Molecural ecological analysis of symbiotic microorganisums of beetle inhabit in forest

日本天牛Ttypoxylus dichotoma(L.)幼虫栖息于红豆杉(Shiia Sieboldii)或红豆杉(Fagus Crenata)的森林中，在森林生态系统中扮演着重要的分解者角色。从幼虫的消化道中检测到各种葡聚糖酶活性，居住在肠道中的微生物似乎至少归因于其中的一部分。此外，中肠的内容物是碱性的，这表明消化道在土壤环境中是相当独特的生态位。本研究采用培养法和非培养法对中华绒螯蟹幼虫消化道、中肠和后肠的微生物区系进行了研究。以葡萄糖或木聚糖为碳源，在中性和碱性条件下分离好氧微生物，并对其16S rDNA进行了部分测定。提取总DNA，用聚合酶链式反应-凝胶电泳法进行分析。通过该培养方法，只属于有限属的微生物是…在碱性和中性条件下生长的菌种有所不同。PCR-DGGE分析表明，在幼虫的后肠中可能以特定的微生物为主，而在中肠中没有或很少检测到优势带。在幼虫的消化道中，对阔叶树的主要半纤维素木聚糖有很强的水解性。由于消化道的内容物是碱性的，因此可能存在嗜碱木聚糖菌。以含木聚糖的碱性培养基作碳源，从幼虫所栖息的消化道、粪便和落叶中分离到木聚糖菌。从中肠分离到7株，从后肠分离到8株，从粪便中分离到8株，从落叶中分离到8株。16S rDNA序列分析结果表明，从幼虫消化道中分离到的木聚糖降解嗜碱菌主要为芽孢杆菌。从落叶和粪便中分离到具有较强木聚糖酶活性的革兰氏阳性菌。木聚糖的水解酶活性在pH为9.3时达到最大值，在pH为5~11范围内稳定。

The larvae of Japanese horned beetle, Ttypoxylus dichotoma (L.), inhabit in forests of Shiia sieboldii or Fagus crenata, and are assumed to play an important role as decomposer in ecosystem of the forests. Various glycanase activities were detected from the digestive tracts of larva, and microorganisms inhabiting the tracts seem to attribute at least some of these activities. In addition, the contents of midguts are alkaline, suggesting that the digestive tracts are quite unique niches in soil environments. In this research, microbial flora of the digestive tracts, midguts and hindguts, of the larvae were examined by the cultivating method and the non-cultivating method. Using glucose or xylan as carbon source we isolated aerobic microorganisms under neutral or alkaline pH, and their 16S rDNA were partially determined. Total DNAs were extracted from the samples and analyzed by the PCR-DGGE method. By the cultivating method, microorganisms belonging to only limited number of genera are … More detected, and the genera grown on alkaline media were different from those on neutral media. PCR-DGGE analysis suggested that the specific microorganisms might be dominant in hindgut of larvae where as no or few dominant bands were detected in midgut.In the digestive tract of the larvae of the beetle detected strong hydrolyzing activity to xylan that is a major hemicellulose of broadleaf trees. Since the contents of digestive tracts are alkaline xylanolytic alkaliphiles might exist the tracts. We isolated the xylanolytic bacteria from the larvae of digestive tracts, feces, and leaf litters that larvae inhabited using the alkaline media contain xylan as carbon source. We isolated seven strains from the midguts, eight strains from the hindguts, and eight strains from the feces, and eight strains from the leaf litters. By the result of 16S rDNA sequence analysis, the majority of xylanolytic alkaliphiles from the digestive tracts of larvae were Bacillus. The gram positive bacteria with strong xylanolytic activity were isolated from leaf litters and feces. The xylan hydrolyzing activity showed its maximum at pH of 9.3, and is stable at a range between pH 5 and 11. Less