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: $ 2.91万
• 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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=668343
• 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将二氧化碳和有毒氨的新陈代谢排泄为最终产物。这是通过其ills或皮肤总体上完成的。这两个分子都充当酸碱等效物，并且可以在气态状态（NH3，CO2）或相应的离子形式（NH4+，HCO3-）中运输。排泄物必须严格调节，因为细胞内pH或细胞外pH（PHI或PHE）可能会导致例如蛋白质功能的丧失和整体生理障碍。另外，外在因素（例如pH，PCO2）可能会影响动物的酸碱稳态和水生物种，而水生物物种目前受到人为CO2排放引起的海洋和淡水酸化的威胁。 迄今为止，无脊椎动物中酸碱调节的分支机制知之甚少。在5个目标中4博士学位。和2 M.Sc.学生将研究采用各种不同卤素无脊椎动物物种的分支和细胞酸碱调节机制，以发现这些过程中的栖息地特定差异。 1）基于我们的初步研究，我们将确定微管网络在三种不同的Haline Crab物种中新发现的微管网络中新发现的作用。除Gill灌注研究外，还将研究蟹Rh蛋白的细胞定位和转运特性，即预测的双NH3/CO2通道。 2）我们进一步发现了2种新型的氨转运蛋白HCN和HIAT，通过异源表达实验和蚊子幼虫的体内研究，将对它们的底物特异性（NH3，NH4+，CO2）进行研究，并使用基因敲除方法（RNAI）和离子选择性微电极进行研究。 3）将在使用pH敏感染料的共聚焦激光扫描显微镜下，将调节PHI调节，该调节将在分裂的马蹄蟹和水ech皮中进行。细胞PHI恢复机制将在顶端或基底外侧pH障碍后进行研究。 4）使用转运蛋白特异性抑制剂和mRNA表达分析的Gill灌注研究将提供有关居住在水热通风孔附近的极端环境如何调节其PHE以及生理可塑性在2周适应PCO2水平后是否发生变化的螃蟹的见解。 5）水中的转录组分析将在一项超过42代的实验中揭示针对预测的未来全球变化情景的适应性机制（2100年）。生理参数（MO2，[Ca2+]等）和可塑性将在42个月长的实验中进行监测。结果将大大增加我们对无脊椎动物酸碱调节的了解，但对于评估全球变化对生态系统的影响也将是至关重要的。 数据对于受威胁物种和无脊椎动物水产养殖操作的重新销售工作也非常宝贵。 *****