| 1 Induction and cytogenetic study of allotriploid scallop in Chlamys farreri♀×Argopecten irradians♂crossPrior to the allotriploid induction, the feasibility of fertilization between Zhikong scallop Chlamys farreri eggs and Bay scallop Argopecten irradians sperm was verified by epifluorescence microscopy. In 20℃seawater, crosses between C. farreri♀and A. irradians♂can be easily made with mature parents.Triploidy and incubation rates of D-stage larvae with an average of 88.56% and 53.52% respectively, were obtained by Meiosis II inhibition, with 60 mg/L 6-deimethylaminopurine (6-DMAP) treatment for 15 min durations when 50% zygotes released the first polar body. The consequent larvae grew very slowly, and reached 148um in shell length, far behind of the control of C. farreri×C farreri cross (173μm) at 14 days post fertilization (DPF). The resultant larvae encountered high mortality when reached 6 DPF, and larvae survival dropped to 0.00067% at 14DPF. Using Genomic In situ Hybridization (GISH), the consequent larvae were validated to be real interspecies hybrids from C. farreri X A. irradians cross. Maternally biased chromosome conversion, including fragment conversion and whole chromosome conversion, were revealed in about 2% of all metaphases. These results might indicate the instability of the hybrid genome, with complicated interactions between nuclear-nuclear or nuclear-cytoplasm, which might give rise to the inviable larvae.2 Induction and growth performance of triploid yesso scallop (Patinopecten yessoensis)Hypotonicity shock, a new method to induce triploidy was tested. The highest triploidy reached 91.2.4% in average with a mean incubation rate 52.94%, with 20‰salinity treatment for 20 min durations, when 50% zygotes released the first polar body.A large-scale population of triploidy induction group was made to test the ploidy alteration and growth performance from D-stage to 24-month age, by the method mentioned above. Triploidy was found to drop significantly from D-stage (50.06%) to 4-month age (12.07%), and then slightly to 8-month age (7.27%). From 8 months on, the ploidy of the induction group changed little.Prior to growth comparisons, ploidy of each individual used was conformed by flow cytometry. No growth advantage of triploid population was found during the cultivation from 4-month to 24-month age. Mean height, length and width of shell, and body weight of triploids were 8.59%, 10.33%, 8.79% and 24.78% lesser than diploids respectively.3 Relative fecundity and tetraploid induction of triploid yesso scallop (P. yessoensis)Triploids reached sexual maturity with apparent gonad development about 1 month later than diploids. Among the 60 triploids sampled, gonad developed worse with various degree. Gonad of males developed prior to female, with cream-white appearance in general, while female gonad possessed orange-red looking. Sex ratio in triploids seemed to be normal, with female to male 1 : 1.18 at 1-year age, and 1:1.50 at 2-year age, with no hermaphroditic individual found.None of the 29 male triploids spawned in spite of artificially thermal stimulation. Gonad from 3 males was investigated by electron microscopy. Many spermatids were found in male gonads, with condensed chromatin of various shape, and lacked endoplasmic reticulum. Mitochondria in some spermatids possessed lacked developed cristae, some of which even turned to empty vesicles. No acrosome was found, nor did sperm.Spawning was successfully induced in 4 (17.39%) of the 23 triploid females, with a relative fecundity estimated to be 3% of normal diploids at 1 year age, and 4% at 2 year age. Mean diameter of these eggs was 86.65±2.53μm, 10.79% longer than that of diploids, and 35.99% bigger for volume than that of diploids. Eggs produced by female triploids were fertilized with sperm from one diploid male to investigate meiotic maturation in the eggs and the resultant larvae. Most of the eggs seemed to complete meioses normally. Aberrant meioses were observed in about 30% of the eggs, with unsynchronization and lagging of chromosomes. Flow cytometric analysis of the blastula stage larvae from the 3n×2n cross showed the same pattern, with one aneuploid peak lying between 2n and 3n possessing about 70% of the total area, and the other peak falling between 3n and 5n possessing about 30% of the total area. Mean survival of the fertilized eggs to D-stage for 3n×2n crosses (0.23%) was significantly lower than that for controls (75.67%).At day 7, no survival was observed.The highest mean tetraploidy reached 75.12%, in 3n♀×2n♂cross by 20‰salinity treatment for 20 min durations, to inhibit the first polar body. The resultant larvae developed extraordinary slowly, and reached trochophore stage at about 65 h, and D-shaped stage 120 h. Incubation rate of D stage larvae was only 0.09%, and survival to 7-day age was only 0.07%.4 MSAP analysis on triploid yesso scallop (P. yessoensis) 13 triploids and 20 diploids used in MSAP analysis were from the same triploid induction group. Using 9 pairs of selective primers, a total of 7206 amplified loci were identified, of which, 1988 loci (27.6%) were methylated including 1095 full-methylation loci (15.2%), and 893 hemi-methylation loci (12.4%).Main conclusions: (1) methylation level was high in yesso scallop. (2) methylation level varied bigger among individuals. (3) triploidy resulted in lower methylation level in yesso scallop. (4) methylation level may be related to gender of yesso scallop. (5) for mean methylation level: diploid female >diploid male >triploid female >triploid male (6) triploidy hindered the development of female gonad in some extent.
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