Microbial food webs in Movile Cave

面向普通观众的项目摘要：1986年，在罗马尼亚靠近黑海的地方发现了一个天然洞穴。莫维尔洞穴是一种极其罕见的洞穴系统，几千年来一直与世隔绝。尽管与地面完全隔绝，莫维尔洞穴拥有丰富的生态系统，拥有近50种不同的洞穴适应的水生和陆地无脊椎动物，包括蠕虫、伪蝎子、蜘蛛、水蚤和蜈蚣。值得注意的是，这些无脊椎动物中有30多种是莫维尔洞穴特有的。这些无脊椎动物通过一种被称为轮回形体的过程适应了没有光线的生活，这一过程从没有眼睛、附肢延长和缺乏色素来证明，这表明了地下进化的漫长历史。洞穴由地下深处的热硫化水供应，光线无法进入洞穴。因此，这种丰富的生态系统必须由洞穴中非光合作用细菌产生的有机碳的初级生产驱动。初步研究表明，洞穴水下湖泊表面和洞穴墙壁上的微生物垫含有活性甲烷和硫氧化细菌，这些细菌必须推动微生物食物链的启动，这些细菌在那里生长并释放养分，供其他细菌和真菌生长。反过来，细菌被蠕虫和其他无脊椎动物吃掉，最终食肉动物通过吃其他无脊椎动物而登上食物链的顶端。这种环境可以被认为是一个极端的环境(如深海热液喷口)，研究移动洞穴可以给我们提供关于生命如何进化的线索。由于维持地下深处这种新型生态系统的所有食物的最初生产都依赖于特殊的细菌群，我们想要研究通过这些细菌进入Movile Cave的食物网的碳流动。我们将使用一些尖端的微生物分子生态学技术来研究负责在甲烷和其他一碳化合物上氧化和生长的不同细菌的多样性和活性，这些细菌固定二氧化碳，不使用阳光，而是使用洞穴热水中存在的无机硫化合物的能量。我们还将研究细菌对氮化合物的固定和循环，这也是维持移动洞穴中所有生命所必需的过程。我们将使用一种名为稳定同位素探测(SIP)的技术来追踪碳从甲烷和二氧化碳通过各种细菌群进入异养细菌和真菌的流动情况，这种技术使我们能够用同位素标记的13-C(重碳)来标记消耗这些形式碳的微生物的DNA和RNA。从微生物垫群落中存在的所有其他核酸中分离出大量的DNA和RNA，使我们能够确定这些特定的碳主要消费者的结构和功能。此外，我们将使用一种令人兴奋的新技术，即拉曼荧光显微镜，在单细胞水平上研究这个生态系统中的微生物。利用我们对微生物席和洞穴水样进行的SIP实验中的DNA和RNA序列信息，我们可以制造特定的荧光探针，专门与微生物席材料中的甲烷和硫氧化细菌结合(这一过程称为荧光原位杂交或FISH)。最重要的是，我们可以在同样的鱼类标记细胞中检测到来自甲烷和二氧化碳的13C，这是我们用拉曼显微镜喂养细胞的结果。这将使我们能够调查包含我们的测试C底物的细菌的确切位置和数量，随着时间的推移，我们可以跟踪碳通过Movile Cave中的微生物食物网的过程。

Summary of the Project for a general audience: In 1986, a natural cave was discovered in Romania near to the Black Sea. Movile Cave is an extremely unusual cave system which had been sealed off from the outside world for many thousands of years. Despite being completely isolated from the above ground, Movile Cave harbours a rich ecosystem with nearly 50 different species of cave-adapted aquatic and terrestrial invertebrates including worms, pseudo-scorpions, spiders, leeches and centipedes. Remarkably over 30 of these invertebrates are endemic to Movile Cave. These invertebrates have adapted to life without light through a process called troglomorphy, as evidenced by absence of eyes, elongation of appendages and lack of pigment, indicating a long history of evolution underground. The cave is fed by thermal sulfide water from deep underground and no light can enter the cave. Therefore this rich ecosystem has to be driven by primary production of organic carbon made by non-photosythetic bacteria in the cave. In preliminary studies, it has been shown that microbial mats on the surface of the underwater lakes in the cave and on the cave walls contain active methane and sulfur oxidising bacteria which must be driving the start of the microbial food chain, where these bacteria grow and release nutrient for other bacteria and fungi to grow. In turn, the bacteria are grazed on by worms and other invertebrates and finally carnivores head the top of the food chain by eating other invertebrates. This environment can be considered an extreme environment (like deep-sea hydrothermal vents) and studying Movile Cave can give us clues as to how life evolves. Since the initial production of all food to sustain this novel ecosystem deep underground is reliant on specialised groups of bacteria, we want to study the flow of carbon through these bacteria into the food web in Movile Cave. We will use a number of cutting edge microbial molecular ecology techniques to examine the diversity and activity of different groups of bacteria responsible for oxidation and growth on methane and other one-carbon compounds, bacteria which fix carbon dioxide, not using sunlight but using energy from inorganic sulfur compounds present in the thermal waters of the cave. We will also investigate the fixation and cycling of nitrogen compounds by bacteria, processes also necessary to sustain all life in Movile Cave. We will follow the flow of carbon from methane and carbon dioxide through the various groups of bacteria into heterotrophic bacteria and fungi using a technique known as stable isotope probing (SIP) which allows us to label up the DNA and RNA of the microorganisms consuming these forms of carbon with isotopically-labelled 13-C (heavy carbon). Isolating the heavy DNA and RNA from all of the other nucleic acids present in microbial mat communities allows us to determine both the structure and function of these specific primary consumers of carbon. In addition we will use an exciting new technique called Raman FISH microscopy to study the microbes in this ecosystem at the single cell level. Using DNA and RNA sequence information from our SIP experiments with microbial mat and cave water samples, we can make specific fluorescent probes which specifically bind to methane and sulfur-oxidising bacteria in microbial mat material (a process called fluorescence in situ hybridisation or FISH). On top of this we can detect in the same FISH-labelled cells the heavy, 13C from methane and carbon dioxide that we fed the cells using the Raman microscope. This will allow us to investigate the exact location and numbers of bacteria that incorporate our test C substrates and over time we can follow the course of the carbon through the microbial food web in Movile Cave.