Acid-base regulation and ammonia excretion in aquatic invertebrates with considerations of future environmental changes: Characterization of novel transporters and mechanisms

水生无脊椎动物需要排出二氧化碳和有毒的氨作为其代谢的最终产物。这是通过它们的鳃或皮肤来完成的。这两种分子都具有酸碱等价物的功能，可以以气态（NH3, CO2）或各自的离子形式（NH4+, HCO3-）进行运输。排泄必须严格调节，因为细胞内或细胞外pH值（pHi或pHe）的变化会导致蛋白质功能的丧失和整体生理损伤。此外，外部因素（如pH、pCO2）会影响动物的酸碱平衡，而由于人为二氧化碳排放导致的海洋和淡水酸化不断增加，水生物种目前正受到威胁。迄今为止，对无脊椎动物酸碱调节的分支机制了解甚少。在5个目标中，4名博士和2名硕士学生将研究鳃和细胞酸碱调节机制，采用各种不同的盐无脊椎动物物种来揭示这些过程中的栖息地特异性差异。1)在初步研究的基础上，我们将确定新发现的微管网络在3种不同盐蟹鳃囊氨和CO2排泄中的作用。除了鳃灌注研究外，还将研究蟹的rh蛋白（一种预测的NH3/CO2双通道）的细胞定位和运输特性。2)我们进一步发现了两种新的氨转运蛋白HCN和HIAT，将通过基因敲除（RNAi）和离子选择微电极在蚊子幼虫体内的异源表达实验和底物特异性（NH3, NH4+, CO2）研究。3)在共聚焦激光扫描显微镜下，采用ph敏感染料，研究将马蹄蟹鳃和水蛭皮分别置于传输腔中，对pHi的调节。细胞pHi恢复机制将在根尖或基底侧pH紊乱后进行研究。4)利用转运体特异性抑制剂和mRNA表达分析进行鳃灌注研究，将有助于了解生活在热液喷口附近的极端环境中的螃蟹如何调节其pHe，以及适应升高的pCO2水平2周后生理可塑性是否会发生变化。5)在一项跨越42代的实验中，对水体的转录组学分析将揭示对预测的未来全球变化情景（2100年）的适应机制。在长达42个月的实验中，将监测生理参数（MO2， [Ca2+]等）和可塑性。结果将大大增加我们对无脊椎动物酸碱调节的认识，但对于评估全球变化对我们生态系统的影响也至关重要。数据对于濒危物种的重新放养工作和无脊椎动物水产养殖业务也是非常宝贵的。

Aquatic invertebrates need to excrete CO2 and toxic ammonia as end products from their metabolism. This is accomplished at large by means of their gills or skin. Both molecules function as acid-base equivalents and can be transported either in the gaseous state (NH3, CO2) or in the respective ionic form (NH4+, HCO3-). Excretion must be tightly regulated as shifts of intra- or extracellular pH (pHi or pHe) can cause e.g. loss of protein function and overall physiological impairment. Additionally, extrinsic factors (e.g. pH, pCO2) can affect the animal's acid-base homeostasis and aquatic species are currently threatened by continuously increasing ocean and freshwater acidifications caused by anthropogenic CO2 emission. To date branchial mechanisms of acid-base regulation in invertebrates are poorly understood. In 5 objectives 4 Ph.D. and 2 M.Sc. students will investigate branchial and cellular acid-base regulatory mechanisms employing a variety of different haline invertebrate species to uncover habitat specific differences in these processes. 1) Based on our initial studies we will determine the newly discovered role of the microtubule network in branchial vesicular ammonia and CO2 excretion in 3 different haline crab species. In addition to gill perfusion studies, cellular localization and transport characteristics of the crab's Rh-protein, a predicted dual NH3/CO2 channel, will be investigated. 2) We further discovered 2 novel ammonia transporters, HCN and HIAT, which will be investigated for their substrate specificity (NH3, NH4+, CO2) by heterologous expression experiments and in vivo studies in mosquito larvae, using a gene knock-down approach (RNAi) and ion selective microelectrodes. 3) Regulation of pHi will be investigated in split horseshoe crab gills and leech skin mounted into a transport chamber positioned under a confocal laser scanning microscope employing pH-sensitive dyes. Cellular pHi recovery mechanisms will be studied after apical or basolateral pH disturbances. 4) Gill perfusion studies using transporter specific inhibitors and mRNA expression analysis will provide insights of how a crab that inhabits the extreme environment near hydrothermal vents regulates its pHe and whether physiologic plasticity changes after a 2 week acclimatization to elevated pCO2 levels. 5) A transcriptomic analysis in water flees will unravel mechanisms of adaptabilities towards the predicted future global change scenario (year 2100) in an experiment that spans over 42 generations. Physiological parameters (MO2, [Ca2+], etc.) and plasticity will be monitored over the 42 month long experiment. Outcomes will significantly increase our knowledge of invertebrate acid-base regulation, but will also be critical to evaluate the impact of global change in our ecosystems. Data are also invaluable for restocking efforts of threatened species and invertebrate aquaculture operations.