Material and methods Directory UMM :Data Elmu:jurnal:A:Aquaculture:Vol185.Issue1-2.May2000:

1. Introduction

Increasing demand for sea urchin roe has led in the last decade to over-fishing natural Ž . populations Conand and Sloan, 1989; Le Gall, 1990 . Several possible solutions have Ž been tested: reseeding natural habitats with farmed juveniles Agatsuma and Momma, . Ž 1988; Gomez et al., 1995 ; mariculture Fernandez and Caltagirone, 1994; Fernandez, . Ž 1996 ; raising sea urchins in immerged cages, alone Fernandez, 1996; Robinson and . Ž Colborne, 1998 ; or with other animals polyculture with salmons notably, Kelly et al., . 1998 ; and finally land-based, closed-system echiniculture allowing control of each Ž phase of the echinoid biological cycle Le Gall and Bucaille, 1989; Le Gall, 1990; . Grosjean et al., 1998 . Whatever the culture method, temperate sea urchins exposed to natural conditions of temperature and photoperiod have a seasonal sexual cycle that restrains the crop to only Ž 2 or 3 months per year Byrne, 1990; Lozano et al., 1995; Fernandez, 1996; Spirlet et . al., 1998a . Many experimental attempts have already been made to manipulate the gonadal cycle by modifying these exogenous parameters. Most of them were successful Ž in obtaining out-of-season gametogenesis Leahy et al., 1981; Pearse et al., 1986; . McClintock and Watts, 1990; Walker and Lesser, 1998 . However, the experiments were always run on specimens collected from the field, and hence, sexually in phase to begin Ž with. Moreover, the shift in the reproductive cycle took at least 7 months Walker and . Lesser, 1998 . Ž . With adult sea urchins originating from cultivation see Grosjean et al., 1998 , the first problem is to obtain individuals in phase with regard to their reproductive cycle. As gonads also act as storage organs, it is possible to induce the consumption of most of Ž . their content by starving the animal Lawrence, 1985; Pearse and Cameron, 1991 . This Ž . attribute, experimentally observed, was successfully used by Spirlet et al. 1998b to study the impact of feeding strategies on gonadal production and to obtain marketable Ž . sea urchins all year long see Grosjean et al., 1998 . However, it has not been used yet Ž to determine the best values of exogenous parameters mainly, temperature and photope- . riod for optimizing the production of marketable gonads. Several criteria have to be considered in echiniculture. The reproductive state of the gonads has to be in the correct range, from stage 3 to stage 5 according to Spirlet et al. Ž . 1998a ; the gonads preferably need to be fleshy and firm without an abundance of gametes spilling when they are consumed; and, as previously stated, they also need to be sexually synchronized to ensure a large quantity of exploitable individuals all at once. Ž . This study has two objectives: 1 to determine the influence of the two major abiotic Ž . parameters temperature and photoperiod on gonadal growth and gametogenesis of Ž . cultured sea urchins in a closed-circuit facility; and 2 to control the reproductive cycle and determine the best combination of temperature and photoperiod to obtain individuals ready for marketing as soon as possible.

2. Material and methods

The Paracentrotus liÕidus used were laboratory reared from two successive fertiliza- Ž . tions Le Gall and Bucaille, 1989; Grosjean et al., 1996, 1998 . The original stock was Ž . collected on the rocky shore of Morgat Brittany, France . The individuals selected were Ž . Ž . 30 2 mm SE of ambital diameter excluding spines and aged 2 years and 3 months at the beginning of the experiment. Indeed, this size class presents the best Ž . gonadal index GI and therefore, a priori, the best potential of growth for the gonads Ž . Jangoux et al., 1996 . The experiment was conducted in four specific rearing structures described by Ž . Grosjean et al. 1998 . They consisted of three superposed toboggans overhanging a Ž reserversettling tank. These experimental structures were thermoregulated one tempera- . ture per unit and each toboggan was isolated so that photoperiod regulation was independent. A centrifugal pump insured circulation of the water. A water renewal of 200 per day maintained seawater quality during the experiment. Ž Sea urchins were starved 2 months beforehand i.e., until most of the nutrients . present in the gonads were resorbed to make sure they were sexually in phase at the beginning of the experiment. Starvation occurred at the lowest temperature used in the Ž . Ž . study 128C , to prevent subsequent mortality Jangoux et al., 1996 . Before initiating the experiment, five batches of six individuals were weighed in Ž . water immersed weight, IW, see below and dissected for control measurements. The Ž . immersed weighed was then standardized Jangoux et al., 1996, Grosjean et al., 1999 as shown in Eq. 1: 2.80 y 1.00 SIW s IW 1 Ž . ž 2.80 y Mdr1000 with SIW being the standard immersed weight in g, Md the water mass density in grl, estimated from sea water temperature and salinity at the time of measurement and 2.80 Ž the mean density of the sea urchin test allometry between the IW and the dry weight of the skeleton calculated on 356 reared animals after digestion of the soft tissues with . sodium hypochloride 10 under gentle agitation . The gonads were extracted and fresh weighed. One was fixed in Bouin’s fluid to be analyzed histologically, while the remaining gonads were dried for 48 h at 708C and dry weighed. The value was corrected for the missing gonad. Ž For the experiment, eight further sets of 30 individuals five replicates of six . individuals were subjected for 45 days to eight treatments involving combinations Ž . Ž between four temperatures 128C, 168C, 208C and 248C and two photoperiods 7 h days . or short day period, SD; 17 h days or long day period, LD . Initial immersed weights were determined for each batch. From the first day on, the individuals were fed ad Ž . libitum with extruded food, in the form of cylindrical pellets 8 = 15 mm . This food Ž contained mainly wheat, fish, soybean, and minerals see Klinger et al., 1998 for exact . composition . New food was distributed every other day after leftovers from the previous distribution were collected. The latter were dried and weighed to estimate the Ž . quantity of ingested food ingestion rate . Additional portions of food were placed in the same experimental structures, but away from the echinoids, and treated similarly to estimate the loss due to their degradation in seawater. The total ingested food was calculated with Eq. 2: F s kÝ P y Ý L 2 Ž . i i with P being the portions distributed, L the leftovers, and k a correction factor that Ž takes into account the partial degradation of the food in the water k was estimated for . each treatment . At the end of the experiment, the immersed weight of the echinoids was determined Ž . prior to dissection. Somatic growth SG was calculated as the difference between the initial and final dry weights. The initial dry weight was estimated from initial standard Ž . immersed weight IW . Eq. 3 shows the relationship between the SIW and the dry Ž . weight of soma Grosjean et al., 1999 is: DW s 1.68 = SIW q 0.21 3 Ž . Ž . soma where DW is expressed in g of dry weight and SIW is in g. soma Ž . The gonadal growth GG in g of dry weight was assessed as the difference between the final measured value and the initial value estimated from the control batch and corrected by the following calculation in Eq. 4: GW b GW s SIW 4 Ž . iniŽest. ini SIW b where GW and SIW are the values for the measured initial batch, SIW and GW b b ini iniŽest. is the estimated dry weight of the gonads in g when feeding starts. Ž . Hence, GG is calculated as follows Eq. 5 : GG s GW y GW 5 Ž . fin iniŽest.. The differential allocation of resources to soma and gonads is evaluated by means of Ž . the final GI expressed in dry weight Eq. 6 : GW GI s = 100 6 Ž . d SW q GW where GI is the gonadal index in dry weight in percent, GW is the dry weight of the d Ž . Ž . gonads g and SW is the dry weight of the soma g . To compare with other studies, Ž . Ž . wet weight of gonadal index GI is also evaluated Eq. 7 : w GW w GI s = 100. 7 Ž . w TFW With GI being the gonadal index in fresh weight in percent, GW being the fresh w w weight of the gonads in g and TFW being the total fresh weight of the sea urchin also in g. As a reminder, total fresh weight is independent of the fresh weight of gonads since the volume unoccupied by the gonads is replaced by coelomic fluid of equal density while total animal volume does not change. As the SIW is independent of the gonad weight and much more accurate than fresh weight, the TFW is evaluated from the Ž . allometric relationship presented as Eq. 8 Grosjean et al., in press : TFW s 4.95SIW 1 .05 . 8 Ž . Ž . Fig. 1. Complete gametogenic cycle of P. liÕidus. In closed-circuit cultivation standard conditions , the growing phase circled by a dotted line is actually by-passed: the gonads tend to start gametogenesis directly once they have enough nutrients, storing them only occasionally. The maturity stages were diagnosed by histology and classified following an eight Ž . Ž . Ž . stage scale Spirlet et al., 1998a . The maturity index MI Eq. 9 of a batch was calculated as: 8 w x MI s C n rn 9 Ž . Ý i i 1 with C the maturity coefficient going from 1 to 8, n the number of individuals i i presenting that coefficient, and n the total number of individuals in the batch. Table 1 P. liÕidus. Two-way ANOVA analysis of the effect of temperature and photoperiod on somatic growth, gonadal growth and food ingestion SS df F-ratio P Gonadal growth Temperature 44.400 3 66.313 - 0.001 Photoperiod 0.736 1 3.299 0.079 Temperature=Photoperiod 4.004 3 5.980 0.002 Error 7.142 32 Somatic growth Temperature 73.123 3 53.857 - 0.001 Photoperiod 2.473 1 5.464 0.026 Temperature=Photoperiod 3.268 3 2.407 0.085 Error 14.483 32 Food ingestion Temperature 537.452 3 34.477 - 0.001 Photoperiod 0.335 1 0.064 0.801 Temperature=Photoperiod 37.904 3 2.431 0.083 Error 166.281 32 As no difference was noted between males and females in previous studies on wild Ž . populations of P. liÕidus in Morgat Spirlet et al., 1998a and cultivated specimens Ž . Ž personal observation , the data were pooled in all analysis Mann–Whitney U-test, . P s 0.05 . The treatments and the effect of temperature and photoperiod were compared using Ž . two-way ANOVA and Tukey test for temperature t-test for photoperiod , food inges- tion, for somatic production and gonadal growth. Normality and uniformity of variances were ensured, respectively, by x 2 and Bartlett’s test. As distributions were sometimes asymmetrical and could not always be considered normal, one-way Kruskal–Wallis and Mann–Whitney non-parametrical U-tests were more appropriate for the gonad index Table 2 P. liÕidus. Kruskal–Wallis and Mann–Whitney U non-parametrical tests of the effects of temperature and photoperiod on gonad water content, GI in dry weight and GI in fresh weight Water content 2 Photoperiod n Rank sum Mann–Whitney U-test P x approximately df SD 20 376 234 0.358 0.846 1 LD 20 444 234 0.358 0.846 1 Ž . Temperature 8C n Rank sum Kruskal–Wallis test P df 12 10 344 31.26 - 0.001 3 16 10 231 31.26 - 0.001 3 20 10 190 31.26 - 0.001 3 24 10 55 31.26 - 0.001 3 Ž . GI dry weight 2 Photoperiod n Rank sum Mann–Whitney U-test P x approximately df SD 20 443 167 0.372 0.797 1 LD 20 377 Ž . Temperature 8C n Rank sum Kruskal–Wallis test P df 12 10 56 30.069 - 0.001 3 16 10 224 30.069 - 0.001 3 20 10 199 30.069 - 0.001 3 24 10 341 30.069 - 0.001 3 Ž . GI fresh weight 2 Photoperiod n Rank sum Mann–Whitney U-test P x approximately df SD 20 406 204 0.914 0.012 1 LD 20 414 204 0.914 0.012 1 Ž . Temperature 8C n Rank sum Kruskal–Wallis test P df 12 10 58 22.594 - 0.001 3 16 10 275 22.594 - 0.001 3 20 10 217 22.594 - 0.001 3 24 10 270 22.594 - 0.001 3 Ž . 2 Ž . GI . Watson’s U -test was used for the polar transformed values of MI Zar, 1996 . The factor we defined as ‘‘temperature’’ is in fact the combined effect of temperature Ž . and rearing structure. Indeed, the number of structures available four did not permit replicates. Thus, although the structures were identical and the seawater was the same, the effect of temperature cannot be technically dissociated from a possible effect of the structures.

3. Results