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。这两种分子的作用相当于酸碱等价物，可以以气态（NH3、CO2）或相应的离子形式（NH 4+、HCO 3-）运输。必须严格调节排泄，因为细胞内或细胞外pH值（pHi或pHe）的变化可导致蛋白质功能丧失和整体生理损伤。此外，外在因素（如pH值，pCO 2）可以影响动物的酸碱平衡和水生物种目前受到不断增加的海洋和淡水酸化造成的人为CO2排放的威胁。 迄今为止，对无脊椎动物鳃的酸碱调节机制知之甚少。在5个目标4博士学位。和2名硕士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，以揭示栖息地在这些过程中的具体差异。1)基于我们的初步研究，我们将确定新发现的作用，微管网络在鳃泡氨和CO2排泄在3种不同的盐蟹。除了鳃灌注的研究，细胞定位和运输特性的螃蟹的Rh蛋白，预测双NH3/CO2通道，将进行调查。 2)我们进一步发现了两种新的氨转运蛋白HCN和HIAT，并通过异源表达实验和蚊子幼虫体内研究，利用基因敲低方法（RNAi）和离子选择性微电极研究了它们的底物特异性（NH3，NH 4+，CO2）。3)调节pH值将在分裂马蹄蟹鳃和水蛭皮肤安装到一个运输室定位在共聚焦激光扫描显微镜下，采用pH敏感染料。将在顶侧或基底侧pH紊乱后研究细胞pHi恢复机制。4)鳃灌注研究，使用转运蛋白特异性抑制剂和mRNA表达分析将提供洞察力的螃蟹，栖息在热液喷口附近的极端环境调节其pH值和是否生理可塑性变化后，2周的驯化提高pCO 2水平。5)在一项跨越42代的实验中，对水蚤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。生理参数（MO 2、[Ca 2 +]等）和可塑性将在42个月的实验中进行监测。结果将大大增加我们对无脊椎动物酸碱调节的知识，但也将是至关重要的，以评估全球变化对我们的生态系统的影响。 数据对于受威胁物种的重新放养工作和无脊椎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.