckfish larvae and the specific dry feed for wreckfish broodstock. The preliminary study on the biochemical composition of some tissues of wild wreckfish shows that wild wreckfish have a big amount of proteins in muscle (84.4%of dry weight, DW) and a low level of lipids (6.9%). Docosahexaenoic acid (26%), was the predominant fatty acid in the fatty acid composition of wild wreckfish muscle, similar to the one obtained by the authors in reference [33] in the Mediterranean wreckfish with values among 24.2–25.7%. Others were oleic acid (18:1), palmitic acid (16:0), EPA (20:5n-3) and stearic acid, representing the major fatty acids in both Atlantic and Mediterranean wreckfish. A high variability among individuals was found in liver and immature gonads composition. Samples of wild males and female mature gonads were taken out showing high values of proteins (60% in females and 44% in males) and the lipid content represented 21% in females and 13% in males’ gonads, n-3 PUFA values varied between 35% and 40%, a high level of ARA was found (7–10% of total fatty acids, TFA) and the EPA/ARA ratio was nearly 1.

3.1. Effectiveness of Live Prey and the Influence of Enrichments The development of enrichment products of live prey is very important for the success in the larval culture. The understanding of the PUFA requirements of marine fish larvae requires the definition of optimal dietary ratio of DHA, EPA, and ARA [29]. 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. Total lipids reached 15–19% of DW in wreckfish eggs and PUFA content reached values of 43–45% of TFA. Similar values were found in 1 to 10 dph larvae (no feeding). Three experimental enrichment products were formulated during 2017 and 2018 to meet the EPA, DHA, and ARA levels obtained from tissues of wild-cached wreckfish. For experimental enrichment preparation, a combination of different products based on microalgae was used. The experimental enrichments were formulated using two different levels of ARA for rotifer (Brachionus plicatilis) and one level for Artemia. The effect of the enrichment of these new products on the biochemical composition of rotifers and Artemia was evaluated and the results showed an efficient enrichment in both preys although the enrichment was less effective in Artemia than in rotifers specially in DHA as it was said recently by the authors in reference [34] who reported that, in general, rotifers assimilated PUFA in a much higher ratio than Artemia. 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.

3.2. Influence of Broodstock Feeds on Fecundity and Spawning Quality Regarding wreckfish broodstock feeding regimes, results obtained showed that most of commercial dry feeds have too much fat for wreckfish. Results obtained with dry feed specifically formulated for wreckfish broodstock (25% fish meal, 34% squid meal, 7.5% krill meal) in DIVERSIFY demonstrated that the diet must contain a big amount of proteins (60%), low level of lipids (12–13%), a high amount of n-3 PUFA (30%TFA), and the EPA/ARA ratio must be similar to that obtained in wild females. A clear relationship between fatty acid profile of broodstock diets (semi-moisture, dry feed, and a mixture of hake and squid) and fatty acid profile of oocytes and eggs from females fed with different Fishes 2019, 4, 14 8 of 14 diets were found. Furthermore, first data on the fatty acid profile of sperm from wreckfish males of different broodstock were obtained.

A relationshipFishes 2019, 4, x FOR was PEER found REVIEW between broodstock diets and fecundity, number of spawnings 8 of 15 of the females, etc. Relative fecundity (n◦ of eggs/Kg of female) and number of spawns per female have been increasingdiets were in found. females Furthermore, fed with first dry data feed on over the fatty the years,acid profile from of 2015 sperm to from 2018. wreckfish males of different broodstock were obtained. 4. Larval HusbandryA relationship was found between broodstock diets and fecundity, number of spawnings of the females, etc. Relative fecundity (n° of eggs/Kg of female) and number of spawns per female have 4.1. Developmentbeen increasing of the in Digestive females fed System with dry in Wreckﬁshfeed over the years, from 2015 to 2018.

Larval4. Larval rearing Husbandry of wreckfish is currently the major bottleneck for the successful culture of this species, due to the low survival rates observed during this period. One of the main scientific goals for wreckfish4.1. Development larval rearing of the Digestive is the System development in Wreckfish of protocols according to the specific requirements of the larvaeLarval during rearing the of early wreckfish developmental is currently the stages. major Thebottleneck study for of the the successful development culture ofof thethis organs related withspecies, larval due feedingto the low behavior survival rates offers observed part of during the necessary this period. information One of the formain the scientific optimization goals of the for wreckfish larval rearing is the development of protocols according to the specific requirements of larval rearing protocols. During larval stages, the systems that were closely related with the feeding the larvae during the early developmental stages. The study of the development of the organs related behaviorwith were larval the feeding vision behavior system, offers by whichpart of the the necessary fish perceived information the for different the optimization feed items of the in larval the rearing environment,rearing and protocols. the digestive During system,larval stages, which the enables systems fish that larvae were closelyto capture, related ingest, with digest, the feeding and absorb nutrientsbehavior from the were feed. the vision These system, two systems by which and the thefish structuresperceived the of different which they feed areitems composed in the rearing are related with theenvironment, larval rearing and feeding the digestive protocols. system, The which vision enables system fish(i.e., larvae the to eye) capture, determines ingest, digest, the ability and of the larvae toabsorb identify nutrients the prey from under the feed. the These light two conditions systems thatand the exist structures in the rearing of which environment, they are composed whereas the are related with the larval rearing feeding protocols. The vision system (i.e., the eye) determines the digestiveability system of the was larvae also determined to identify the by prey the qualitative under the light and conditionsquantitative that composition exist in the ofrearing the feeding protocolenvironment, that was used whereas during the digestive rearing. system was also determined by the qualitative and quantitative Incomposition wreckfish, of the the ontogenesisfeeding protocol of that the was digestive used during system rearing. was considered a slow procedure in comparisonIn with wreckfish, other species. the ontogenesis The development of the digestive of the system digestive wassystem considered was a controlled slow procedure by endogenous in factors andcomparison generally with it other was geneticallyspecies. The programmed, development of but the the digestive time ofsystem appearance was controlled of the by digestive endogenous factors and generally it was genetically programmed, but the time of appearance of the system structures could be influenced by a number of exogenous factors, with temperature being one digestive system structures could be influenced by a number of exogenous factors, with temperature of the mostbeing important one of the [most35]. important The ontogenesis [35]. The ofontogenesis the organs of relatedthe organs to therelated digestive to the digestive and the and vision the system was notvision completed system untilwas not 23 completed dph (Figure until6 ).23 dph (Figure 6).

Figure 6. Schematic representation of the main structures of the digestive system identiﬁed. The time of appearance of each structure is presented with a black solid circle as a function of days post hatching (dph, horizontal axis). Fishes 2019, 4, x FOR PEER REVIEW 9 of 15

Figure 6.Schematic representation of the main structures of the digestive system identified. The time

Fishesof2019 appearance, 4, 14 of each structure is presented with a black solid circle as a function of days post 9 of 14 hatching (dph, horizontal axis).

MostMost of of the the organs organs (except (except for for the the maxillary maxillary teeth teeth at at the the upper upper jaw jaw that that became became visible visible at at 19 19 dph) dph) appearedappeared at at 8 8 dph. dph. TheThe ontogeny ontogeny of the retina ofof thethe wreckfishwreckfish was was found found to to be be similar similar to to the the general general pattern pattern shown shown in inmost most fish fish species. species. At hatching,At hatching, the retinathe retina was was an undifferentiated an undifferentiated and non-functional and non-functional tissue, tissue, as occurs as occursin most in marine most fishes marine with fishes pelagic with early pelagic life stages early [36 life– 41 stages]. Cone [36–41]. cells were Cone the cells first photoreceptors were the first photoreceptorsthat appeared (6 that dph). appeared This fact (6 indicatesdph). This that fact at indicates this developmental that at this stagedevelopmental wreckfish larvaestage wreckfish were able larvaeto see differentwere able items to see in different the rearing items environment in the rearing only environment during daylight only hours.duringThus, daylight it is hours. necessary Thus, to itprovide is necessary light intothe provide rearing light tanks in ofthe wreckfish rearing tanks after of 5dph wreckfish to facilitate after that5 dph the to fish facilitate are able that to seethe feedfish areitems able like to rotiferssee feed and items Artemia like rotifers nauplii. and Artemia nauplii.

4.2.4.2. Optimum Optimum Conditions Conditions for for Larval Larval Rearing Rearing TheThe main main objectives objectives were were to to develop develop a a culture culture protocol protocol and and study study the the influence inﬂuence of of different different sea sea waterwater temperatures temperatures on on larval larval survival survival and and growth. growth. After After incubation incubation and and during during the the autotrophic autotrophic stage, stage, ◦ ◦ temperaturetemperature was was maintained maintained at 16.016.0°C,C, and and gradually gradually increased increased afterwards to 17.517.5°C.C. A A common feedingfeeding protocol protocol with with rotifers rotifers and and Artemia Artemia was was applied applied and and larvae larvae survived survived until until 20 dph. Growth Growth performanceperformance until until 22 22 dph dph was was obtained obtained (Figure (Figure 77),), withwith similarsimilar resultsresults forfor thethe MediterraneanMediterranean andand AtlanticAtlantic stocks (MC2, CMRM andand IEO)IEO) (Figure(Figure8 ).8). Larval Larval length length was was 4.70 4.70±0.27± 0.27 mm mm at at 1 1 dph, dph, yolk yolk sac sac consumptionconsumption occurred occurred at at 11 dph at 14–1714–17°C◦C and and 8 8 dph dph at at 17–20°C, 17–20 ◦C, and and the the mouth mouth opened opened at at 77 dph dph andand 4 4 dph at 14–1714–17°C◦C and and 17–20°C, 17–20 ◦C, respectively respectively [42]. [42]. The The large large yolk yolk sac sac and and the the large large oil oil droplet droplet indicatedindicated the the presence presence of of a a long long autotrophic autotrophic larval stage.

Fishes 2019, 4, x FOR PEER REVIEW 10 of 15

Figure 7.Figure Images 7. ofImages wreckfish of wreckﬁsh larvae from larvae 1 dph from to 1 22 dph dph to reared 22 dph at reared the IEO at thefacilities. IEO facilities.

8.0

7.0

IEO HCMR 6.0 TL(mm)

5.0

4.0 0 2 4 6 8 10 12 14 16 18 20 22 24 Days post hatching Figure 8. 8.Total Total length length curves curves (TL, (TL, mm) mm) for Instituto for Instituto Español Español de Oceanograf de Oceanografía-IEOía-IEO (11 dph) (11 and dph) Hellenic and CenterHellenic for Center Marine for Research-HCMR Marine Research-HCMR (22 dph) larvae.(22 dph) larvae.

No statistical differences (p > 0.05) in size or weight were observed in the larval growth at different temperatures (Figure 9).

Length Weight 1.2 (mm) (mg) 6.0 1.0 LENGTH (18-20ºC) 5.0

0.8 4.0 LENGTH (15- 17ºC)

0.6 3.0 WEIGHT (18- 20ºC) 0.4 2.0 WEIGHT (15- 17ºC) 1.0 0.2

0.0 0.0 0 5 10 15 20 25 30 DPH Figure 9. Mean growth (dry weight, squares) and length during temperature trials. The highest temperature is represented with the red color while the coldest one with the blue. The bars indicate standard deviation of the values.

The average length increased from 4.56±0.08 mm (0 dph) to 6.02±0.27 mm (26 dph). This development occurred mainly during the first 10 days of life, staying relatively constant afterwards. Growth showed a different pattern with an irregular distribution with the larvae age. Newly hatched larvae weighed 0.34±0.07 mg (dry weight) and maximum values of 0.47 mg were registered at 10 dph and 26 dph. Some malformed individuals were observed in the rearing trials (Figure 10). The problem was identified as similar to a syndrome related to swollen yolk sac (SYSS) described in Murray cod (freshwater fish in Australia), that it is related to inadequate nutrition of the broodstock [43]. Furthermore, similar appearance has been also described in the Blue Sac Disease that is common in trout [44]. Toxicity from nitrogen compounds such as ammonia or oxidative stress have been suggested to play an important role in the appearance of this type of deformity. A situation of SYSS could be happening in wreckfish larvae but further studies are required to confirm this conclusion.

Fishes 2019, 4, x FOR PEER REVIEW 10 of 15

8.0

7.0

IEO HCMR 6.0 TL(mm)

5.0

4.0 0 2 4 6 8 10 12 14 16 18 20 22 24 Days post hatching Figure 8. Total length curves (TL, mm) for Instituto Español de Oceanografía-IEO (11 dph) and Fishes 2019Hellenic, 4, 14 Center for Marine Research-HCMR (22 dph) larvae. 10 of 14

No statistical differences (p > 0.05) in size or weight were observed in the larval growth at No statistical differences (p > 0.05) in size or weight were observed in the larval growth at different different temperatures (Figure 9). temperatures (Figure9).

Length Weight 1.2 (mm) (mg) 6.0 1.0 LENGTH (18-20ºC) 5.0

0.8 4.0 LENGTH (15- 17ºC)

0.6 3.0 WEIGHT (18- 20ºC) 0.4 2.0 WEIGHT (15- 17ºC) 1.0 0.2

0.0 0.0 0 5 10 15 20 25 30 DPH FigureFigure 9. 9. Mean growth (dry (dry weight, weight, squares) squares) and and length length during during temperature temperature trials. trials. The The highest highest temperaturetemperature isis representedrepresented withwith thethe redred colorcolor whilewhile thethe coldestcoldest oneone withwith thethe blue.blue. TheThe barsbars indicateindicate standardstandard deviationdeviation ofof thethe values.values.

TheThe average average length length increased from 4.564.56±0.08± 0.08 mm mm (0 (0 dph) dph) to to 6.02±0.27 6.02 ± mm0.27 (26 mm dph). (26 dph). This Thisdevelopment development occurred occurred mainly mainly during during the the first first 10 10days days of oflife, life, staying staying relatively relatively constant constant afterwards. afterwards. GrowthGrowth showedshowed aa differentdifferent patternpattern withwith anan irregularirregular distributiondistribution withwith thethe larvaelarvae age.age. Newly hatched larvaelarvae weighedweighed 0.340.34±0.07± 0.07 mg mg (dry (dry weight) weight) and and maximum maximum values values of of 0.47 0.47 mg mg were were registered at 10 dph andand 2626 dph.dph. SomeSome malformed individuals individuals were were observed observed in in the the rearing rearing trials trials (Figure (Figure 10). 10 The). Theproblem problem was wasidentified identified as similar as similar to a to syndrome a syndrome related related to swollen to swollen yolk yolk sac (SYSS) sac (SYSS) described described in Murray in Murray cod cod(freshwater (freshwater fish fish in Australia), in Australia), that that it is it is related related to to inadequate inadequate nutrition nutrition of of the the broodstock broodstock [43].[43]. Furthermore,Furthermore, similarsimilar appearanceappearance has been also described in thethe BlueBlue SacSac DiseaseDisease thatthat isis commoncommon inin trouttrout [44 [44].]. Toxicity Toxicity from from nitrogen nitrogen compounds compounds such as such ammonia as ammonia or oxidative or oxidative stress have stress been have suggested been tosuggested play an importantto play an important role in the role appearance in the appearance of this type of of this deformity. type of deformity. A situation A ofsituation SYSS could of SYSS be happeningFishescould 2019 be, happening4, inx FOR wreckfish PEER in REVIEW wreckfish larvae but larvae further but studies further are studies required are torequired confirm to this confirm conclusion. this conclusion. 11 of 15

Figure 10. DeformedFigure individuals 10. Deformed collected individuals during larval collected rearing. during larval rearing.

During the last year (2018), experimental larval rearing trials were carried out at IEO, MC2, and Instituto Galego Galego de de Formaci Formaciónón en en Acuicultura Acuicultura (IGAFA) (IGAFA) facilities facilities focused focused on the on use the of usedifferent of different culture systemsculture systems such as ﬂow-through,such as flow-through, mesocosm mesocosm and aquaculture and aquaculture recirculating recirculating system (RAS). system The (RAS). best results The werebest results achieved were with achieved the RAS with conditions the RAS conditions in two different in two batches different of batches larvae fedof larvae with rotifers,fed with Artemiarotifers, naupliiArtemia and nauplii enriched and metanaupliienriched metanauplii at the IGAFA at the facilities, IGAFA wherefacilities, both where reached both the reached juvenile the stage. juvenile stage.The study was approved by the Committee of Research Ethics of the University of Oviedo (Project identiﬁcationThe study code was ES360570189801/15/FUN.01/FIS.02). approved by the Committee of Research The animals Ethics of used the in University all the experiments of Oviedo described(Project identification in this manuscript code have ES360570189801/15/FUN.01/FIS.02). been treated with the strictest respect The and animals in accordance used in with all the experiments described in this manuscript have been treated with the strictest respect and in accordance with the ethical norms approved at both the State and European level in the following legal regulations: “Royal Decree 53/2013, of February 1, which establishes the basic rules applicable for the protection of animals used in experimentation and other scientific purposes, including teaching” and “Directive 2010/63/EU of the European parliament and the council of 22 September 2010 on the protection of animals used for scientific purposes”.

5. Conclusions Reproduction in captivity: The spawning season covers the months of January to June, and occurs sequentially in batches, with spawned eggs having a diameter of 2 mm, as also described in other studies [17]. The males produce large amounts of sperm with a maximum in April and June, which overlaps with the period of maturity of the females. Despite the easy handling of this species in captivity, its large size requires large volumes of sea water for its welfare in captivity and avoids stress that would affect gametogenesis and, therefore, the achievement of maturation in captivity. The stripping method seems unfavorable for wreckfish because it entails a regular handling of the breeding specimens at the time of spawning, and especially when it has been verified that the spontaneous spawnings in large-volumes tanks occurred at the scheduled time. The positive response to the induction trials only took place in females with oocyte sizes >1200 µm. This study complements the previous observations on the biology of this species described for wild populations [8], and brings new knowledge about their reproduction in captivity. Sperm characteristics: Wreckfish males produced a high volume of easily expressible milt with a concentration considered as medium range for marine fish, and much higher than flatfish. On the top of those general features, the setup of a CASA protocol adapted to wreckfish sperm demonstrated that wreckfish sperm exhibits a high percentage of motile cells at activation, and one of the highest initial speeds recorded for fish sperm. This high speed was associated with a long swimming duration compared to other marine fish. The long duration exhibited a double trajectory shape. The first trajectory was straight (associated with the search of target eggs) and then the trajectory began bending, which was interpreted as a phase of searching for the micropyle on the egg surface.

Fishes 2019, 4, 14 11 of 14 ethical norms approved at both the State and European level in the following legal regulations: “Royal Decree 53/2013, of February 1, which establishes the basic rules applicable for the protection of animals used in experimentation and other scientiﬁc purposes, including teaching” and “Directive 2010/63/EU of the European parliament and the council of 22 September 2010 on the protection of animals used for scientiﬁc purposes”.

5. Conclusions

Reproduction in captivity: The spawning season covers the months of January to June, and occurs sequentially in batches, with spawned eggs having a diameter of 2 mm, as also described in other studies [17]. The males produce large amounts of sperm with a maximum in April and June, which overlaps with the period of maturity of the females. Despite the easy handling of this species in captivity, its large size requires large volumes of sea water for its welfare in captivity and avoids stress that would affect gametogenesis and, therefore, the achievement of maturation in captivity. The stripping method seems unfavorable for wreckﬁsh because it entails a regular handling of the breeding specimens at the time of spawning, and especially when it has been veriﬁed that the spontaneous spawnings in large-volumes tanks occurred at the scheduled time. The positive response to the induction trials only took place in females with oocyte sizes >1200 µm. This study complements the previous observations on the biology of this species described for wild populations [8], and brings new knowledge about their reproduction in captivity.

Sperm characteristics: Wreckfish males produced a high volume of easily expressible milt with a concentration considered as medium range for marine fish, and much higher than flatfish. On the top of those general features, the setup of a CASA protocol adapted to wreckfish sperm demonstrated that wreckfish sperm exhibits a high percentage of motile cells at activation, and one of the highest initial speeds recorded for fish sperm. This high speed was associated with a long swimming duration compared to other marine fish. The long duration exhibited a double trajectory shape. The first trajectory was straight (associated with the search of target eggs) and then the trajectory began bending, which was interpreted as a phase of searching for the micropyle on the egg surface.

Nutrition: Results obtained from tissues composition of wild wreckfish were very useful to advance the knowledge of nutritional requirements of this species showing a big amount of proteins and low levels of lipids in muscle. Gonads from females of wild wreckfish have a high level of proteins, n-3 PUFA and ARA. The EPA/ARA ratio was nearly 1. Enrichment products for live prey (rotifer and Artemia) were developed and wreckfish larvae exhibit, in general, a good acceptance of the enriched live prey. The first results of larval culture are promising, but it is necessary to continue with the research on nutritional requirements and their impact on the growth, survival, and larval quality. A specific dry feed was formulated for wreckfish broodstock with a big amount of proteins, low level of lipids, and a high amount of n-3 PUFA. The EPA/ARA ratio must be similar to the one obtained in wild females’ gonads and a relation was found between fatty acid profiles of diet and those of the oocytes and eggs. Relative fecundity and number of spawns per female have been increasing in females fed with dry feed over the years, from 2015 to 2018.

Development of digestive and vision systems: Wreckfish larvae were also characterized by the large size of the yolk sac and oil droplet. During the autotrophic stage the digestive system and the vision system of wreckfish larvae were Fishes 2019, 4, 14 12 of 14 developed to such an extent that larvae were able to identify, capture, and assimilate zooplanktonic organisms and this should be included to the feeding rearing protocol. As the main organs such as the gastric glands did not appear until the length of 5.5 mm, a combination of easily captured and more digestible preys as rotifers or different types of copepods in different developmental stages, have to be included in the larval rearing feeding protocol of wreckfish. The above, in combination with the optimization of the rearing conditions, such as the tank hydrodynamics, the temperature protocol during the rearing procedure, and the photic conditions in the rearing tank, is considered necessary for the development of the wreckfish larval rearing protocol. Summarizing the results of this study, it appears that after the first 23 days of rearing, the digestive system and the eye of the larvae were developed to such a degree that by that time fish were, in principle, able to detect, capture, and utilize the different types of zooplanktonic organisms.

Larval husbandry: Results thus far suggest that the optimal water temperature for artificial incubation of wreckfish eggs is in a range of 16.5–19.5 ◦C. Lower temperatures (14.0 ± 0.5 ◦C) promote higher number of deformed larvae and lower hatching rates, with more egg mortalities during the first three days of incubation. There are 25 juveniles that reached 150 dph that were cultured at approximately 18 ◦C at the IGAFA facilities and it represents a significant step forward in wreckfish larval culture and provides a basis for further studies. This was the first time in the project that we succeeded in producing juveniles weaned to feed, and it signifies a milestone in the efforts to produce wreckfish under aquaculture conditions. This trial achieved important data on growth and increased our knowledge about the feeding protocol and the specific behavior and metamorphosis of wreckfish larvae.

Author Contributions: Conceptualization, C.C.M., J.B.P., and F.L.; methodology, C.C.M., J.B.P. and F.L.; software, M.P., I.F. E.P.R.; validation, C.C.M., J.B.P., B.A.-B. and F.L.; formal analysis, M.P., I.F., E.P.R. and C.C.M.; investigation, M.P., I.F., I.S., J.L.R.V., E.P.R., N.L., C.C.M., J.B.P., B.A.-B., A.V, J.R. and F.L.; resources, C.C.M., J.B.P., B.A.-B., F.L., and A.V.; data curation, M.P., I.F., A.V., J.B.P., B.A.-B. and F.L.; writing—original draft preparation, E.P.R, B.A.-B. and F.L.; writing—review and editing, M.P., I.F., C.C.M., B.A.-B., A.V., J-.L.R.V. and F.L.; visualization, M.P., I.F., C.C.M.; supervision, C.C.M and B.A.-B.; project administration, C.C.M., J.B.P., B.A.-B., M.P., A.V. and F.L.; funding acquisition, C.C.M., J.B.P., A.V., B.A.-B. and F.L. Funding: This research was funded by the EU 7th FP project DIVERSIFY (KBBE-2013-07 single stage, GA 603121) titled “Exploring the biological and socio-economic potential of new/emerging fish species for the expansion of the European aquaculture industry” awarded to C.C.M., J.B.P., F.L. and A.V. Acknowledgments: We would like to acknowledge to all of the staff of different facilities that were involved in this project. Conflicts of Interest: The authors declare no conflict of interest.

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