| As we know, sea cucumber industry is meeting its increasing flourish in chinaeven worldwide. Unfortunately, it also meets some problems, such as low rate oflarval settlement and metamorphosis and disease, which hinder its developmentseriously. Studies have demonstrated that the settlement and metamorphosis of marineinvertebrate is affected by two factors: settlement stratum and biofilm, bacteriaespecially attached to them (Zobell, C. E，1935). In China, polyethylene corrugatedplates (PCP) are often used in A. japonicus breeding farms as stratum. But it oftenresults in low percentage of settlement and metamorphosis and high morality. In thisstudy, we changed the superficial material of PCP by sticking oyster shell powder(OSP) or bagasse, which were both separated into three size groups:250-330,330-450,450-100μm, to that with the help of epichlorohydrin. Besides, we pickedout bacterium which can induce A.Japonicus`settlement and metamorphosis highlyfrom bacterium scraped from the surface of seashore rocks and stones in seacucumber culture ponds.At first, PCP is attached by three size groups of OSP or bagasse, such as,250-330(S),330-450(M),450-1000(L)μm, which was called improved PCP (IP).Then these IPs were immersed into sea cucumber culturing ponds for0,5and10daysbefore they were used for laboratory experiment in sterile glass Petri dishes. Thepresent result showed that the percentage of larval settlement and metamorphosis wascorrelated with the immersion days. And the OSP(S) showed the highest inductiveactivity[25.00±3.30%（0d）65.00±6.38%（5d），70.00±6.67%（10d）； PCP：15.00±3.33%（0d）40.00±3.33%（5d），46.67±5.44%（10d）]. The applicationexperiment practiced in A.Japonicus breeding ponds also showed that OSP(S) couldresult to percentage of larval settlement and metamorphosis of A. Japonicus as 78.43%higher as that of PCP. For each selected nature bacterial strain, a loopful ofbacteria grown on an agar plate was scraped off and subject to the DNA extraction bya Takara genomic DNA extraction kit. Then the16S rDNA gene sequence of thegenomic DNA was amplified by PCR using primer sets of27F and1492R. AmplifiedPCR products were extracted and purified before being determined by a commercialcompany (BGI, Beijing). The obtained16S rDNA gene sequences were analyzedthrough the BLAST program of GenBank. At last16bacteria strains were selected toperform the following experiment. The experiment result showed that four bacteriastrains could induce larval A. Japonicus’ settlement and metamorphosis effectively.Three of these belongs to Pseudoalteromonas, the other one belongs to Alteromonas.The percentage of larval settlement and metamorphosis were32.5-37.5%、30-47.5%and57.5-60%, respectively, which were higher than the PCP (25.00±10.00%)whenthe density of four monospecific bacterial bioflims were （1.07-7.84）×10~6、（6.95-8.48）×10~7and （1.01-3.61）×10~8cells/cm~2.Then the OSP(S) and PCP were immersed into the four monospecific bacterialmedium so that monospecific bacterial films can attached to the surface of them.These stratum mentioned above were applied in the following experiment. Accordingto the result,we could conclude that these OSP(S) and PCP attached by monospecificbacteria films showed higher activity than PCP and OSP(S) without biofilms. Thethree bacterium belonging to Pseudoalteromonas didn’t make percentage of larvalsettlement and metamorphosis on OSP(S) higher than that on PCP， while theAlteromonas sp.1made it. Besides,the Alteromonas sp.1biofilm on OSP(S) induce77.50±2.20%larval A. Japonicus settled and metamorphosised which was higherthan that of any other monospecific bacteria biofilms on OSP(S)or PCP. So we canbelieve that Alteromonas sp.1biofilm on OSP(S) would be the best choice for theimprovement of stratum. In order to confirm the feasibility of the IP in breeding ponds,we examined it in a5l beaker. It showed that the number of pentacula onOSP(S)+Ba.[(75.00±3.82) ind/dm~2] was larger than that on PCP+Ba.(64.00±3.26ind/dm~2), PCP(49.00±6.83ind/dm~2) and OSP(S)(54.00±4.00ind/dm~2). In sum,250-330μm OSP and Alteromonas sp.1could be possible applied together in sea cucumber breeding farms.In conclusion, the stratum improved by oyster shell powder(250-330μm) andAlteromonas sp.1together is beneficial to resolving the low percentage of larval seeacucumber settlement and metamorphosis. And the sea cucumber industry will getgreat profit from this. |
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