Effect of manganese accumulation on the survival of juvenile short neck clam, Ruditapes philippinarum in the sandy tidal flat of Ariake Bay, Kyushu, Japan.

1 .野外研究在砂锰含量较高的Arao潮滩3个研究区（A：近海采沙覆盖区、C：未覆盖区、C: A与C之间的中间区）对菲律宾蛤幼蚌的沉降、生长和存活进行了追踪。在沉积物和间隙水中采集的Mn中，对可溶性Mn进行了定量分析，可溶性Mn的浓度与海鲢幼鱼的死亡率呈显著相关。估计间质水锰离子浓度阈值大于60 mg/ l。通过饲养试验缩小死亡因素。即使锰离子浓度超过60 mg/L，对幼蛤也没有致死作用。但在饲养水中加入锰离子后，其成活率下降。此外，用最上层透明层的沉积物-锰离子混合水饲养，其死亡率提高了50%以上，而用有机自由砂饲养则无致死效应。这些结果表明，蛤蜊幼鱼的死亡是由沉积物中可溶性锰与某些有机物的反应引起的。三、细胞培养的生物测定为了随时对野外研究或饲养试验中缩小的因素进行暴露试验，我们尝试建立细胞培养的生物测定系统。然而，脊椎动物的分流细胞培养技术在一定程度上对蛤蚌无效。另一方面，爪蟾肝细胞侧支实验表明，Mn离子对爪蟾卵黄形成有一定的抑制作用。虽然在光镜下进行了详细的观察，但幼蛤的切片未见明显的病理改变。我们正在持续进行透射电镜观测。另一方面，我们准备利用MALDI-TOF ms对幼蛤分泌的肽进行鉴定。如果在高Mn浓度的荒尾潮滩和低Mn浓度的池池江口采集的幼蛤分泌的肽有差异，则用原位杂交法检测相应的分泌细胞。在上述试验中，我们发现饲养试验中高死亡率组的壳变得脆弱，等等。认为在高锰条件下，壳的形成过程中出现了问题。我们目前正在进行将重点应用于壳形成的检查：壳强度，壳的微观结构，地幔中的壳形成细胞。少

I.Field researchSettlement, growth and survival of juvenile clams (Ruditapes philippinalum) were traced at three research areas (A : covered with sand collected from the offshore, C : uncovered area, C : medium area between A and C) in Arao Tidal Flat where Manganese concentration of sand is very high. Among the Mn collected from the sediment and interstitial water, soluble Mn was quantified, and there was significant relationship between the concentration of soluble Mn and the mortality of juvenile calms. The threshold Mn ion concentration of the interstitial water for the extinction of juvenile clam was estimated to be over 60 mg/L.II.Narrowing down the death factor by rearing experiment.Mn ion did not have a lethal effect for the juvenile clams even if the concentration was over 60 mg/L. However, the survival rate decreased when the sediment was added to the rearing water with Mn ion. In addition, the mortality raised when reared with top clear layer of the sediment-Mn ion mixed wat … More er, whilst no lethal effect when reared with organic free sand. These results suggest that the death of juvenile clams are caused by the reaction between the soluble manganese and some organic matter in the sediment.III.Bioassay by cell cultureIn order to carry out the exposure experiment about the factors narrowed down by field research or rearing experiment at any time, we tried to establish the bioassay system by cell culture. However, diversion cell culture technology of vertebrates was partly ineffective for clams. On the other hand, the collateral experiment using Xenopus hepatocyte suggested that Mn ion had some inhibiting effect on the vitellogenesis of Xenopus.IV.Pathological study of juvenile clams.Although detail observation was carried out under the light microscope, no obvious pathological change was detected in the sections of juvenile clams. We are continuously carrying out TEM observation. On the other hand, we prepared to review secreted peptides in juvenile clams by using MALDI-TOF MS. If any difference of peptide secretion was detected between the juvenile clams collected from Arao Tidal Flat (high Mn concentration) and Kikuchi River Moouth (low Mn concentration), corresponding secretion cell will be detected by In situ hybridization. During the above experiment, we found that shell of the high mortality group in the rearing experiment became fragile, and so on about Arao population. It is thought that trouble is caused in the process of the shell formation under the high Mn condition. We are currently carrying out the examination that applies the focus to the shell formation : shell strength, microstructure of the shell, shell formation cells in the mantle. Less