Aquaculture 496 2078) 130145 Contents lists available at ScienceDizect Aquaculture ELSEVIER Journal homepage: www.slsevier.comilocate/aquaculture Breeding, larval development, and growth of juveniles of the edible sea urchin Tripneustes depressus: A new target species for aquaculture in Ecuador i Jorge I. Sonnenholzner-Varas"*, Noelia Touron”, Maria Manuela Panchana Orrala” ean Spo Potente del a BSFOL Congo Nacona Acct vesigertnes Mars, Facade lca de L Vd, 305, Perea “toes Sipe oltre del eral ESPOL, Crary Nana deAcclare e veigacione Maria, km. 305, i Peel Gosyagal Bus ower Prowse dese Laetele| Sve Semancing ‘Sea urchin Fishery har gradually expanded in eiferent countries and tetris around the worl, including South Ameria, and the sea urchin Trpneustes depress isa feasible eanldate for aquaculture. The aim of this seudy was to prepare « bioteehnealprotocl for rearing larvae and production of uvenles. Large T. depress ‘were collected at monthly terval at subtidal rocky cos ff Palmar, Santa Elena, Ecuador rom Janie) 2015, tw December 2017. T. depressis spawns when exposed to thermal shock in low iuination room. Seven spawning events were recorded eoween March and May and between July and August. On average, females spanned approximately 3.60 = 083 « 10” oocytes (mean diameter 78.4 = 21m) and males spawned ap prowimately 560 = 1.10 x 10” spermatozoa respectively). A cohort of May 13, 2017 was followed for 250 days. Embryo and larval stages were completed within 48 and 21 day, respectively, at 265 = 05°C. Larvae were fed a mixed det of Tyochrit hte, Chartoceros rats and Rhodomenas sp. at concentrations ranging fom25 x 10" to 20 x 10"eells ml as they increased in sae. Larve were measured considering Tota Length (TL), Medium Base Line (MBL) and Base Width (BW). On day 2 alter feriizaton (AP): $11.7 = 2.04%, 158.9 * 10 and 2707 = 3.4 (n= 10), respectively, On AF 548.7 + AOpm, 2736 69 and 355 = 35 (@ = 10), On AF, larval rudiment size war equal in ie to that ofthe stomach and larvae were competent. ‘Their body measuremens, and speefelly total length at that food conceatation, showed no phenotypic plasty. Thirty two ys post spawning (DPS) all avers had open mouths snd a fuetional gut for feeding (ie: 0.40 = 601 mm DI and at DP5y thre size categories were diflerentated, Sea urchin ses by categories 2 DPSy and DPSie, were, respectively small (2.20 + 01mm DT and 239 = 045mm TD), medium (4.90 =" 0.08mm DF and 353 = 0.3mm TD); and laige (7-20 * 0.11 mm DT and 488 = 05mm DI) with zero morthty.T. dees reach 4em diameter at 188 ys after metamorphoss and ean reproduce afters ‘months of life, when it reach approximately 42cm elameter,alkhough the gonads are not yet commercial in ‘ze, Tals protocol is aa inital perfec protocol for sca urchin aquacuturing. However, we have made re- ‘commendations for consideration thet inelue inreasng survivorship and supplying postlarva for juveale production 1. Introduetion ‘This has triggered conceras about structural and functional changes of shore reefs through trophic cascades (Okey etal, 2004; Glynn, 2008; Sonnenholener et al, 20118), and decreasing the income of coastal ‘communities. The fishermen have sought to diversify its fishing by targeting a wider range of marine benthic animals and exploring fishing grounds along the mainland of Ecuador and around the Galapagos Ecuador (in the eastern tropical Pacific, ETP) is a country with a long tradition in coastal-artisanal fishery on rocky-reet ecosystems, for some marine invertebrates, such as lobsters, crabs, mollusks and sea ‘cucumbers (Alava etal, 2015; Henchion etal, 2017). ‘The high demand for consuming high-quality animal protein of ‘marine origin, consequently has made some natural populations das tically diminish by overfishing (Manabu, 2007; Hear, 2008; Woltt etal, 2012; Castej6n and Charles, 2013; Martnez-Ortiz otal, 2015), Archipelago, But, paradoxically, aquaculture is poorly developed in Ecuador (FAO, 2016; Pullin, 2017), ‘A comprehensive spatio-temporal dynamics on basic biological and ecological issues (embryonic phase, larvae and juvenile) for potential + Gonresponding author a: cute Superior Politeniea del itr, Campus “Gustavo Galindo Kin. $0.5, Va Perineal, Casilla 09-01-5865, Gusyagul,Eevadr. mall dérees soonenoleespol.edace L Sonnenholaner-Vars), olouronierpoLeduce (. Touro), mpaschanG@espol eh. ce (MM. Oral) hep: //dotorg/10.1016/}aquacltre-2018.07.018 Received 1 Apri 2018; Received in revised form 2 July 2018; Accepted 11 July 2018 ‘Avalable online 20 Sly 2018 0044.8486/ © 2018 Elsevier BV. Al rights reserved‘new marine resources for fishery and aquaculture i lacking. Due tothe decline in some fisheries (Le, sea cucumber, Sonnenholaner et al, 2017), interest as developed in a sustainable and altemative agus: ‘culture production (supported with population restocking) in order to diver fishing pressure away from traditional fisheries. In this sense, se urchin aquaculture is very pertinent Sea utehin fishery has gradually expanded in diferent countries and territories around the world (including South America, Anew eal. 2002). At least twenty-one species of sea urchins have been heavily harvested and extensively implemented in the sea urchin trade, espe cially for some Asian countries (a total production of about 73,000, metric tons, Casilla-Gavilén etal, 2018). This rade represents an es Uimated at about 200-300 million US$ (Shimabukuro, 1991; Sun and Chiang, 2015; Castlla-Gavilin eta, 2018). > 70% of this production ‘comes from six different areas from the Pacific region (Misael eta, 2008; PAO, 2016), Japan has long been the major luerative market for slobal sea urchin production (annual domestic demand of about 50,000 metric tons whole weight) and isthe major driver of global sea urchin prices. A top-quality (as fresh, dried, salted or brined sea urchin roe) ‘ean sel for over USD $60/g (Sun an Chiang, 2015). Roe is part of a special gastronomy part of fish markets and are prized in delicacy cuisine (in sushi or sashimi) for their sensory at ‘utes, in terms of palatability, favor and eolor(Keesing and Hall, 1998; ‘AAFC, 2002; Devin, 2002; Muthiga, 2008; Rahman etal, 2012). They also are consumed forthe high-quality protein content, polyunsaturated fatty acids; and potential use in medicine because of its rich chemical constituents and biochemical compounds, such as polysaccharides, PUFAS, B-Carotene, xanthophylls and others nutrients (Kaneniva ane “Takagi, 1986; Lawcence etal, 1997; Yureva et al, 2003; Archana and Babu, 2016). Nonetheless, the supply of domestic sea urchins is limited and the feasibility of this industey is largely dependent on sulicient natural populations. At this juncture, this important industry is kept ‘virtually unstable. Because several sea urchin populations have diminished as result from ineffective fisheries policies and management (2 declined pattern ineatches has been noticed in several countries in the Pacific region, as Chile, China, Japan, New Zealand, and in many areas of Southern and ‘Western Australia), interest in sea urehin aquaculture has developed rapidly and has grown steadily over the past few years (Lawrence, 2013; Lawrence and Agatsuma, 2013; Paredes eta, 2015). Sea urchin aquaculture can help alleviate the pressure for overexplotation of wild sea urchins (Barker et al., 1998; Hugget et al, 2006; Liu et al, 2010; Kayabs et al, 2012; Eddy etal, 2012; James etal, 2016). Trpneustes is 4 genus of toxopneustid echinoids that recognizes three extant edible species with commercial value, T: ventcosus (Camarck, 1816) from both sides of the Atlantic, T. grail (Linnaeus, Rhy Oy Aone 4962018) 136145 1758) in the Indo-West Pacific, and T. depressu (Agassiz, 1863) inthe central eastern Pacific (ig. 8-e)-A study of the phylogeography ofthe pantropical sea urchin Tripmeustes by Lessios etal (2003) revealed that based on morphology, COI, and bindin daca, T. depressus from the eastern Pacific Is in fact the same species aT. gratia from the western Pacific. Inthe intemational market the gonads of T. venrcosus and T: rari ate good vist-vis quality competitive in the trade with other species (Scheibling and Mlndenov, 1987; Rahman et al,, 2009; Pena ral, 2010). A number of studies have examined the biology, ecology, fisheries and aquacultute relevant issues of T.gralla (Shimabukuro 1091; Cyrus etal, 2013; Lawrence and Agatsuma, 2013) and T. ven- tricosus (Keesing and Hall, 1998; Andrew et al, 2002; Robinson, 2004; Woleott and Messing, 2005), but no detailed information exist for T: Aepressus (Sonnenholzner et al, 2013). T, depress is a very desirable species for fishery and isa feasible candidate for aquaculture in Ecuador for is bological atsibutes, such, as: largesize, short time to maturity, high growth rate, high re- productive effort and output, and short life span (lavrence, 1990; lawrence and Bazhin, 1998; Lawrence, 2013), They are part of a con- spicuos assemblage of coral refastociated communities that inhabits moderate hydrodynamic leeward rubble and open sloping rocky shores ith interspersed coral-sandy rubble pockets (but never on pure sand or ‘muddy sand) on encrusting coralline, calcareous articulated algae on shodolith beds and on extensive and dominant soft, fleshy brown ‘acroalgee (varieties inclade Padina and Sargassum species) that sip- port their grazing activity (Sonnenholener and Lawrence, 2002; Lawrence and Sonnenholzner, 2004; Foster et al,, 2007; Irving and Witman, 2008; Uthicke et al., 2009; Sotelo-Casas et al, 2016) in shallow waters (preferentially at average 12m depth) to 73m depth, from the Gulf of California, México to Lobas de Afvera islands, Per, Including the Galipagos and Clarion Islands (Malu, 1988; love and Sonneniolzner, 1994) (ig. 1a-c). In Galépages, T. depresus is spread, ‘out throughout all five eco-regions with diferent sea temperatures ranging from 20.3-27.1°C (Banks, 1999; Brandt and Guarderas, 2002), and bathymetricaly between 3 and 40m depth, it ranges from 173-28.5°C, in the central coast off Ecuador (Datos de la Estacion Oceanogrifica H Pelado del Cenaim-Espol 2013-2017). Traditionally, this species has been locally consumed as food by fishers in Manabt (on. the mainland coast of Eeusdor) and Galspagos. Since 2014, considering the importance of T: depress, the Centro, Nacional de Acwicultura © Investigaciones Marinas of che Escuela Superior Politécnica del Literal of Ecuador (CENAIM-ESPOL) is pro- mating research studies on T: depressus diversity aquaculture. These suds wil coneibute for future mangement plans (as alternating new fisheries based on aquaculturing), leading to the sustainability of benthic resources in Fevador. The aim of this study was provide Fig 1. View of large sa urchins Trpmettes pests in open rocky and coral ree habitats; ) specimen at 12 depth, Bl Pla inland, Santa Elena, Eevador;b) a ‘covered specimen with foliose macroalgae Uta lcticaat 3m depth, Bahia Sata Hlena, Guanacaste, Costa ica and «) specimen at 2m dept associated with large Pocilpora verucosa, Punt Galera, Baja California Sur, México2 Someone Vara et Agua 496 2018) 134-145 Fig. 2 Aboral view of large Tripmetes depres spawning at laboratory conditions in May 13,2017; 2) female releasing ooeytes (14cm TD) and) mal sperm (13 TD), protocols for rearing competent larvae and producing juveniles of 7: depressus on a small seal. 2, Materials and methods 2.1. Btbical procedures (i vivo and experiments) All the procedures realized considered in this study followed the ‘guidelines for ethical and responsible researches using in vivo animals snd experiments for echinoderms (Kilkenny et s), 2010; Rubilar and Crespi-Abri, 2017). 2.2, Sea urchin collection Large sea urchins, 7: depressus (mean test diameter: 125 + 0.Sem land wet weight: 759.4 = 9.1 g: N = 200) were collected by diving at high tide, between 10 and 12m depth, early in the morning (before (0900 h) at monthly intervals along the subtidal rocky coast off Palmar, Santa Elena, Ecuador (15'59'00"S 80°44'00"W) from January 2015 to December 2017 (Pig, 1a) ‘Specimens were transported individually in double plastic bags with multi-track zipper (29.9 x 26.8cm, 3.81 size, Diamond), filled with seawater using styrofoam containers Kept at 22°C to the aquaculture (Centre (CENAIM) in San Pedro de Manglaralto, Santa Elena, Ecuador. After collection, sea urchins were gently rinsed with sterile and filtered seawater (SPSW) and kept them individually in fla-bottomed 401 rectangular tank. Test diameter and body weight were determined Using a caliper (in centimeters) and a spring seale with 0.1-g accuracy, respectively, 2.3, Culture sytem, 2.3.1. Seawater quality and environmental conditions “The seawater used during the spawning, hatching and larval rearing was 1 and 5 jm filtered and UV treated (Seawater flow rate used as kept constant 11min“, as sterile and filtered seawater: SESW), tem- perature between 22 and 27°C, salinity 35 ppt, pH of 82, dissolved ‘oxygen of 56mg 0; 1-1, and kept in semidarkness condition (05uEm™*57), 23.2. Maincenance of large and juvenile animals In this study, we defined juveniles are individuals after metamor phosis, before production of gametes, and also have had opened its mouth with functional jaw mandible for initiating feed. And we ut lized the term large sea urchin, instead adult since these rapi-growth sea urchins begin producing gametes when they are small sze, and they ould be adult by definition at 0.4 + 0.01 mm diameter. The large sea urchins (from the natural population and after spawning) and the juvenile sea urchins (cultured at CENAIM) were in ‘outdoor conditions. Large sea urchins were Kept in 5001 flow-through stock tanks (approximately, 501 per animal, N'= 10 per tank), open circuit (water exchange rate in 5B, 100%), aeration and no direct lighting. Juveniles were separated by three size-classes (by their test iameter: (9 1.1-3.9 mm; (i) 4.1-6.0 mm and (i) 6.1-8.1 mm. During this time, both were fed on the brown algae Paina durvilaet. Twice a week the tanks were cleaned removing uneaten fod, fecal pellets and ebris by siphoning. Seawater temperature and dissolved oxygen were recorded daily by a multi-parameter probe (YSI-S50A, USA). 23.3. Spawning induction and observation Laboratory scale observations were firstly made for providing an indirect and rapid assessment inthe application of stimuli for spawning. in mature sea urchins, as follows: ) haphazardly sampled urchins with 11.5 ml of 0.5 M KCl injections with a 26 gauge needle into the coelom, via the peristomiel membrane; i) manval shaking ofthe animal, and i) thermal shock (water temperature raised 45°C for 10 min) in Tow il. lamination room condition 23:4. Spawning and fertiization Seven cohorts of sea urchins T: depressus were produced, but cohort of May 13, 2017 of was reared over a period of 250-days in captivity Mature oveytes (bright orange: Fig. 2a) were collected through 30 um, [Nytex mesh net, and washed with SESW, then were pooled (approxi= ‘mately 400 to 600 eggsmi~*) and shed into a4 jar. Sperm (semen off. white creamy streams; Fig. 2b) was dry-collected, Afterwards, 2m of diluted sperm (1 drop of dry semen with 10ml of SESW) were added and gently stirred with a plastie paddle for 1 min (pooled sperm: oocyte 20:1). After three minutes the ferilzation membrane was asessed and the spermstozoa removed by three consecutive washes with SFSW (with a 30m sieve). Sperm concentration and motility (as rapid sperm) were selected forthe pools (three individuals per pool) prior to oocyte fertilization, 23.5. Embryonic development, larval rearing, and microagal diets Eggs were incubated in SESW for 48h at 25°C until the pluteus stage in 5001 fiberglass cylindroconical tanks in static SPSW without aeration ata density of 0.5 eggs ml". Embryo development was scored as: fetlized egg; unequal cleavage: equal to or > 16-cell stage; equal 0 for > 32-ell stage, hatching blastul, blastula, and gastrula. Larvae were reared for 20 days until competency in 5001 fiberglass cylindroconical tanks in aerated salle SESW, with three paral (50-75%) water changes per week (larvae were collected with a2 Someone Vara et Fig. 8. Digital image of « twosrmed Tripetstes depress phtews larvae de isting the tnee morphometric characteristics sed to compare larva growth pesfocmed by TL ~ total length, MSL ~ mid-body line length, and WS ~ width ‘ase (40% magnification) a suggested Schl al. (20). 100-125 um mesh). A complete water change and thorough cleaning tanks were done once every ten days. Prism larvae were stocked at a density of 1 larvae ml"'. Density was estimated for each batch as the proportion of swimming larvae (up to the eightarmed pluteus stage). Larvae stages were scored as prism, two-aem, four-atm, six-arm, and cight-arm. Once larvae were staged, the percentage of larvae in each stage were calculated and averaged among the replicate tanks. Larvae were fed (86h after fertilization, when the functional gut appeared) ‘vice a day with a mixture of three live microalgae epecies: Tysochrisis ea (=Lsoschrys galbana, strain CI/03), Chaetocers gracilis (seain ‘CCH2-2/06) and Rhodomonas sp (strain CR16/03) at 2 concentration ranged from 2.5 x 10° to 2.0 % 10" celle mi of seawater, by ae: justing the food level (dictated by the daily checking of the gut with @ dissecting microscope forthe flines, color pigmentation and presence ‘of algae) every day until attaining metamorphic competence, During larval rearing, information was compiled to measure the larval per formance: survival counts assessed volumetrically on days 7, 14 and 21, and at the end of rearing and measurements (maximum dimensions, Fig. 9, 2.3.6, Settlement and metamorphosis Seven days after reaching competence (Indicated by larval mor phology and tactile behavior), larvae with a large juvenile rudiment ‘were placed (with a density of 1 larval ml~") in 10 days pre-condi \oned $000 fiberglass rectangular flat bottom tanks with an inoculum ‘of the benthic diatom Navicul sp. (Strain CN13/03 at 1310° cll m=") forming @ biofilm, as a sertlement inducer for competent larvae. Seawater was 20% replaced per day and gently aerated. Juveniles were ‘at 22 days port-settlement » DPS, with a size of 0.1 em diameter. They foraged upon a lush green carpet of the algae Cladaphora sp, after ‘wards, semi-dried small pieces of Padina duvllae’ were introduced. 2.3.7. Counting, morphometric and growth measurements ‘Sperm and oocytes densities were determined by means of a he: -mocytometer (Neubauer counting chamber) and a six mult-well plate (area: 95cm’, diameter: 348mm, Cellgro Coming), respectively, Agwaenr 496 2018) 134-145 under a compound microscope and a stereoscope microscope using a 4x and 40% objectives, respectively. Measurements of oocytes, 25 goles, embryo and larvae were made wnder a compound microscope and test diameter of juveniles (DPS; se) was made on freshly prepared samples using a profile projector microscope (Mitutoyo). All materia photographed was fitted with an image analysis software (Works 2.0. Larvae were place in a flat focal plane for measuring them on total Tength (TL), base width (BW), and medium base line (MBL) (Scholtz ct al, 2015, Fig. 3). Measurements were made every day by image analysis of fresh samples of 10 larvae. Compecence vas considered a8 achieved when a least 80% of the observed larvae showed that the rudiment was at least equal or larger in size than the stomach. The following calculation (Ghe midpoint method) was made to determine the percentage change for growth uniform differences over time: a = [(6,— S18, + $02)] x 200 where So represents the initial size and S; i the total final sizeof larvae fn each measurement by category. All values are expressed in mil meters. The denominator is the average of the stating and ending va- ues. [AC DPS, of culture, juvenile sea urchins were separated into three size-categories: (9 small 1.1-3.9 mm; (i) medium: 4.1-6.01mm and (if) large: 6.1-9.1 mm and maintained in independent tanks for avoiding the intraspecific competition between large and small individuals (ae suggested Grosjean etal, 2003), Size is expressed by the ambital test diameter (ID) the external diameter ofthe test at the ambitus excluding the spines, TD was measured with « manal sliding caliper at the ‘nearest 0.1 mm, Specific growth rate (SGR) was caleulated, as follows SGR (Rday~) [Qn TD, ~ In TD)FAX] x 100 where In is natural logarithm, TD; and TD, are the average test dia- meters of the sea urchins (in millimeters) at time 1 and 2 in days, re- spectively, and At isthe growth interval in days (t, ~ ). SGR is ex pressed as a percentage of sea urchin size increase per day. 24, Statistical analysis Significant differences in the growth of juvenile sea urchins were tested using one-way ANOVA and post hoc Tukey HSD test. Assumptions of normality and homogeneity of variance were confirmed prior to the analysis using Levene test. Differences were regarded a5 significant at P= 0.05, All results are given as mean * SE, Statistical analyses were carried out using Statistica 10.0 software 3. Results 3.1. Spawning OF the three methods tested to induce spawning in T: depresus, increasing seawater temperature 4 to 5°C in in the dark was the most efficient and non-invasive method for obtaining gametes 20 min ater animals arrived inthe laboratory (see Subsection 2.2, Fig 2a, B. Seven spawning events mortly occurred between March and May and between, July and August (2015-2017). The spawning by females and males was continued intermittently for approximately 30min. No spawning oc curred after intracoelomic injection of 1 ml of KCI 0.5M and manual sacking, Several sea urchins died after potassium chloride injection, ‘The mean diameter of unfertilized oocytes war 78.4 2.1 4m, (N= 110, 6 females, 50 a0cytes of each, Fig. 4a). The mean of the spermatozoa head and tal are 15.0 + 0.1 um and 45.0 = 02pm, re- spectively. In average oocyte and spermatozoa number spavned by females and miles were 360 = 0.83% 10” oocytes and 5.60 = 1.10 x 10” spermatozoa, respectively.Someone Vara eat Agwacnr 496 2018) 134-145 Fig 4. Barly cleavages of Trpmeutes depress under light microscopy 2) Oseyte xg: embry with two cell before the second cleavage; haline layer is ible ‘between cells; d animal pole view of an embryo with eels) embryo with 16 cells arrangement of mieromeres and macromeres onthe vegetal pole ofa 16 ell, ‘embryo vegeta ple ofan embryo wih 5 cls: lateral view ofan embryo with 108 cei) embryo during blastla formation: embryo during gastrulation as Dochutching swisuing Mastula with thickened vegetal pole epithelium and fal ‘Not: black arrow indicates elite band of gusta 3.2, Fertlization and embryonic development Dynamics of T. depress cell divisions is shown in Table 1. The ‘embryo stages were completed within 48h at 25.1 + 0.7°C (ig. db. “The fertilization envelope was observed with the release of pola bodies (ig. 4b). The eggs exhibited holoblastic cleavages (until 32-cell stages) ‘Table Development of neuter depress from frclzation to 40 um ameter ju este. Tine sk hours after friction. Sages Deserion Tine size en) nbwe Frled eggs with cnpleeeulaten—SI2min 55 ‘membrane etd epson paler beds oem eal stage) temn 1900 Sted lvsion eel Sage) 3emin 1070 Td evn el sage) SSmin 1090 oar ein (el sag) IniBmin 1150 Fah vio (2a tage) TheSmin 1200 Nora 2hiamin 0 sein las ahsema 1230 Daal Pearman ta00 Gaorale Fhaomn 1300 2am pleas 2 mas ‘arm tear ‘ eo (arm pltear be asso ‘arm pltesr wa 70 ‘Soap le compte dion, na a0 ves (a1 with open mut) Ha S500 Sea (boy40) 180 Sau tay5d 40 Svea days) 5200 Svea (ey) 12,000 Svea (845) 25,000 Sea (618) 49000 ‘stage of archenteon invagination touching the anterior pole, k) pass larvae at approximately 27-60 min after fertilization (AF) (Fig. 4e-e, Table D. ‘The morula (128.cell stage) occurred 2h AF (Fig. £&, Table 1). The embryo hatched and developed into the late blastula and an cary ci- jated gastrula stage 7h AF (Fig. 4h, i, Table 1), An early fullsize spherical gastrula occurred 20h AF (Fig. j, Table 2) and a bilateral pyramidal prism larva witha complete digestive tract occurred 48h AF igs 4k, Table D. 3:3, Larval development and growth ‘The echinopluteus larval stages were completed within 11 days AF (Pig. 5a, Table 1) 26.0 = 015°C, On day-2 AF, the size ofthe early feeding larvae of two-armed stage (Fig. a) was TL: 3117 + 2.0um, MBL: 158.9 = 10um and BW: 270.7 + 3.4ym, N= 10 (ig. 0). These larvae showed fenestrated rods and the esophagus, sphincter, igestive wact and anus were clearly visible (Fig. 53-0). On day-9 AF, the size ofthe sixatmed stage (ig, 9) was TL: 5045 3.7 um; MBL. 265.1 + 35um; BW: 2707 = 35ym; N= 10 Gig. 6) with the highest growth increment variations at 479, 50M and 49%, respec tively Fig, 6), On day 11 AF, the size ofthe elght-armed stage (Fig. 58) wasTl:524.1 © 4.3ym; MBL: 258.4 5.1 ym; BW: 3228 + 3.5 um: N= 10 (ig. 6). Their growth Increment variation decreased at 4%, 3% and 18%, respectively (Fg. 6). On day 16 AF, the sizeof the larvae was Ts 544.7 + 4.0um; MBL: 273.6 + 6.9um; BW: 355.0 + 3.5m; N 10 (Pig. 6). The anterolateral arms were reduced in length, the larval body extensions were bent towards the previous end, and its body shape was rounded (ig. 59 with very low increment variations at 4%, 696 and 9%, respectively (Fig. 6). The rudiment grew and at- tained approximately one third the length of the stomach (Fig. 5f ‘Table 1). On day 20 AF, the larval rudiment reached a sie-equal to that of the stomach and occupie a large part ofthe larva body, constricting the stomach to the left side, The lava developed cilia over its whole body Gig. Sgh, Table 1). On day 21 PF, the movement of the first pedicellaria, which were emerging from the larval rudiment, was ob- served (Fg, 5), This indicated that larvae were ready fr the settlement and metamorphosis (Fg. 5j, Table 1)2 Soman ra ab Avene 4962018) 136145 Fig. 5, Laval development and juvenile of Dipnewstes depress. a) easly two-armed pluteus; b) two-rmed: «) fourarmed: d) sixarmed:«)eightarmed: D a ‘competent eighcermed putes lava with juvente rudiment occupied mest of the body and displaced the larval gut: )pre-metamerphic larvae: b) pre-netamorphie larvae with pedicels arval dane regresion of the premetamorphlc larvae with ambulara et) aboral view of «recently metamorphosed ea urchin showing odin and spines; Ken) oral ad aboral views of veils at labarsory. Arrow indicates a well-developed rimect(. 100 [hn stays o care al juveniles were separated in three sie to Eine segs) small'220°2 007mm 10,1) medium 480 2 008mm TD and dp lrg 720 = 011 am TD, and mina in independent seo Wve, Ghar crip sot. Ater ths ant Bad-ape of cat) the pve of 400 300 200 100 Aepressus was synchronous and alternated (as rapid and slow-moderate growth episodes) forall chree sea urchin size-ategories (Fg. Se). During the slow-moderate growth episodes, small individuals grew faster (0.6 + 0.06% d=") than large individuals (ar) = 3.7544, P = 035; Fig 9, but during the rapid growth episodes, all individuals grew fast and. there were no differences between categories (2ay) ~ 0.2282, P= 798) The higher amounts of growth were re- cored at one event syneheonically for all size-categores of sea rehias, Larval size (jn) 0 2 4 6 8 10 12 16 16 18 20 Time (cays) betiveen days 160 and 180, but medium and large individual showed & Fig. 6 Larval morphomesiee of Thputes depress, TL =total length, high-frequency of sine increments (Figs Sed). Te final sizes for small, [MBL = mid-ogy line length and WB = with base Error bars éenote se medium and large sea urchins were 23.9 = 045mm 1D, 323 = 03mm TD, and 428 # 05333 = 0.3mm TD, respectively 5.4 Sie variably an growth of jens Gigs Be 9 Size variability and growth performance for juvenile ae given in the Figs Skom, and 7 lo 9. AU DPS jvenile fed Navewa sp. 35. Survival Gigs. 5) 78). AUDPSys, 08 urchins without opened most pated to juvenile stage with an average size of 0.40 + 0.01 mm TD (Figs. sl, Based on all our record ofthe culturing of 7 depress between Sah; Table 1), At thie size, animals opened theicmouth with a movable 2015 and 2017, during the first day pos-hatching (rom fertilization to jive structures, as Arsole's lantern, and developed a functional gut. *€ prism larvae stage) survivorship wes between 97 and 95% is 10). They iil fed on rls of Enteromorpha sp. and pasted to blades of Before metamorphosis, from the prism tothe eightarm larvae stages, Patina duriaet (Figs. Sm 76.0). Two remarkable rapi-growth epi- _ *8Fvivorships were around 75 and 80% (Fig. 10). We reported high codes occurred (SGR ranged from of 08 t0 4.2% "2, hes fa, 9) and mortalities between 80 and 93% after metamorphosis and setlement were intercalated by a long slow-growth episode between 53 and 82- Process (Fig. 10), Overall in the cohort of 13 of May 2017, approxi- Tay of caltre (SGR ranges from 81 and 03% "Mew Sab 9), at mately 280,000 larvae were seed in the culture tank, and of them, DPSy.-5, growth rates of juveniles were well differentiated into three 18,000 reached the juvenile stage. At DPSes of culture, the data for size groups at (Fron) = 961.63, P = 0000, Fig. 9. juveniles in Fig. 10 indicates survival was ca, 10%2 Someone Vara et 7. calving of juveniles of Trpnesterdepresesinoxtdoor tanks. 1) 8 ‘urchin barren 74mm TD, DPS) feeding Marca sp Sim with other sessile iatoms;b) stall juveniles (220 = 0.07 mm TD, D?Syq) fading the brown rmacrslgse Podina durvilae and «) exhibiting exypie covering behavior (For Interpretation ofthe references to color inthis figure legend the reader i Terre tothe web version ofthis atle) 4, Discussion ‘The results of this study promise an advance for production of ju veniles (a sed) ofthe edible and native sea urchin Tripneustes depressus in Feuador. Our protocol is based on larval and juvenile culture over Uhree years WJanvary 2015 to November 2017) It provides base in {formation and timing schedules on hatching and development of the larvae and juveniles, diets for promotion of growth increasing their survivorship, and coasideration of systematic broodstock selection ‘effective spawning under laboratory scale conditions. Our results wi be useful for culturing T: depressus, but i€ must be calibrated for the different regions ofthe ETP. 4.1. Spawning and reproduction of targe sea urchins ‘Although KCl isan effective chemical used to induce spawning in mature echinoids during the breeding season (Giudice, 1973; ‘Thompson, 1983; Levitan et al, 1992); it was not for T. depessus. An mals were not stimulated for spawn and can die. A similar unfavorable ‘ase wat reported in for Gisdice (1973) who noted that Arbacia can release a fertilization inhibitor from the epidermis or from the per viseeral Nuid when they were stimulated with KCI, which needs to be carefully washed away from the eggs to prevent unfetiliztion. Based ‘on our laboratory seale observations, T: depressus is very sensitive to {emperature sess (4-5 °C) and light change as stimuli (See Fig, 16) © induce spawning. This reaction to light was reported for T.esculentus Agwacnr 496 2018) 134-145 (= T. ventions) where Lewis (1958) determined that it shows re- sponses to change in light intensity. Therefore, we decided co apply & non-invasive and non-destrcive inducer instead of the classical in- ‘raoral injection of 0.5 M KCI method. For this reason, large zea urchins ere transported tothe laboratory ina thermastatic hath in the dark to ‘maintain their pre-spawning state until their arrival to the laboratory (see sessions 2.1, 2.2). T, depressus does not show a restricted spawning period with vari= able intensities. We observed seven spawning events between Mareh-May and July-August, corresponding to warm/bumid anc’ cokd/ Ary periods, respectively, from January 2014 to November 2017. This suggests that T. depress has a continuous, non-dserete and synchronic gametogenic activity throughout the year, with two peaks of gonadal evelopment for reproduction, where the mean monthly seawater temperature fluctuated from 19 to 30°C, and rainfall was < 150mm, per year (information based on the meteorological station at CCENAIM-#SPOL). Similar pattems of an extended, nearly continuous period of reproduction with peaks (as a seasonal cycle of gonadal growth in the winter and the suramer) have been reported for T. gratia Jn Madagascar, Kenya, and Philippines (Muthiga, 2005; Toha et al. 2017), and T. vertricosus in coasts off Florida (McPhesson, 1965) Nonetheless, both species there seems to be a tendency for semiannual reproduction into any broad geographical pattem or any one seasonal temperature regime (Pearse, 1974), Based on histological examination of gonads (data unpublished), T. depresus may reach their sexual ma- tusity in less than one year, patiearly the males at sixth months-old, and females one month after. Ths is particularly similar to T. gratia reared in hatchery in the Philippines at 27°C, which reached sexual ‘maturity in six to seven months (Creswell, 2011), ‘Although, the factors that control the reproduction of T depresus for producing successful larval supply through the year were not su died here, we consider that some conditions required for T. grailla in the westem tropical Pacific may apply for T. depessus in Revador. arse (1974) concluded that reproductive effort in T:gratilla does not correlate with temperature, Contrary to this Muthiga and Jecearini (2005), Muthigs (2005) and Chen sind Chang (2012) determined that the reproductive cyle, gonad development and spawning evens ofthe Kenyan and Indonesian populations of the sea urchin T. gratila are affected by annval and intradaily environmental conditions, as sea temperature, daily ight intensity, phases of moo, and benthic pro- ductivity (a5 food avilable offer). Its well known that both direct it. Jumination and changes in day length are corcelated with sea tem- perature in tropical shallow-wates, and both factors may affect sexual reproduction in marine invertebrates (Coma eta, 2000; Shpigel eal. 2004; Farhadian et al, 2014). Lodeiros eta. (2018) reported a good environmental data for mariculture ofthe oyster Previa stema at sites here large T: depressus were mostly collected in ths study (between, almar and Ayangue sites). They determined that temperature, salinity, concentration of seston and chlozophy/l a were highly variable all year Jong, and suggested that high temperatures during the reproduction period of P.stema can be the most negative influential trait since the dally temperature range was > 6°C (ald-March 2015), Higher inteaday temperature variation was observed during Jan-Feb (°C), Feb-Apr (up £0 6°C), and Jun-Jul (4.3°C). Variation during the test of year was < LC. Although, it is not clear whether the environment variables ‘mentioned here may contribute to the reproductive pattern in T. de- pressus, & bigh variation in sea temperature and non-uniform light conditions by turbidity occur. In addition, this variation is often ex acerbated by season. T, depress (as shortliving sea urchins) that inhabits shallow rocky shores off Palmar, and typically lives in a group with patchy distribu tion patter between $ and 12m deep (\drovo and Sonnenholzner, 1994; Sonnenholzer and Lawrence, 2002; Lawrence and Sonnenholzner, 2004). This distribution pattern reported to the long lived Strongyocentrotus franciscanus for increasing their fertilization suceess (Leahy etal, 1981; Levitan, 2004; Mercier and Hamel, 2009).2 Someone Vara et 95 39 46 53 60 67 76 “Time (days of culture) 2 89 oa 07 ° 06 $05 ° x 04 ra Eos B O02 a oo , ; Smal Medium ‘Lage em conuie Fig. 9. Specie growth rates (SGR) during slow-growth episodes by sae eate fies in jveniles ofthese urchin Thprewtes depress. Error bars denotes In the case of T.depresus, female's location in the reef and her prox rity to males in high environmental variable conditions are erucal for ‘the survival and production of new recruits, Nojima and Mukai (1985) and Shimabukuro (1991)fide Toha etal. (2017) determined that some T. graulla have a tendency to live in couples, while populations that showed no individualto-individual body contact were observed, even, with high density. These issues ace very important, since another in teresting finding was that atthe end ofthis study, we were challenged tw collect sea urchins in the field to evaluate their gonad production. the capture effort increased and the number of animals collected pro- sressvely decreased throughout three last years (between 2014 and 2017). This explains to us that their densities apparently have a high ‘variability at very. short time periods at intervals of perhaps between four and five years until the next recruitment event in 2018, This study TD (mm) SGR (% x day") Agwacnr 496 2018) 134-145 50,0 45.0 40.0 36,0 30,0 25.0 20,0 95 110 123 187 165 130 194 207 214 228 242 “Time (days of culture) ath increments in ie (ext diameter, TD) and specific rom rates in juveniles ofthe sea urchin Trpnewstes depress, respectively; 39) DPS, and cd) nt grey bars in the x axis mean rapid and slow-growh phase, Data are expressed as mean = se. (W = 50}. 100 %0 80 = 7% =o zs 5 4 ? x» 20 10 ° Embryo Larvae sivenile Stages ig, 10, Survivorship ofthe sea urchin Prpneutes depresss by stages as em bye, larvae and juvenile coincided with a similar natural event noticed in the Galépagor Ar. chipelago, during 2015-2017. Reported densities of T. depressus in Ecuador ate approximately “0.1 to 5.0 individuals per m® and vary considerably from year to year (Brandt and Guarderas, 2002; Lawrence ane Agatsuina, 2013). Based on our results itis well known that po- pulation densities of 7. depressus in cuador can rapidly diminish oF ‘completely disappear at monthly intervals, 42, Lanieulure Comparatively, the mesn egg sizeof the tripneustids echinoids, T Aepressus from Bevador (78.4 + 214m), T. verricoss from the Caribbean and Panamanian coasts (80.0 1.1 um) and. grail from,the Indo-Pacific (85.2 = 134m) seems different in size, but their planktotrophic larvae are competent to feed at 2.5, 2.0 and 35 day post-fertlizaion, respectively (Lewis, 1958; Lessios, 1987; Byrne etal, 2008). Lessios (1987) studied temporal variation in mean egg sizeof T Verricosus from different females colleted from the same locality on the same day. He determined monthly and interannual differences, and suggested that size of mature eggs isnot determined by reproductive state ofthe parental population. In a Tater study, Byrne etal. (2008) ‘demonstrated thatthe growth éynamic larvae of T: gratia larvae is not divergent under unfed and fed conditions (with no evidence of phe rnotypical plasticity in growth). They suggested that larvae maintain 2 nutrient storage buffer against starvation, which may exceed & days, a¢ a facultative feeding period, considerably longer than that of other ‘echinoids with similarly sized eggs, emphasizing that egg quality may ‘be more important than egg size ‘Our findings are important to understand the Ife history strategy of| 1 depress and determine their resistance to starvation, risks of mor tality, successful metamorphosis, and an optimal quality production of| juveniles, considering two factors, high sea temperature and low food ‘conditions; which are characteristic at tropical waters (Reitze! ct a, 2005). In our laboratory, we pre-evaluated the feeding of T depressus larvae (at 2 density of 1 larva per milliliter) with low concentration of microalgae (with five diferent species with < 500 cell m~). Larvae died in les than five days (data unpublished), This suggests that larval development T. depressus (with small egg) proceeds immediately after {he third day post-fertilization and the larvae eannot survive more than five days in very low food conditions. They do not have the ability to use maternal reserves (as endogenous energetic lipid reserves, part- culary tiglycerides) as was reported for T: gratia (8yrne etal, 2008), Inthis context, the tr-algal det used here (Tysochrs lutea = Ioschrsis albana, Chactoceros gracilis and Rhodamonas sp) at concentations ranging from 25 x 10° to 20 x 10* cells mI”? were adjusted by feeding rations to ensure apparent satiation forthe non-fcultative T. depresses larvae, This combined diet positively contributed to meet balanced nutritional needs in some of the essential polyunsaturated fatty aeids (PUPAS and DHA) for achieving the competence with ‘continuous growth performance, non-delayed metamorphosis, and no abnormalities, with high survival rates of larvae. Therefore, although ‘he final output in term of larval ssevival was high after rearing, but it ‘cannot be affirmed that this combined diet leads to success in sete ment. Further research is needed for this stage. With supporting data {rom larval morphometric, the larvae did not exhibit phenotypic pls ticity. Indeed, the phenotypic plasticity of the post-oral arm size (as Total Length) makes It good indicator of the morphologial devel ‘opment of larvae in response of the food type and availability (Gerathmann et al, 1992; Soars et sl, 2009). Gallager et al. (1985) suggested that larvae fed with Rhodomonas sp. produce the highest lpi content, as PUFA content (22:6n-3 and 20:5n-3), and high levels of ‘earbohydrates, which is « good physiological condition indicator and the capacity for successful metamorphosis. Therefore, the specific growth of T. deprescus larvae reported here could be explained by the ‘carbohydrates ingested (Brown etal, 1998), Other authors have con firmed that Rhodomonas sp. alone may provide the critical nutrients or ‘energy (for their higher digestibility) for echinoderm larval develop ment (Castll-Gavilén otal, 2018), ‘The larve of . depressus is intermediate temperature tolerant, with wide temperature range, between 23 and 29°C, and has a relatively short larval stage lasting between 25 and 30 days at 26°C. Our results agree with those of Rahman et al. (2008), who experimentally studied the early development to T.graila at Okinawa Island, Japan. Our re sults indicate that the optimum range of temperature for T. depressus was between 25 and 27°C for normal development. 43. Postsetlement and growth of jivenles {In our study, the high mortality in the transition between larvae and, Aone 4962018) 136145 juveniles is remarkable, which would be a major problem in the pro- duction seeds, and this represenss a limitation of our study. Therefore, four high rate of morality will be discussed based on the positive findings of Rials et al. (2018) who determined a good inducer for set- tlement, and high growth and survival rates Rlals etal. (2018) experimentally evaluated the suitability of if. ferent cues (treatments as uniaigal films by Tetraselmis marina, Nitschia sp, Oylindrotheca closterum, and mixed) as inducers of settlement and et after metamorphosis for promoting the growth as increase in test iameter and survival of juveniles of the slow-growth sea urchin Paracenromus lividus. They monitored metamorphosed individuals for 32days on these treatments and found one treatment (C.closteium) that showed higher survival rates (confidence intervals: 74-100%), igh setlement and growth rates (DPS): 73.4um /day). We used @ iret film of the pennate diatom Naviula sp for costing the tank walls asthe substrate, as inducer and diet for pose-settlement of T. depress. [AC DPS, survival was low ca., 10%, but the calculated daly growth rates was 60.0 1m /day. Rlals et al. (2018) determined thatthe released dissolved organic ‘matter, mainly fee amino acids, with higher protein conten, a lipid and fatty acids (hat represent concentrations at 14:0, 160, 16:1(0-7) 18:1 and 20:5(n-3)) and nitrates ofthe pennate diatom C.clstertum (as ‘unialgalbiofins) in P. tvidus, is nutritionally very important due is high digestibility, contributing on the development of the life stages fom the larvae to adults for different species of sea urchins (Salas {Garza etl, 2010; Lawrence etal, 2013; Watts etal, 2013). Taken this {nto account, we recommend an experimental test with C. closterium for improving general performance ofall these phases as an inducer film and as the primary food for postsettlement juveniles of T. depessus to ecide the proper moment (probably occurs for individuals between 3 and 6 mun TD, according to Onitsuka etl, 2015) to start a maezoalgal dict (Rials et al, 2018) ‘There are very few studies on fisheries ecology of fast growing echinoids (Lawrence, 2013). Therefore, our study contributes to the understanding of growth patterns for ruderal echinoids, which has been associated to a “bethedging” strategy, where large-size, high growth rate, short ie span, short time to maturity, ahigh rate of adult survival, fand high annual reproductive effort and output are adaptations to compensate for the low and highly variable survival rate of first-year juveniles (Stearns, 1975; Rotf, 1992), Lawrence and Bazhin (1998) pointed out that ruderal species with economic interest, such a8 T. de- pressus are more susceptible to stress. Therefore, we must coasider more ‘are forthe management ofthis species in the wild T. dpressus, because of its biological characterises, must be included as an interesting species for the global urchin roe industy, but based on aquaculture and restocking operations, and not asa direct fishery. In this study, early stage (as post metamorphic juveniles) T. de pressus (0.1 mm TD) opens its mouth with fonctional gut at 5 wk. of age after postsetlement (0.4mm TD). They continue developing thelr podia, spines, and pedicellarise at long ar the thermal (26-28 °C), salinity (31-35 pp0, pH (8.2), dissolved oxygen (5-8 mg 0, I~") and food (Padina durvilae) regimes are appropriate, In this study, we used selected dry cut and eaves of the brown macroalgae P. dura, since ft laboratory seale experiment it showed that t dominantly consumes P. durallaei than Sargassum ecuadoreanum, Gracilaria lemaneiformis, tava laenca, and Codium fragile (unpublished data). 1 is well known {hat some species can grow rapidly to total diameter of 1-2em in the first year (\loore, 1966) but T. depressus ean reach Lem at 152 days ler postsettlement. Many sea urchin species can reproduce in their second year of life, But T depressus ean reproduce after six months of life. These results are in agreement with other observations in the Philippines that T.gratila may spawn throughout the year (Fob el 2017) ‘Our results contrast with the study of Mille and Emlet (1999) who studied envionmental effects on early development of two slow- growing. sea urchin species (Stronglocentomus francscanus and S.purpura), focusing on ther larval feeding condition (based on food ration regimes) and different temperatures. They determined that larval food-rations significantly affect the development of trails in spines, Podis, and pedicellarise in juveniles up to and alter the onset of Feeding, which begins @ days after metamorphosis at 14°C. Juveniles from the high-ration larval culture were significantly larger after me- tamorphosis and grew faster than ones from the low-ration culture Other differences relate to time of development of jaws and pedi ‘ellariae. Juveniles from the highfood larval cultures were comparable in size to juveniles collected from the field at 14 days of age, but ones from the low-food larval cultures were much smaller. Here, we report growth rates of juveniles of T. deprescus which seems that the nut onal requirement for somatic growth was satisfactory with a high survival rate (90%). Nonetheless, the growth rate of juveniles at 95 days post-settlement was affected by an emergent and divergent separation ino three size categories (large, medium and small individuals). Where, the access of smaller sea urchins to food wae mosty limited by large ‘ones generating a chronic growth inhibition, sbert (1975) determined a strong positive correlation between growth rate and mortality in nat ‘ural populations of fast growing sea urchins that may have higher rates, ‘of natural mortality ee, given this, at 95 days after postsetlement of T.deprescus (juvenile culture ofthe same cohort of May, 13 May 2017) ‘we reported three subpopulations of new recruits with differentiated size structure (as small, medium and large individuals), which was mostly composed of small inhibited-growith individuals (60%) and with an evident retarded growth, Cannaism was mostly by medium-size ‘congeners to small-size individuals. Therefore, subpopulation of small individuals ean be drastically reduced. Sonneninolzner etal 20118),) reported recurrent cannibalism events by conspecifics in small sea urchins of Songyocentouus purpwatus under starving conditions. T. depresus i an appropriated species for aquaculture, because here we showed that when animals are early isolated (before 95-days after post-settement) into diferent tanks by size-lasses, intravsize cana Dalism does not occur, and then mortality is reduced to zero. In this sense, T depessus has 2 ruderal strategy for allocating more energy to production (for growth and reproduction) than to protection and ‘maintenance, as consequence, they grow rapidly and have great roe production at an early age (Lawrence, 1990; Lawrence and Bazhin, 1998), Therefore, they would have to have a predictable recruitment to persist (Ebert, 1975, 1982; Rowley, 1990). This pattern is contrary to ‘those commercial species with unpredictable reeruitmens, which must live longer to persist, and may invest more resources in body con struction - growing heavier skeletons, more slowly, while stil allocating resources to repeoduetion, such as the sea urchins feom temperate ‘ecosystems, eg, the family Strongylocentrotidae (Hbert et al., 2018). (Our results indicate that the changes in growth rate (as an alternated biphasic growth pattern, suchas, rapid and slow-moderate growths (as ‘was shown in igs. 8 and 9) in all three sea urchin size groups are not size dependent and this suggest thatthe changes are based on a cul turing system change. Finally, for the aquaculture of juveniles of 7 depressus, che brown ‘macroalgae P. duville can be applied as functional food (Vailas eal, 11982) that promote heath condition for growing and protection of their beneficial intestinal microbial flora (Fence! and Harrison, 1976; Lawrence and Lane, 1982). P. duit dominates inte- and subtidal flora, especially at sites where large sea urchins were collected. This ‘alga has a high content of ashes (relatively high concentrations of SO,~*, calcium carbonate as extracellular calcium precipitated in the form of aragonite and organic matter (34.4%), carbohydrates (pr marily mannitol as the major carbohydrate of low molecular weight, 442%) and total dietary fiber (7.6%), but low in. proteins (6.24 + 0.1%) and lipids (essential fatty acid lke omega-S, omege-6 ‘and PUFAS, with 0.69%) as usual for these macroalgae (Goceke eta. 2012), Therefore, our seed production protocol provided a good en. vironment to cultivate juveniles for commercialization and stock en- hancement. But further research on natural and formulated diets for Aioinre 4962018) 136145 juvenile sea urchins 7. depressus is needed, 44, Perspectives Due to the decline in ea cueurnber and spiny lobster fisheries, in terest has developed in Heuador for new fisheries (Wolff etal, 2012; Ccastrején and Charles, 2013; Alava et a, 2015), in order to divert fishing pressure away from traditional sheries. For example, in 1998, local fishermen of Galépagos requested the Autoridad Inerinttucional de Manejo ofthe Galipagos Marine Reserve (AIN-GMR, Resolucién No. (003-2002) for permission to open the fishery of T.depressus. The AIM GMR suggested realizing biological and ecological surveys of T. de- pressus to synthetize information of their population state in the Are chipelago price for establishment a nev non-traditional fishery in Ga- ipagos. Therefore, this initiative continues to be pertinent based on this newly reported information. T: depressus is occasionally caught for local consumption by fishermen on the Ecuadorian mainland coast and Galapagos (Luna and Darwin, 2000; Sonnentoner et a, 2013). So, there is interest in consumption and aquaculture ofthis native marine invertebrate species in Ecuador Aware ofthese conditions the Eevadorian Government developed a ‘numberof laws to regulate this sector. In 2012 the Acuerdo Ministerial # 458 was published for the regulation and control of aquaculture Teases, whieh could be awarded in areas with rocky bottoms where the cultivation of marine invertebrates may be technically feasible for re- production and restocking natural populations (LipesAlvarado etal 2016), Beside of that T. depressur hae a high importance as human food Recently, Sslas-Rojas esl (2014) reported that T.deprescus has im- portance for human health. This species produces antimicrobial pep- fides with a direct and stable activity against Suid herpesvirus type 1 and/or rabies virus, They have been characterized as new molecules with antiviral activity for designing new drugs from the coelomic uid ofthis commercial and edible sea urchin species Consequently, this study has developed a perfectible new culture technology for supplying juvenile seed ofthe native sea urchin T. de- resus or sea urchin aquaculture purposes in Ecuador. In other parts of, the world, there are producers who grow species of slow-growing sea urchins. For this reason they have to face high investments and require waiting approximately three years to achieve sea urchins of 55 mm DT (uch as Paracentrots lividus in Europe). In contrast with this, T. de- pressus isthe opposite, Itis a species of sea urchin of rapid growth, and therefore, the investment of money for its cultivation will be much smaller. Additionally in its first year of cultivation the animals will be larger in size with larger gonads Acknowledgements ‘This work was supported by the Secretaria Nacional de Educacién, Ciencia, Teenologiae innovacién of Beusdor (SENESCYT) for providing funds fo the project tiled: "Desarrollo de protacolos de domesticacién, para el uso sostenible de nuevas especies marinas para consumo de alimentos y repoblaci6n de baneos naturales” (grant number: PIC-2014- CENAIM-002). We are grateful to John Lawrence and John (Chamberlain for their comments and ercicisms. We also thank to Juan Carlos Navarro, Daniel Gonzélez and Manvel Gonzilez for coatsibuting photographic material t0 Fig. 1 References Sy telcos Uline ae. 82 Soman erat at steneck RG, Vadas, RL, Woody, D.A. Klee, 2, 2002, Stats and management of trot se chin shee. Ocemng Mat lal Aa Rev 40, 345425. Arcs, As Bab KR. 2016 Nubiecomposion a tise acy of nas of ‘shin Stmepttes yr Poe Chem. 197 (RA) 597-502 “aton an Vana ees the Galpags Mare Reserve Se thes Southampton Oceanographie ene, Uk po ‘ever ME Keaph, fh Lawrence JM Lae AL, 1996 Feed ae, sion ‘etn fects Valenciennes hire: Beamer od tural et. Sel Res 17, 1980-1590. Bn Mt Carers, P2002 Enos Mars: Data Edge as), Reseva sin de Calipags Linea Suse dela Boveri Funds Chats Drwi Serio Parque Nena Glspago, Sata Cu, Clipper, Cinder pp. 394-418, own MUR, Mecauand ALA. Kowa 1988 The atonal alae four "isvalan moll stan edo Pai eyter Cassoea ge rt. baer yt Srl ALA Pr, FAA 2008 Nun alge hn ae cau ryt gr, eet el 22 te ‘Optimising eng lets nthe Se ehh Pare as ite 6 promot uaciretveieation Agee #90, 251-259, ‘cutis ML Cheer, A 215 groin bere oomanaeren rough caste Sued patil anopeer te Calapagr Manne Rene Mar Pai 38. 235-245 ‘chen CP. Chang Rt, 2012. Reade perc ofthe ea ein, Teper ‘Palo awa compared ty the eons I ner. Reprod 3.308313 cont RRs Me Chi J, Zabel M2000 Sasol i eos Dene eco ‘remell RL, 201, Detling chinederm eutce fr enmnpon an std ‘incre in he Cadbean i Lvs, R Sci Sth} A Renal Sellen they Tor the Wier Cabbean ASesing 1 Peasy and Sisal FAO Eegonl Tene Wakchop. 18-21 Oxebes 2010, Rago, aniee FAD Fetes and Aguaalere Proceedings. Ne. 19 TAO, Re po. =I, ‘ys MD baton 12, De Weel Mace M2012 The devtlepen a a formated Teed containing iva (Chloaps) to ponte apd rm and embed prose hon of high qi Toe inthe sea rehi Tpetes lean) ee Re Devin ALG, 2002 Land-bred ecole: novel eye to cure dul se chins, Ie ehata Matranga, Smolesies, 2 tas) The Sea Urehn rom base Blatogyt Agee. AN ble Poshes Sets & Penge, Le ‘er TA, 1975. Growth and meta of oslarvlechneds. Am, Zo, 18, 75.775 Ber, TA 1962 Lnogeiy a ie hry date yl ae chi oer TA, Hat LAF, Bodin, J. Barcham, D. Brea, D. Cason, HS, Cars, Ni, ‘ele JH, Chis, IT. Cleese S, Das, Ket PO Dios, JD Degens D0, Eagle J, Ets, 1A, Groth SD: Gripe, Ne aim Peder, Kos ieenindee Sc, Jurgens LJ Kats 9 Keer MLC” Kane une, Da; lee Le Lighten, bi, Monae sete, Gin, igen. Palle, 1S, ale, 0, Pas, 1, Poel 0. Rogerseoet tn ehveee, 5, Shon, £0, SononboenesJ1, Taig, 1K, Vana. GR, Watan, JE! Westman, 82, Willams 19, Yabimsbye hang Z, 2018 Se grow ‘isda 19bs-2016teloy 9,701, bse én eg ooow/ec alo 4g), 8D brown UP Kings Avs Wat Sa amen, A, 212 Cronth of vende een searching, Sroncenens dracon fede edith 98 Protein evel compare with + macroagal det sha commer sblone fed J eed Agus Se 43 158-173 ‘0, 2016. FAO yeabeck I Fey and Agus Stine 2014 Rome Fadian ©, Yaea shad. hy 008 tec fay temperate, tn Teri nd light egies on predean roth and repredcte patel ‘Apocpos desis ray PS 15. 304 ‘eotel Tlkstbas P1976 The iano acral gaing and mines eg i: he dconosn oper ci fara J, an, Bacewel, oxo Foster MS. econo, tune, Wadsworth, Kimball Babs Weiner M, Rirmeneodgus Re Hterninde Carmona. Verge zen, Ai, Schon, 5 Sele Di, 2iU7 Diverse e hori ar! de na ernie Lashom Sagas ard el all e Calor. ‘Gallager, SM. Mana, R, Ssh, G.C, 1986 Lipid san index of growth and viability in hee specie of ae lata Ages 5, 8-103 ‘iudice 197s Derclogenal sag fhe Sea Ure Embry. Acad res fe ‘iyen PW, 2008 fone Web Srear ane Dye of Eaner Tope Pace Cord Rete anand and Galipaes inden lanaan, TR, Bratch, a ee), ood Web en the Dyas of Matin Rees Oxford erty Pres, pp. 185208 \coete,F Heobar Hale 6.2012, Chemie! campeon of Pain oman ese, Dita) ram Juan Fenn Aocipelaga Cle Re ‘rasan Phy Spe ch Jango M2005 fants growth med with in ‘especie compesion apie to «see cia Fearne lis. Ca, eta Se 257-246, ‘Aquarians 496 2018) 136-145, "008 Thea pth tina i maces and omen Heoehion, M, Hayes, M. Mullen, AME, Fenelon M, war, ®, 2017. Face prosein ‘ply ed cand sues ab ats Lafentng «sable ql get MJ, Wlamson, J. de Nye. R, Kleber. 8, Seber, PD, 2006 Laval Sieman ofthe common Atal son techn Hehe enreprana nf {one co Seer fram the aac of earaline alae Oeclogia 49, 09-519, icoo,FSontanbstnr, J, 1984 ibn y succeded A gui 108) y Dad conan A Agus 185 (Eoknoderma Eeoles «8 Pina as rales, Porous Navona Machi Mana, Leader Report eco Se ‘nvetiacign No. 72 Facultad de Genes Nature, eal de Bl ntesided de Guay pp. 2. tong AD. Wtman JB, 200 ostve lc of amu nei negate ecto Janes P edie Clason tno, 6, berarzuds, , Slane R, Zee, achad Jo 2018. Se rhin Serer agement ad pkey rei (ety Nea ef the URCAIN project), Rafa Repert192016 pe ane taka T1986 at cds ep of fond pres ay see rein. ayaba TT Hawa, Kae, ¥, Kawabata, Ke, Ota, Watanae,T, 2012 le fir temperate earn, wing deepsea wae, o8 fade mars Son fhe shartapned en rn Sranglacemarmomed Ress olinie Fh sr Pa, 1231272, eesing. J Ha KC. 998 Ree of atest ed stat of weld se chia bere Sintec research eperng the ARRIVE iis or teparting anal sere asa 8-3 lawrence. JM, 2990. The eect of es and dstrbenee on Feinodems Zea Se 7, Lawrence Ly 2012 Chape 2: sen whine ory stg: Lawrence 2 ‘Ga Se Ue: logy ane Besos. Hele BV, pp. 1523. tawrece J, Agana, ¥ 2018. Chap 52 Trott Lawes, 52), See his lala ad Eesogy. here BV. pp. 891-07 awe JM Buin AT Ltr ees pte of a chine Lawrence. JM, Lan 1M, 962 The latin af ret by ponetamorphie echinoderms. sangah Lene, Jt (Es), Bhiude Nuon Buena, Rotem he Neca, pp 3316571 "chines ind hlatheris in Galpagor Ie caer T- Nebel J (Ps), eknedemne, nchen roc leh ne Fined Cat Tayler and anc Grp, eoéen yp 230244 lawrence Jie Lawrenge, i Was $A 2933, Chpter 9 fing, gion and ‘gest of ven wns a Job, ML Ba), Devapala quae ab iter Stance, Eee pp 195-104, aly, PSs Houghbvas, BR, ben RI, Dasiion, EEL, 198, Syackony of 2 ese erty. anne ad wl popuins oe papl se een ‘Sroncowrn para) xp. Yok 9,722. Lusi, fi 1987 Tenor and patil variation Ing of 13 Panamanian eh sis LA Hane, J, Robeon, DA. 2005 Phylogeography a he pascal ea hn Fg cnn ptr of pope ae een se Lust, Bi 2004 Detydependens el eletin in external fret variance hae ced femal ean sucess lon the ests ae sper ots fx relent i ie cin Senne fe, Na 1 erlation sees nthe sa ch Semper franca, slg) 73, ew 195TH of he ope i pat cee sce In Chine Bll Auneule Ass Can 18, 3637 Laden C- Rage Psat Dy Marge A Reva J eles, Lees. ‘ena tema (Goi) pended eaten ropa Eatern Pe Auenc of eoveomental far, heaae Rar #9, 822-838 LUpetivacede 1 Rul Wy Monaye, E2316 Ofere auacaledeslopmen in candor int Ren Ede 21s ‘hu blanca Inponate deems (ichinoeermat:ehnade) ess Glipegar eects Tes Univernded Sap Hane de Quite, Qu asad a, 1988. Compeston and Dato ofthe Cel Ese Pace "ehaodens. Teele Reps, Munbes 2 Naural str seam of oe Angeles Manabu E2007 Repo: f Stay of Fubuie Products Dutibsen and Cansumption “Sue Sema Cre ian EA/Paberis an Aguas nena Co, Mater On, 3, AesDusiva, AML. LenneCody, CE, Mande, MN, 2015. The ‘sevaeran areal ery fr arg plage species conposton and spat2 Soman erat at tempo jamie: Pla One 10, «0135136 eperan BE 96s, Contbutin’ othe Bog af esx urn Tyee ven er Bl Mar S15, 28-248 Mere A Hane, 17,2009, Coupe 2 Capetgeoes a Meer A. Hae J (tas), Endogenous and Exofnmes Contra of Gumelognes ad Spang lcd, iver Me Cot, AC, Jone, MB, 2008 Bplaton an envertion of ‘hinodems. Oceanogr Mar Bil Ar Ret #7, 391-28 Der BA, He RB, 199 eelpet of nny mecamerpbosed ave see ‘teh (5 rewucane and purpura marholgy, te lec of epee della drain sedate or deeming ap. a Bl 5, Mare P1966, Te te of aber banda rth ndestors nies fe se ‘chin Meiners enya Rs S629, 52-5, big, WA. 2005. Tsun othe lets f sane ad una pedo tee Poducon of the aes rh Ieee) Kenyan er el fons tydoiolaga St, 57 shin sks Daca 300 eof easnaty and opsaian desi an he "eprducian a he Indo-Pee ec Elona re i Kenyan cree ‘Oke TA, Bake, Sor AB, Bupamant Ri, Cavpia. Me ger C.J. pins, Efe i, Carte, LE” eck Gk Saatar,S,Sepher Sy Toran Wall. P2004 opie model ef aliags subs ocky te for eaatg Miners and eoservaon satis. Bak Mode 172, 383-40 ‘ona T Ny Unum, Ue, 2015 Diary sat a the fv ea ‘rehn Seogfoecim ne neaed wit tne development of Sgene ym Mar ha 162 69-86, res tbls 3, Cats, D 2015 Se bin Peraenra vu lave — clus om cryopreserved enbgon Agence 87, 386-90, Pease, J, 1974 Reproductive pars of Wpies ela hee pets a ‘agement ofthe white sea urehin, Dpmenses vencoss, in the eastern Caribbean, FAO Rsheres and Agunutire Cet, 86 1056 Rone FAO, 9p 8. ‘Pula RS.V. 2017 Diveseaton ia aghacuce specie ated pes adele ‘eens inc avy, 8. Sta, Caraee J, Bevis Me Bae, Da (Es), Peon for Rguestre Dersler Te inporans of Cate Change at ‘thr Divers FAO Teche! Workshop, 2:25 ne, Rome fa pe 1236. Rahman, 6, Tachija Uehara, 2008 Pes of temperate ohana, tet ep ayia es hn, tn abman MA. Yor FM, Ache, A, Samadin MN, Amin SAN, 2012, Timbyon aval nd eas joven developmen’ ofthe wpe sea chin, Stns ‘hae Eehinocermat: Bdgoen) Ser Woe Jo hip ea. er 10, ‘ete AME es, CAC, Hela A, Cavare 2D, Metre, LR, 2005. Te ae [ial DR PCa Coxe, N Costas Dy 2018 Ingen of steer ted tiv ot jovnes f Prartarons iar, Aguas 48, 16-20, ‘hin Is Lawrence J Gan, © (ba) Sea Urehin Mahe ad Sela. Dustech pubbessens tances USA, pp 382-957, tA, 1992 The rain a he ie eres Theres and Analy, Chapan and ial Landon, New Yrk, 9p. 1-55, Roviey, RJ, 1960 Newly ed su chine nal ed an cin ben ground: aaron of gh a moray Mat ea Progr Se, 2, 20820, ‘sdecation? Rev. Bik. Trop. 65, 811-22 snal-Grta hy Psst PooedMugiet MA. 2010, Culture of bebe dons lect of dived expe carbon on mennerpas ef x wissen tein, Fecha un (er. Agane Res #1 35-58 saaroj Maver Romer, Asso Plma, By Acevedo, , lana, ‘nsletn A 2014 The cna ld af sen ehin Trp deprsae ‘Sows envied atity aut Sd bepestus type 1 (SH ana iss ‘Aquesnne 496 2018) 134-145, (2). Poh Shel een 36, 158-163 shin Maceo Po, 1987, Th dec ofthe ss urchin, Tents vn ‘rea fry of Barbar savy of hee ad canines Mar he Sebel Bon J. Macey, BM, 2015. es of deren ong eds ad hl ne nia eres nthe espn cha Tyree simabokar, 5.1991 Teper risen phi) Sok, S., Kaas, “Torani Toma, T, Yamaguen, M (as), Acts t Topiea! Aes Ma Shab, Toko, pie pp 8-228. Stig Mee 8 Mac $Lepuch 1208 The lc peti ane ‘eucuare 282, 345555 Som Nh, Prowse TAA, Bn, M, 2008, Ovi a phono plasty nec lie spl aia ev pe esa a Ea Pg Hewat B Beattopie 36, 25-5, Sonnenolne J, taller, lash 1.8, 20114 Food webs an bing ee paee ‘tom of th ee ct sigs in Cage Eig 32 Senteholne 3, Monase.aceis,, Sesry-Bemal R201 le of ere hoe det and na so on she svete rom of een Sepia pri mtry eprint sos Ppa Sencenbte, 1, Brandt M, rani, V, Hear, A. Laigs M. Gaba “var, ¢, 2013 Chap esinader of ted Alvarado 3, Sle ‘Mari, Ai, Eeinogm Revech and very In Ltn Ane, Sig ‘eg. bein zeberg pp. 00-99, Sonneholne Ji, Seary-Setal,H.Puncats M2017 Te patente propagation tthe commercial ser camer Iho cs dg. 1679) By nau ‘Saver fin Reg St Mat Sh 3-40 SeteloCaas, CapabMage, A, Rodger Zaagons, PA. Safar, FA ‘osrgce roo, AP. 2016 Scie sd enone elec ah e+ een wince coy ha ne Steam S197 lifer ac: reve of eas, QR. Bl 1, 47. Serbia, RR, Feat, Sathoann, ALF, 192, Htrehone develope peli es cis ad pans fer sluts of sotetingahae sunt Chlng #5 2015: Chapter 2 Use and explain of chine I ron, ibs Fdoy SD (es), Ehinader Agere Wiley So ie, 9p 3545 Pointe Then, 1989. The eaon bene fod rtin and reader in he ‘est AiiAy Smo, Ts Wed in, Rae Agger, A ‘anaes 1758) (eile Enteral). Ocean Le. 10. nice 5 State 8 yin M2009 A boars phy? Elgin ee vedas is Fenehel Foggen 16,1962 bale sor one ses in, “hecho vargas an the la segras comets ofthe Maia aye Niarngin Ast hot 14 1994128 Gi), Developments ia Aquaculture and Fisheries Science. levies, pp. 135-169, ‘lvl ctr fhe ee ea rch, rpm owrcons (thine chines) ball Mar. 177-190) Wall Scuba A Cat M201, A reve ey fr he motoring 8 ‘orn ly itvsays, Oy A, UI Rapin A, 062 Coed