Study On Artificial Induction And Early Growth Of Ruditapes Philippinarum Clam

Ruditapes philippinarum clam is nutritious and delicious.Domestic and foreign consumers love rudiment.In China,the annual output of clam is about 4 million tons,accounting for more than 90%of the world’s output.It is an important beach economic shellfish widely cultivated in the South and north coasts of China.Taking clam as the research object,this thesis finds out the characteristics of artificial induction and early growth and development of clam from four aspects:artificial induction the spawning of clam,muscle development,growth markers and optimal growth environmental factors.The main research results are as follows:1.Effects of 5-HT and thermal stimulation on artificial induction the spawning of clamTaking clam with mature gonads as the research object,the induction effects of5-Hydroxytryptamine（5-HT）and thermal stimulation or their combination on spawning Oocytes and sperm of clam parents were investigated.The results showed that the spawning rates of females and males within 20 min respectively were calculated as 52.4%and 50.0%after 5-HT(2 mmol·L^-1)was injected into the gonads of clams.The efficiency time for 5-HT was more than60 min.In contrast,the spawning rates of clams within 20 min achieved 80.9%in females and91.3%in males,which were induced by the combined effects of 5-HT injection and thermal stimulation.The response time for the combined induction was generally less than 20 min in both females and males.During the whole experimental period,the highest spawning rate（85.3%）was obtained by the combined induction of 5-HT injection and thermal stimulation.The spawning induction effects of 5-HT（75.0%）were remarkably superior to the single induction of thermal stimulation（less than 5.0%）.Therefore,the combined induction of 5-HT injection and thermal stimulation is served as the best way to induce the spawning of clams.2.Phalloidin fluorescence and confocal microscopy reveal the musculature development of clamKnowledge of early development in bivalves is of great importance to understand the function of animal organ systems and the evolution of phenotypic diversity.Clam is an economically important bivalve living in marine intertidal zones.To determine the pattern of muscle development in the clams,we investigate the characteristics of musculature development utilizing phalloidin staining and confocal microscopy.Myofilaments first appear at the early trochophore stage,and gradually become orderly arranged during the transition from trochophore to veliger.For veliger,larval muscle system is mainly composed of dorsal velum retractors,medio-dorsal velum retractors,ventral velum retractors,ventral larval retractors and anterior and posterior adductor muscles.After metamorphosis,the muscle system of late veliger has been reconstructed,showing the irreversible shrink of velum retractor muscles,the rapid growth of wedge-shaped foot and mantle margins.One of the most striking changes in settled spats is the development of sophisticated architecture of foot musculature,which consists of transverse pedal muscles,anterior foot retractor and posterior foot retractor.These findings will not only provide the basis to understand the dynamic pattern of myogenesis in the burrowing bivalves,but also provide valuable information for comparative analysis of muscle development among bivalves.3.Applicability of three fluorochromes as in situ growth markers in Manila clam Ruditapes philippinarumMarking technology is essential to evaluate growth performance for shellfish species in aquaculture.Some fluorochromes have been widely used in shellfish growth studies,but their marking quality remains unresolved.In order to establish an effective marking technology in bivalves,three fluorochromes are used to evaluate the suitability of in situ growth markers in spats of commercially important bivalve,Manila clam Ruditapes philippinarum.The different concentrations of calcein（Cal;25 mg/L,50 mg/L and 100 mg/L）,alizarin red S（Ars;5mg/L,10mg/L and 20mg/L）and oxytetracycline hydrochloride（OTC;250mg/L,500mg/L and 750mg/L）have been used to examine their potential effects on marker quality,growth and survival of spats.In general,marking quality scores for three fluorochromes have a positive correlation with their increasing of concentrations.Cal（50-100 mg/L）and Ars（10-20 mg/L）have produced healthy spats with a bright and clear fluorescent shell mark after the recovery culture for 69 days.In contrast,OTC appears to have the weak staining signals at all concentrations.The growth and survival rates affected by fluorochrome emersion are negligible at all concentrations.The present findings indicate that in situ Cal-or Ars-marking of spats with moderate concentrations are non-invasive and effective method for growth studies in clams.The suitability of Cal and Ars as in situ growth markers in clams will provide new marking techniques to distinguish cohorts or families in aquaculture.4.Effects of temperature,salinity,density and their interaction on the growth of clamThe experimental design scheme was carried out by using design expert 12 software and box Behnken design center combination,and the clam growth model was constructed to find the optimal combination of growth conditions.Three factors and three levels were designed.The factors were temperature（15℃,23.5℃,32℃）,salinity（14,20.5,27 psu）,density（10000,30000,50000 ind/m²）;The level was（-1,0,1）.The combination was cultured for 73 days.The final shell length was measured,and the juvenile shell length was taken as the response value.The results showed that the secondary effects of temperature,salinity and density had a significant effect on the growth of juvenile clam（P<0.05）,and salinity had the most significant effect on the growth of juvenile.The interaction of various factors had no significant effect on the growth of clam（P>0.05）.The three factors have a certain positive correlation with the growth of clam within the suitable range,and the shell length can reach the extreme value at a special value.After software optimization,when the temperature is 24.32℃,the salinity is 21.13 psu and the density is 28800ind/m²,the maximum theoretical value of juvenile shell length growth is 4.07 mm.