| Sea cucumber (Apostichopus japonicus) is one of the most important aquatic animals in Northern China. The artificial breeding technology of A. japonicus was preliminarily established in the early 1980’s. After decades of development, a relatively complete technology system has formed in recent years, and new problems and challenges also emerge. In view of the problems existing in current A.japonicus seed production, study on reproductive conditioning and fertilization biology of A.japonicus, effects of Nannochloropsis sp. on growth and development of A. japonicus larvae, and immune response of A.japonicus juvenile to handling stress were conducted, and the main results were listed below:1. Effects of different diets and temperature regimes on reproductive conditioning in A. japonicusTwo experiments were conducted, water temperature experiment and diet experiment. In water temperature experiment, A.japonicus broodstock was conditioned with two different conditioning temperatures (12℃ and 15℃). In diet experiment, A. japonicus broodstock was conditioned with three different microalgae diets (Sargassum thunbergii Kuntze power+sea mud= S, Laminaria japonica power+sea mud= L, and S. thunbergii Kuntze power+L. japonica power+sea mud=SL) Biochemical composition of body wall during conditioning and female gonad after conditioning at the different treatments were examined. Gonad index and spawning rate of female animals, egg diameter, fertilization rate of egg, hatching rate of larvae, body length of early auricularia were also evaluated. In the end of experiment, spawning rate, gonad index and fecundity of females conditioned at 15℃ were 6.87±2.02%,16±2.31%, and 12±59.3×104, respectively, and significantly higher than that at 12℃(P<0.05). Although broodstock conditioned with SL diet showed the highest gonad index, spawning rate, and fecundity of females, there were no significant differences in these parameters among broodstocks conditioned with different diets. Hatching rate of larvae and body length of early auricularia in SL treatment were significantly higher than that in L treatment. The results indicated that in the same conditioning duration, A. japonicus conditioned under 15℃ showed higher fecundity than under 12℃. However, there was no difference in spawned egg quality under these two temperatures. The broodstocks supplied with different diets showed the same fecundity, but the egg quality was best in broodstock supplied with mixed macoralgae power and sea mud.2. The cytological observation of fertilization and early cleavage in A. japonicusDuring fertilization and early cleavage in A. japonicus, a series of characteristic cytogical events, including sperm’s penetrating in egg, emission of polar body, association of the male and female pronuclei and polysperm, were studied by the fluorescent microscope with HOECHST 33258 stained. The results indicated that unfertilized mature eggs of A. japonicus were globular and remained at the metaphase of meiosis I. At water temperature of 22-23℃ and salinity 30, sperms quickly attached to the surface of the egg after mixing of sperms and eggs. At 12min after insemination, the first meiosis is finished, with release of the first polar body. Most of fertilized eggs released the second polar bodies after 20min. About 35min, the male and the female pronuclei associated with each other after their chromosomes formed respectively in the center of egg. The first and second cleavage finished at 80 min and 100min respectively in fast developing fertilized eggs. Polyspermy phenomena was observed in the fertilization process in A. japonicus.3. Effects of Nannochloropsis sp. on growth, survival and settlement of A.japonicus larvaeNannochloropsis sp. isolated from the East China Sea was tested as food for A. japonicus larvae in three feeding trials. The first trial evaluated the influence of seven microalgal diets (C= Chaetoceros muelleri only, D= Dunaliella tertiolecta only, N= Nannochloropsis sp. only, CD= C. muelleri+D. tertiolecta, CN= C. muelleri+ Nannochloropsis sp., DN= D. tertiolecta+Nannochloropsis sp., and CDN=C. muelleri+D. tertiolecta+Nannochloropsis sp.) on larval growth, survival, and settlement. The larvae fed CD had the greatest body length (1032.10±16.50μm) and highest percentages of survival (87.4±2.06%) on the ninth day 9 after fertilization. The larvae fed a mono-species diet of N showed a body length of 901.37±25.59μm and a settlement rate of 32.67±2.21% at the end of the experiment. In the second trial, the effects of different rations (5,000,20,000, and 40,000 cells mL-1 day-1) of Nannochloropsis sp. were tested. Larvae fed 20,000 cells mL-1 day-1 were significantly larger than larvae in other groups and experienced the highest level of survival. There were no significant differences in settlement between larvae fed 20,000 and 40,000 cells mL-1 day-1, both of which were significantly higher than larvae fed 5000 cells mL-1 day-1. In the third trial, the effects of feeding frequency (two, three, and four feedings per day) of Nannochloropsis sp. were tested. The greatest body length was observed in larvae receiving three meals daily and the lowest in larvae receiving two meals daily. There were no significant differences observed in survival and settlement between the groups fed three and four meals daily, both of which were significantly higher than the group fed two meals daily. These results suggest that Nannochloropsis sp. can be used as a diet for the large-scale production of A. japonicus seed. The optimum feeding ration and feeding frequency for growth and development of larvae were 20,000 cells mL’1 day-1 and three meals per day, respectively.4. Mechanical stress induces neuroendocrine and immune responses in A. japonicusThe present study investigated the impact of a 3-min mechanical perturbation, mimicking the grading procedure, on neuroendocrine and immune parameters of the sea cucumber A. japonicus. During the application of the stress, concentrations of noradrenaline and dopamine in coelomic fluid increased significantly, which indicated that the mechanical perturbation resulted in a transient state of stress in sea cucumbers. Coelomocytes concentration in coelomic fluid increased transiently after the beginning of the stress, and reached the maximum at 1h. Whereas, coelomocytes phagocytosis at 3 min, superoxide anion production from 3 min to 0.5h, acid phosphatase activity at 0.5h, and phenoloxidase activity from 3 min to 0.5 h were all significantly down- regulated. All of the immune parameters recovered to baseline levels after the experiment was conducted for 8 hours, and an immunostimulation occurred after the stress considering the phagocytosis and acid phosphatase activity. The results suggest that, as in other marine invertebrates, neuroendocrine/immune connections exist in the sea cucumber A. japonicus. Mechanical stress can elicit a profound influence on sea cucumber neuroendocrine system. Neuroendocrine messengers act in turn to modulate the immunity functions.5. Transport stress induces neuroendocrine and immune responses in A. japonicusThe total coelomocytes counts, phagocytosis activity, reactive oxygen species production, acid phosphatase activity, phenoloxidase activity and catecholamine levels were measured in A. japonicus following both damp and water transportation methods. With both transport methods, stress resulted in significant elevation of catecholamine levels. Total coelomocytes counts increased following transportation, whereas phagocytic activity and reactive oxygen species production decreased. Damp transport significantly depressed acid phosphatase and phenoloxidase activity in A. japonicus. However, there were no significant differences in acid phosphatase and phenoloxidase activity after water transportation. These results show that transport stress induces modifications in the immune system of A. japonicus and that damp transport elicited more severe stress than water transport. This study increased understanding of the mechanism of transport stress inducing neuroendocrine responses and immune modification in A. japonicus. The information obtained should be helpful to optimize transport process in sea cucumber culture. |
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