瘤棘砂壳虫是东亚特有的原生动物,广泛分布于长江与珠江中下游以及福建地区的湖泊和水库,作为水生态系统的捕食者,在维持水生态系统的结构与功能方面发挥着重要作用。利用18S rrnA基因PCr扩增和克隆文库测序等分子生物学技术方法,从基因水平研究瘤棘砂壳虫的食物组成。结果表明:所获得的46条序列在97%相似度水平含有11类OTuS(OPErATIOnAl TAXOnOMIC unITS,操作分类单元),其中包括轮虫6个,桡足类5个,说明瘤棘砂壳虫的捕食类群以轮虫和桡足类为主,同时也证明单细胞生物可以直接以多细胞的后生动物为食。此外,通过克隆文库测序技术分析原生动物的食物组成比例,不仅是一种方便、高效、快速,重复性高的方法,同时也为分析原生动物的生态功能提供了一种新的视角。Difflugia is a morphologically diverse genus of the free-living shelled amoeboid protozoa that are important components of freshwater ecosystems and play crucial roles in nutrient cycles and energy flow through food webs.Difflugia tuberspinifera is an endemic species of East Asia and is widely distributed in freshwater lakes and reservoirs in China.Clearly,this species plays a critical role in maintaining the structure and function of freshwater ecosystems.However,little is known about its diet composition and predatory behavior at both species and gene levels.This testate amoeba( D.tuberspinifera) was first found in the Wujiang River,Guizhou Province,China.Subsequently,more detailed studies on its morphology and biometry have been done on natural populations from Yangtze River and Pearl River valleys,and Fujian reservoirs.Recently,Han et al.illustrated that D.tuberspinifera is an active and agile hunting carnivore that can capture swimming prey including micro-particulates,rotifers,and other metazoans.In previous studies,however,the specimen identifications were done by microscopic examination,which requires a broad and deep taxonomic knowledge and is verylaborious,time-consuming,and somewhat variable because of insufficient taxonomic resolution.In order to facilitate the identification of diet composition in D.tuberspinifera,an inexpensive,efficient,rapid and easy-to handle gene clone library has been developed for diet detection and analysis.The plankton samples were collected from the Hubian Reservoir in Xiamen city in September of 2010.Individual cells were isolated using a glass capillary under an inverted microscope and washed 3—5 times with distilled water before DNA extraction and PCR amplification.The 18 S rRNA gene was amplified by the universal eukaryotic primers,and the purified PCR products were ligated into the p GEM-T vector and transformed into Escherichia coli DH5α-competent cells.In total,46 plasmids containing target gene fragments were successfully identified and sequenced.Each sequence was compared with sequences available in the Gen Bank database using BLAST,and the closest relatives were identified for diet or food composition analysis.Finally,11 OTUs( operational taxonomic units) were identified at 97% sequence similarity level,and they belonged to either Rotifera( 52%) or Copepoda( 48%).Our results,combined with existing data,suggested that:1) the diet composition of Difflugia tuberspinifera is composed of both rotifera and copepoda species;2) unicellular protozoa are not only the food of metazoa,but they can also prey on multicellular micro-metazoa;3) molecular methods are universally applicable,and the SSU rRNA( small subunit ribosomal RNA) gene clone library is an efficient,rapid,and repeatable approach to study the diet composition of protozoa.国家自然科学基金项目(31172114;30800097); 福建省杰出青年科学基金项目(2012J06009
