| Japanese parrotfish, Oplegndthus fasciatus, belonging to the family Oplegnathidae, is a high-quality and high-value marine economic fish, and is distributed in Yellow Sea, East China Sea and Taiwan coast. It inhabits coastal rocky reefs, and feeds on bentic animals. The project observed those morphological, ontogenetic and ecological characteristics in the early life stages of 0. fascia tus and detected the population genetic diversities by using those techniques like chromosomal karyotyping and band-patterning, and those DNA molecular markers like AFLP, mitochondrial DNA CO Igene, Cyt b gene and D-loop fragment. Moreover, according to the results of this study, the techniques for production of seed and breed of O. fasciatus have been developed and establish a reliable method for seed production of this species. The following eight aspects of achievements were obtained:1. FAM-amplified fragment length polymorphism (FAM-AFLP) was used to analyze the genetic diversity and population structure among populations along the coastal waters of China. A total of 1264 loci ranging in size were detected from 58 individuals, of which 1248 were polymorphic. The proportion of polymorphic loci, the Nei genetic diversity and Shannon genetic diversity index of Jiaonan, Zhoushan and Taiwan populations were 80.21%.91.01%and 77.81,0.196,0.199 and 0.173,0.301、0.312 and 0.251, respectively. Gst value, STRUCTURE and AMOVA analysis indicated that the genetic variation mainly came from individuals between populations (59.55%). The NJ tree based on FAM-AFLP data supported this result. Our results provide information on the genetic structure of O. fasciatus, which can be utilized to make informed management decisions. However, AFLP markers are inherited as dominant markers, further studies utilizing co-dominant markers are needed for a better understanding of the genetic diversity of this fish.2. Partial nucleotide sequences of mitochondrial DNA control region fragment were sequenced and analyzed to estimate genetic differentiations between cultured (F1 and F2) and wild O. fasciatus populations. The result showed that the haplotype diversity of cultured population (F1) was just a little below that of wild population, but which was significantly higher than that of F2 based on the length of 484bp sequence. Significant decline trend of nucleotide diversity was detected from wild population to cultured population F1 and F2, the same results were also checked in the Mismatch analysis and average number of nucleotide differences analysis in the present study. Significant population genetic differentiations were detected between cultured population (F1 and F2) and wild population based on the analysis of AMOVA, conventional Φst and the exact test. No significant genealogical structure corresponding to the cultured and wild populations was detected in the species based on the MST and NJ analyses.3. Partial nucleotide sequences of mitochondrial DNA COI gene, Cyt b gene and D-loop fragment were sequenced and analyzed to estimate the relationship between O. fasciatus and O. punctatus. One hundred and three nucleotide substitutions were checked in the total 1107bp sequences analyzed, and most of them were synonymous transitions at the third codon positions in the two protein-coding genes. Nucleotide composition analysis indicated a strong bias against guanine (G) in the three fragments, especially in the third codon positions of COI and Cyt b genes. The mean genetic distances based on the COI, Cyt b and D-loop fragments between O. fasciatus and O. punctatus were 0.097,0.080 and 0.172, respectively. The average genetic distances showed interspecific differentiation within genus. There were obvious differences in the rates of nucleotide substitution between the two species in the three mtDNA segments. In contrast with previous research, the rate of nucleotide substitution for COI gene fragment was higher than that of Cyt b fragment. The estimated divergence time between O. fasciatus and O. punctatus based on Cyt b and ND5 genes was about 3.85～5.3 million years, which indicated a Early Pliocene divergence of the two species.4. Karyotype analysis showed that the chromosome number was 48 and their chromosome formula is 2n= 2m+46t, NF=50, in the females. But the chromosome number in the males was 47 and the chromosome formula is 2n= 3m+44t, NF=50. A large metacentric chromosome observed exclusively in the males was heterotypic chromosome, and no other chromosomes paired with it. The C-banding analysis indicated that heterochromatin stained 41.49±0.71% of the chromosome surface in the females and 40.56±0.38% in the males. The heterotypic chromosome stained by C-banding exhibited 1 centrometric bands and 2 telomeric bands. According to the present study, the unique heterotypic chromosome in the males was likely to be Y chromosome. On the basis of previous references, the sex determination of O.fasciatus was predicted to be multiple sex chromosome system.5. The morphological and histological characters of embryo, larva, juvenile and fingerling stages of Japanese parrotfish were described. We obtained the fertilized eggs by the artifical fertilization. The fertilized eggs were spherical, separate and buoyant with a diameter of 0.79-0.90mm, containing a bright yellow lipid globule that the diameter ranged 0.15-0.20mm. There was the chap on the yolk at the animal pole. At 22±0.5℃ and salinity 30, the first cleavage takes place about 45min after fertilization, the embryo developed to the stage of blastula, gastrulation, cephalization, tail lift, heart beat and hatching at 272min,441min,896min,1393min,1560min and 1876min after fertilization. The total length of the newly-hatched larvae ranged from 1.66mm to 2.20mm. There were 4-5 black melanophores on the dorsal fin drap, and that disappeared in 3dph larva. The formation of the cross-band is commenced with the larva of about 9mm in total length about or 20dph juvenil, and completed at about 35mm or about 50 days after hatching at the water temperatures of 22±0.5℃6. The threshold of temperatures are 15℃ and 33℃ separately on embryonic development of Japanese parrotfish. The suitable temperatures for hatching range are from 21℃to 27℃ and the most suitable temperature is 24℃ with high hatching rate over 96%. The initial feeding time and PNR(the point of no return) are 3dph and 5～6dph respectively, the highest initial feeding rate is 76.67% at 4dph at water temperature 24℃.7. Histogenesis of the immume system and activity of SOD were studied in O.fasciatus from fertilization to 50dph.The pronephric tubule primordium developed in the embryo,14h30min post fertilization.The spleen anlage was observed between the swim bladder and the intestine at 5dph,and the thymus was formed as a paired structure under the pharyngeal epithelium above the gill arch at lOdph.The order of the immune organs becoming lymphoid was the pronephric kidney(10dph), thymus(15dph) and spleen(21dph). As the embryo developed,the specific activity of SOD gradually increased until hatching,but subsequently SOD activity continuously decreased to a minimum at 14dph. After the spleen became lymphoid,the specific activity of SOD was relatively stable.lt is suggested that the immaturity of the lymphoid organs and low specific activity of SOD was the cause of the high mortality of fingerlings 12 to 16dph.8. This paper reviews the techniques developed for Japanese parrotfish including broodstock maintenance, egg incubation, rearing water treatment and management, feed regime, live food preparation, and aquaculture production. The established techniques for Japanese parrotfish seed production in China include hygienic condition of larval rearing water, eggs and live food and rearing in a closed system. The strict hatchery hygiene control measures are effective in avoiding the spread of viral and other diseases. |
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