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新陈代谢的最终产物。这在很大程度上是通过它们的鳃或皮肤完成的。这两个分子都具有酸碱等效物的功能，可以在气态(NH3，CO2)或各自的离子形式(NH4，HCO3-)中传输。排泄必须严格控制，因为细胞内或细胞外pH(phi或phe)的变化可能导致蛋白质功能丧失和整体生理损伤。此外，外在因素(如pH、PCO2)可影响动物的酸碱平衡，水生物种目前正受到人为二氧化碳排放导致的不断增加的海洋和淡水酸化的威胁。到目前为止，无脊椎动物的酸碱调节机制还知之甚少。在5个目标中，4个博士学位和2个硕士学位学生将利用各种不同的盐生无脊椎动物物种研究鳃和细胞酸碱调节机制，以揭示这些过程中特定于栖息地的差异。1)基于我们的初步研究，我们将确定新发现的微管网络在3种不同种类的盐生蟹鳃泡氨和二氧化碳排泄中的作用。除了鳃灌注研究外，还将研究螃蟹Rh蛋白的细胞定位和运输特性，这是一种预测的NH3/CO2双通道。2)我们进一步发现了两个新的氨转运蛋白：HCN和HIAT，将通过异源表达实验和在蚊子幼虫体内的研究，利用基因敲击法和离子选择性微电极来研究它们的底物特异性(NH3，NH4，CO2)。3)在装有pH敏感染料的共聚焦激光扫描显微镜下放置的运输室中，将在裂开的马蹄蟹鳃和水母皮中研究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)利用转运蛋白特异性抑制物和mRNA表达分析进行的鳃灌流研究将为深入了解生活在热液喷口附近极端环境中的螃蟹如何调节其PHE，以及在适应高二氧化碳水平两周后生理可塑性是否发生变化。5)在一项跨越42代的实验中，对水中逃逸的转录分析将揭开适应未来全球变化预测情景(公元2100年)的机制。生理参数(MO2、[Ca~2]等)在长达42个月的实验中，将对可塑性进行监测。结果将大大增加我们对无脊椎动物酸碱调节的了解，但也将对评估全球变化对我们生态系统的影响至关重要。数据对于濒危物种的重新放养和无脊椎动物水产养殖作业也是非常宝贵的。

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.