The results of the study to standardize the larval feeding regime, for the mass production of sea cucumber juveniles through hatchery system are presented.' Auricularia larvae after 48 hours of fertilization obtained from the induced spawning of Holothuria spinifera were used for the experiment to ascertain the effect of feed concentration on larval quality, growth, survival and development. The flagellate Isochrysis galbana at O,1,2,4,and 8 x 04cells/ml were provided as the feed for a period of ten days. The growth rate of the larvae was more or less similar in

1 x lo4 and 2 x lo4 cells/ml. However the percentage of survival, rate of stomach growth, percentage of late auricularia developed and the occurrence of normal symmetrical shape indicated that 2 x 104cells/ml as the optimum larval feed concentration for the species

Asha, P S

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J. mar. biol. Ass. India, 46 (1) : 80 - 86, Jan. - June, 2004 Effect of feed concentrations on larval growth, survival and development of Holofhun'a ( Theelofhun;?) spinifem Thee1 P.S. Asha Research Cenfre of Central Marine Fisheries Research Institute, Tuticorin,Tamil Nadu- 628 001, India Abstract The results of the study to standardize the larval feeding regime, for the mass production of sea cucumber juveniles through hatchery system are presented.' Auricularia larvae after 48 hours of fertilization obtained from the induced spawning of Holothuria spinifera were used for the experiment to ascertain the effect of feed concent~ation on larval quality, growth, survival and development. The flagellate Isochrysis galbana at O,1,2,4,and 8 x 104cells/ml were provided as the feed for a period of ten days. The growth rate of the larvae was more or less similar in 1 x lo4 and 2 x lo4 cells/ml. However the percentage of survival, rate of stomach growth, percentage of late auricularia developed and *the occurrence of normal symmetrical shape indicated that 2 x 104cells/ml as the optimum larval feed concentration for the species. Key words: Effect of feed on sea cucumber larvae Introduction vae of S. rnollis was got reduced. Rearing The availability or concentration of feed plays an important role in the larval growth and survival. Microalgae, espe- cially the nanoplankters are being pro- vided as feed for rearing the larvae of sea cucumbers. The algal concentrations of 2 to 4 x 104cells/ml were indicated as the optimum in larval rearing of Holothuria scabra, H. atra and H-spinifera and Stichopus japonicus, (James et al., 1994; Battaglene,l999; Ramofafia et a!., 1995; Asha and Muthiah, 2002 and Ito, 1995). the larvae of H.gcabra at concentrations of 1 to 8 x lo4 ceIls/ml, Morgan (2001) also observed that high algal concentration inhibited the larval growth and develop- ment. Though the larvae of H. spinifera were successfully reared, the effect of algal concentrations on larval shape, growth and survival have not been attempted so far. Hence experiments were conducted with larvae of this species at different con- centrations of Isochrysis galbana and the results are discussed below. In none of these studies, the effects of The author is thankful to Prof. (Dr.) various feed concentrations on the larval Mohan Joseph Modayil, Director and Dr. growth and survival were attempted. But M.Rajagopalan Head, FEM Division, Archer (1996) stated that at high algal CMFRI, Kochi for their interest and concentration, the ingestion rate of lar- encouragements, to Dr. P.Muthiah, Prin- Effect of feed concentrations on larwal growth of seacucumber 8 1. cipal Scientist, for his guidance and also by observing hydrocoel, stomatocoel and for correcting the manuscript, and to Sri. lateral folds as observed by Smiley (1986) J.X. Rodrigo, Technical Officer, TRC of and Dautov and Kashienko (1995). - CMFR1 for providing the micro The mean size and number of the lar- feed. vae from 4,6,8 and loth day were recorded. Material and methods The mean differences in the size and Auricularia larvae, of 48 hours old and number of ten day old larvae from the having an initial size 362.k 8.4pm (Mean initial values of two day old larvae were + SE), were utilised for the experiments. considered for each treatment in the one They were obtained from a spawning way analysis of variance (ANOVA). The experiment carried out in January 2002. differences between treatments were The larvae were reared at '/, ml in 3 litre tested for significance.. by multiple com- plastic aquarium bowls containing sand- parisons using SPSS 7.5 programme. filtered seawater of salinity 34.8-35.5 ppt Results and discussion and temperature 2B°C to 30°C. Seawater High survival rate of 73.3% was ob- was completely changed On days . served for the larvae fed with 2xl@cells/ and the quantity On other days. ml of Igalbana followed by 58.9% for 4x10' On water the cells/ml and 52.6% for lx  1O4cells/ml on larvae were retained in a 40pm sieve and the loth day. The survival rate of the lar- transferred to 3 1-glass beaker. After vae fed with 8x104cells/ml gradually through mixing, lml sub sample was taken decreased and came down to 25.7% on in a counting chamber for estimating the loth day. The lowest survival of 6.3% was survival rate. For ten larvae, the total observed in the control (Table I). length and stomach length were measured - - using a microscope fitted with a microme- ter. Then the larvae were transferred to their respective bowls. The micro algae I. galbana was provided at concentrations of 1,2,4, and 8x10' cells/ml as feed. No feed was given to the control larvae. The maintenance and mass culture of the al- gae were carried out following the serial dilution technique (Gopinathan, 1982). For each concentration, triplicates were main- tained and the experiment was conducted up to loth day, as the larvae metamor- phosed into non-feeding doliolaria stage. At the end of the experiment, the larval stages in each treatment were also noted On the 4th day, the unfed larvae showed a growth rate of 83.6 pm/day, where as, it ranged from 90 to 158.5pm/day in other concentrations. On the 8th day, maximum growth rate (80.5pm/day) was observed for the larvae fed with 2x10' cells/ml, followed by 77.9 and 69.4pm/day in 4 and 1 x 10'-cell concentrations respec- tively. On the loth day, the larvae reared in 2x104cells/ml registered a growth rate of 59.5 k1.9 pm/day and those reared in lx  104cells/ml showed 59.1+1.9pm/day. The larvae fed with 4 and 8 x 104cells/ml showed a growth rate of 54.8k7.6 pm/ day and 50.3&6.0pm/day respectively. The Table 1. Mean survival percentage (n=30) of the auricularia of Holothuria spinifera at 0, 1, 2, 4 and 8 x lo4 cells/ml of Isochrysis galbana (mean + S.E). Days Survival rate (%)at different concentrations of 1.galbana (mean + S.E). least growth rate of 19.0+6.6 pm/day was Table 3. Mean increase in length and growth rate observed among the control larvae (Table of stomach (n=30) of auricularia larvae of H. spinifera at 0,1,2,4 and 8 x 104 cells/ml 2). of I. galbana (mean + S.E). The highest mean stomach length in- Feed Length increase Growth rate Concentrations on loth day (mean -c S.E). crease of 128.4pm with a growth rate of (Cells/ml) (mean + S.E). (~m/day)  16.lpm/day was observed in the larvae ( ~ m )  fed with 2x104 cells/ml. In 1x104 cells/ml, 0x10~ - 6.9+ 1.4 - 0.9+ 0.2 the stomach Iength increase was 100.4pm 1x10~ 100.45 1.9 12.7-c 0.2 with a rate of 12.6pm/dayf whereas in 4 and 8x104cells/ml, the length of stom- 2x 104 128.4+ 0.53 16.15 0.1 ach increase was 77.7 and 70.7 pm with 4x104 77.7-c 3.5 9.7+ 0.4 a growth rate of 9.7 and 8.8 pmlday 8x104 70.7+ 4.2 8.8-c 0.53 respectively. The unfed larvae, due to the shrinkage of stomach showed a value of larvae fed with Sx104cells/ml concentra- - 6.9 pm with a growth rate of -0.9pm/ tions (Fig.lb). On the loth day, the larvae day (Table 3). fed with 2x104 cells/ml had maximum percentage (80%) of late auricularia stage, In the the larvae de- 15% and 5% mid and earlv auricularia .' velo~ed in to the late auricularia stage respectively (Fig.1~). In concentration of (Fig.1a). Seventy Percentage larvae 4x104, 45% were late auricularia, 35% mid attained late auricularia stage among the auricularia and 20% early auricularia. Table 2. Mean growth rate (n=30) of the auricularia of Holothuria spinifera at 0, 1, 2, 4 and 8 x lo4 cells/ rnl of Isochrysis galbana (mean -c S.E). Days Growth rate (pm/day)at different concentrations of 1.galbana (mean + S.E). Effect of feed concentrations on larwal growth of seacucumber 83 Table 4. ANOVA table on numbers of H.  spinifera larvae surviving on 10fh day at 0,1,2,4 and 8 x 104cells/ ml of I. galbana (p<0.05). One way ANOVA Treatments Sum of df Mean F Sig. Sauares Sauares Between groups 1761626 4 440406.4 9.023 .002 Within groups 488084.0 10 48808.400 Total 2249710 14 (Fig.ld). Only one percentage of larvae at parison analysis, it was observed that the 8x104cells/ml attained late auricularia differences in the survival between con- stage and majority of them (65%) remained trol and larvae fed in 1 2  and 4 x l(r cells/ in the early auricularia stage itself (Fig.le). ml were significant (p<0.05). Significant The differences in the mean survival rates in different microalgal concentra- tions by one way analysis of variance indicated high degree of significance (p<0.002) (Table 4). In the multiple com- differences were also observed between larvae fed with 2x104 to 4x104 and 8 x104cells/ml and also between lx lo4 with 8x lo4 cells /ml. At 8x104 cells/ml, no significant differences were noted ei- Table 5. Multiple comparisons on numbers of H .  spinifera larvae surviving on 10fh day at 0,1,2,4 and 8 x lV'cells/ml of I. galbana (p<0.05). Multiple comparisons Dependent Variable : Concentrations ISD Between Mean 95% Confidence Concentrations Difference Std.Error Sig. Interval (I-J) Lower Upper Bound Bound 0 and 10.0 0 and 20.0 0 and 40.0 0 and 80.0 10 and 20.0 10 and 40.0 10 and 80.0 20 and 40.0 20 and 80.0 40 and 80.0 Control Earlv 1  x l04cellslm l Early 2 x 1  04cellslm l Early C Late 80% Early Late 1 % Fig. 1. Proportions of auricularia (early, mid, and late) larvae of Holothuria spinifera in dif- ferent feed concentrations. ther with control or with 4 x lo4 cells/ml (Table 5). Similarly high survival percent- age of H .scabra larvae was observed in 2x104 cells/ml than the control and other concentrations of 1,4 and 8 x104 cells/ml (Morgan, 2001). Analysis of variance on the differences of mean growth rate among the various algal concentrations were highly signifi- cant (p<0.05) (Table 6). The multiple com- parison carried out indicated that high significance ip<0.05) between the growth rate of the larvae fed with 2x104cells/ml, the control and other concentrations (Table 7). The growth rate of H. spinifera larvae at 1 and 2 x104cells/ml were more or less similar as Morgan (2001) reported for the larvae of H.scabra. When compared to the larvae reared in other algal concentrations, those fed with 2x104cells/ml had a spherical shaped gut. Moreover, high significance ( t = 7.78; n =4; p<0.001) was observed in the mean stomach length increase at this concen- tration, which indicated a better larval quality as pointed out by Ito (1995) that the length and width of stomach are important indicators of quality of the lar- vae of S. japonicus. Similarly, Morgan (2001) noticed spherica1 gut in H. scabra fed with moderate amount of algae and the gut was contracted and variable in shape in higher algal concentrations High percentage (80%) of late auricu- laria with normal symmetrical shape oc- curred on day 10 among the larvae in 2x 104cells/ml, whereas more asymmetry and irregularity in larval shape oc- Effect of feed concentrations on larwal growth of seacucumber 85 . , Table 6. Anova table on growth rate of H. spinifera larvae on 10Ih day at 0,1,2,4 and 8 x 1 0 ~ e l l s / m l  ofI. galbana (P <0.05) One way ANOVA Treatments Sum of D f Mean Squares Sauares Between groups 3431.463 4 857.866 29.600 .OOO Within groups 289.822 10 28.982 Total 3721.285 14 curred in other concentrations as Morgan (2001) observed in H.scabra lar- vae. Well developed lipid spheres in late auricularia reared in 2x lo4 cells/ml indicated the larval competency and readi- ness to metamorphosis as envisaged by Battaglene (1999) in H.scabra. The low survival, low growth rate and asymmetri- cal shape in larvae reared in 8~10~cells;/ ml might have intercepted in the filtra- tion, ingestion and digestion of auricu- laria as l organ (2001,) pointed out for H.scabra larvae. Archer (1996) similarly indicated that larvae of Stichopus mollis eventually stopped feeding when algal concentrations continuously exceeded 0.6 x104cells. Table 7. Multiple comparison on growth rate of Hspinifera larvae on 10fh day at 0,1,2,4 and 8 x I @  cells/ ml of I .  galbana (Pc0.05) (Dependent variable: Concentrations) LSD Between Mean Std. Error Sig. ' 95% Confidence C~nCentra t i~n~ Difference Interval (I-J) Lower Upper Bound Bound 0 and 1.0 -40.043* 4.396 .OOO -49.837 -30.249 0 and 2.0 -40.433* 4.396 .OOO -50.227 -30.639 0 and 4.0 -35.807 4.396 .OOO -45.601 -26.013 0 and 8.0 -31.260, 4.396 .OOO -41.05 -21.466 1 and 2.0 -.3900 4.396 .931 -10.1841 9.404 1 and 4.0 4.237 4.396 .358 -5.5574 14.031 1 and 8.0 8.783 4.396 .074 -1.018 18.578 2 and 4.0 4.626 4.396 .317 -5.1674 14.421 2 and 8.0 9.173 4.396 .063 -.6207 18.967 4 and 8.0 -4.547 4.396 .325 -5.2474 14.340 * R0.05 Various concentrations of micro algae were provided in larval rearing of sea cucumbers. Optimal concentrations of 2 to 3x104cells/ml for H.scabra larvae (James et al., 1994) ; lo4 to lo5 cells/ml for Actinopyga echinites (Chen and Chian, 1990); 0.5 to 3x104cdls/ml for Stichopus japonicus (Ito,1995), 20 to 40,000 cells / ml-' for H. scabra (Battaglene,l999) and 2 to 4 x lo4 cells/ml for H.spinifera (Asha and Muthiah, 2002) have been reported. In the present study, the growth rate of H.spinifera was more or less similar in 1x104cells/m1 (59.lpm /day) and 2x104 Battaglene, S.C. 1999. Culture of tropical sea cu- cumbers for stock restoration and enhance- ment. NAGA, 22 (4): 4-10. Chen, C.P and C.S. Chian. 1990. Short note on the larval development of the sea cucumber Actinopyga echinites (Echinodermata: Holothuroidea). Bull. Inst. Zool, Academia Sinca., 29 (2): 127-133. Dautov, S.S and S.D.Kashienko. 1995. Hyaline spheres in auricularia of Stichopus japonicus. J. Invert. Rep. Develop., 27 : 61-64. Gopinathan, C.P.1982. Methods of culturing phy- toplankton. In: Manual of research method for fish and shell fish nutrition. Spl. Publ., 8: 113- 118. Central Marine Fisheries Research Insti- tute, Cochin. cells/ml (59.5pmlday). ~~t the highest Ito, S. 1995. Studies on the technological develop- ment of the mass production for sea cucumber rates of (73.3%), stomach growth juvenile, Stichopus japonicus. Saga Perfectural (16pm/day), percentage of late auricu- Sea Farming Centre, Japan; 87 pp. laria and the Occurrence James,D.B., A.D. Gandhi, N. Palaniswamy and J.X. of normal symmetrical shape of the larvae Rodrigo.1994. Hatchery techniques and culture indicated that the 2x10~~el ls /ml  of I. galbana, as the optimal algal feed concen- tration for the species. References Archer, J.E. 1996. Aspects of the reproductive and larval biology and ecology of the temperate holothurian Stichopus mollis (Hulton). M.Sc Thesis, University of Auckland, New Zealand, 189 pp. of 'sea cucumber Holothuria scabra. Spl. Publ ., 57: 1-40. Central Marine Fisheries Research Institute, Cochin. Morgan, A.D. 2001. The effect of food availability on early growth, development and survival of the sea cucumber Holothuria scabra. (Echinoder- mata: Holothuroidea). SPC Beche-de-mer Infor- mation Bulletin., 14 : 6-12. Ramofafia, C., M. Gervis and J. Bell. 1995. Spawn- ing and early larval rearing of Holothuria atra SPC Beche-de-mer Information Buletin., 7: 2-7. Asha, P.S and P.Muthiah. 2002. Spawning and lar- smiley, S. 1986. ~ ~ ~ ~ ~ ~ ~ ~ h ~ ~ i ~  of ~ ~ i h ~ ~ ~ ~  val rearing of the sea cucumber Holothuria californicus (Echinodermata: Holothuroidea) and (Theelothuria) spinifera Theel. SPC Beche-de-mer its phylogenetic implications . Biol. Bull., 171: Information Bulletin, 16: 11-15. 61 1-631. 

Effect of feed concentrations on larval growth, survival and development of Holothuria ( Theelothuria) spinifera Thee1

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Effect of feed concentrations on larval growth, survival and development of Holothuria ( Theelothuria) spinifera Thee1

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