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

考虑未来环境变化的水生无脊椎动物的酸碱调节和氨排泄：新型转运蛋白和机制的表征

• 批准号: RGPIN-2018-05013
• 负责人: Weihrauch, Dirk
• 金额: $ 5.83万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744857
• 关键词: Acid; base; regulation; ammonia; excretion

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.

水生无脊椎动物需要排出二氧化碳和有毒氨作为其新陈代谢的最终产物。这主要是通过鳃或皮肤来完成的。两种分子都充当酸碱等价物，并且可以以气态（NH3、CO2）或各自的离子形式（NH4+、HCO3-）传输。必须严格调节排泄，因为细胞内或细胞外 pH（pHi 或 pHe）的变化可能会导致排泄。蛋白质功能丧失和整体生理损伤。此外，外在因素（例如 pH 值、pCO2）会影响动物的酸碱稳态，而水生物种目前正受到人为二氧化碳排放导致的海洋和淡水酸化持续增加的威胁。 迄今为止，人们对无脊椎动物酸碱调节的鳃机制知之甚少。 5 个目标 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来研究鳃和细胞酸碱调节机制，以揭示这些过程中栖息地的特定差异。 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 种不同盐蟹物种鳃泡氨和二氧化碳排泄中的作用。除了鳃灌注研究之外，还将研究螃蟹的 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2种新型氨转运蛋白HCN和HIAT，将通过异源表达实验和蚊子幼虫体内研究，使用基因敲低方法（RNAi）研究它们的底物特异性（NH3，NH4+，CO2） ）和离子选择性微电极。 3) 将在安装在运输室中的分裂鲎鳃和水蛭皮肤中研究 pHi 的调节，该运输室位于使用 pH 敏感染料的共焦激光扫描显微镜下。在顶端或基底外侧 pH 值紊乱后，将研究细胞 pHi 恢复机制。 4) 使用转运蛋白特异性抑制剂和 mRNA 表达分析进行的鳃灌注研究将提供有关栖息在热液喷口附近极端环境的螃蟹如何调节其 pHe 以及在适应高 pCO2 水平 2 周后生理可塑性是否发生变化的见解。 5) 在跨越 42 代人的实验中，对水流逃亡的转录组分析将揭示对预测的未来全球变化情景（2100 年）的适应性机制。将在长达 42 个月的实验中监测生理参数（MO2、[Ca2+] 等）和可塑性。结果将显着增加我们对无脊椎动物酸碱调节的了解，但对于评估全球变化对我们生态系统的影响也至关重要。 数据对于受威胁物种的放养工作和无脊椎动物水产养殖作业也非常宝贵。