Molecural ecological analysis of symbiotic microorganisums of beetle inhabit in forest

日本角甲虫（Ttypoxylus dichotoma， L.）的幼虫栖息于三叶石竹（Shiia sieboldii）和油桐（Fagus crenatata）森林中，在森林生态系统中起着重要的分解者作用。从幼虫的消化道中检测到各种聚糖酶活性，并且居住在消化道中的微生物似乎至少归因于这些活性的一部分。此外，中肠的内容物呈碱性，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，分离出中性或碱性条件下的好氧微生物，并对其16S rDNA进行了部分测定。从样品中提取总dna，采用PCR-DGGE法进行分析。通过培养方法，只检测到有限属的微生物，并且在碱性培养基上生长的属与在中性培养基上生长的属不同。PCR-DGGE分析表明，特定的微生物可能在幼虫后肠中占优势，而在中肠中没有或很少检测到优势条带。在甲虫幼虫的消化道中检测到对阔叶树的主要半纤维素木聚糖有很强的水解活性。由于消化道内容物是碱性的，消化道中可能存在水解木聚糖的亲碱菌。在以木聚糖为碳源的碱性培养基中，从幼虫的消化道、粪便和凋落叶中分离出木聚糖分解菌。从中肠分离出7株，从后肠分离出8株，从粪便中分离出8株，从凋落叶中分离出8株。通过16S rDNA序列分析，发现幼虫消化道中的木聚糖水解嗜碱菌以芽孢杆菌居多。从凋落叶和粪便中分离出革兰氏阳性菌，具有较强的木聚糖分解活性。木聚糖水解活性在pH = 9.3时达到最大值，在pH = 5 ~ 11范围内稳定。少

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