Award details

Novel integration of gas exchange osmotic and acid-base regulatory functions of the gill and gut of fish in hypersalinities

Reference	BB/D005108/1
Principal Investigator / Supervisor	Professor Rod Wilson
Co-Investigators / Co-Supervisors
Institution	University of Exeter
Department	Biosciences
Funding type	Research
Value (£)	300,485
Status	Completed
Type	Research Grant
Start date	17/10/2005
End date	16/07/2009
Duration	45 months

Abstract

The present study is novel in using an integrative, comparative and molecular approach to address previously unrecognised physiological responses (in acid-base regulation and gas exchange) that are secondary to, and consequences of, the more traditionally studied osmoregulatory adaptations to elevated environmental salinity. In order to survive at high salinities, teleost fish drink copious amounts of their hyper-calcaemic external medium. Once imbibed this fluid is processed through the intestine, including pronounced alkalinisation (up to pH 9.2) and the secretion of large amounts of HCO3- ions (produced within the intestinal cells from metabolic CO2). This generates extremely high concentrations of HCO3- (up to 200 mM) within the intestinal fluid which precipitates imbibed Ca2+ in the gut as insoluble calcium carbonate (CaCO3) which is then excreted. This secretion and precipitation in the intestine plays novel roles in 2 fundamental processes, net water absorption and calcium homeostasis. Whilst intestinal excretion of HCO3- and CaCO3 are crucial adaptations to survival in high salinities, they simultaneously result in a substantial net excretion of base (derived from metabolic CO2) via the intestine. This has important physiological repercussions for other organ systems for which we can form 2 new hypotheses: Hypothesis 1 - Intestinal excretion of HCO3- and CaCO3 represents a novel route of excretion for metabolic CO2 which challenges the dogma of an exclusively gill excretion of this respiratory gas. Hypothesis 2 - It also requires that the fish must excrete an exactly compensatory quantity of acid from the gills, for acid-base homeostasis. Gas Exchange - The fraction of metabolic CO2 excreted via the intestine (as HCO3- + CaCO3) is predicted to increase dramatically in hypersalinities, e.g. more than 50 percent at 90 ppt salinity in flounder. In Aphanius dispar, a killifish which lives in ponds surrounding the Dead Sea, salinity can be 175 ppt and Ca2+ is extraordinarily high (approximately 170 mM). We predict that the majority of its respiratory CO2 may be excreted via this route all the time, rather than the conventional route via the gills. The effects of such an unusual anatomical separation of these two gas fluxes on haemoglobin function could be large. For example, we might expect a very small, or even a total absence of the Root, Bohr or Haldane effects (basis for the normal interaction between O2 and CO2 exchange) which would be highly unusual in teleost fishes. Arterial and venous blood gas and acid-base status, and the transbranchial driving forces for gas exchange in hypersalinities will also be of great interest. Acid-Base Regulation - In Aphanius dispar, the amount of compensatory net acid excretion required continuously via the gills of this extremophile species is predicted to be 5000-9000 uEq/kg/h. This would represent massive acid excretion under resting, steady state conditions (e.g. 5-10 times higher than the maximum rates in seawater fish during recovery from acute acidosis). Hypersalinity therefore represents a unique comparative physiology tool for studying the mechanisms and potentially novel genes involved in gill acid-base regulation in epithelia. This proposal has 2 major objectives, using hypersaline tolerant fish (European flounder and the Aphanius dispar, the Dead Sea Killifish) as ideal non-model species to study these phenomena. Through collaboration with several project partners (in Israel, Portugal, Canada, Liverpool and Manchester) we will use a combination of in vivo and in vitro physiology, together with immunohistochemistry, protein expression, and microarray technology, to establish the novel integration of these fundamental biological processes, and to quantify the up/down regulation of known and novel genes in response to the unusual requirements of acid-base transport and anatomical separation of O2 and CO2 exchange within the gills and intestine of hypersaline fish.

Summary

It is well established that animals take up essential oxygen (O2) and excrete unwanted carbon dioxide (CO2) through their respiratory organs. In humans this is our lungs, but in aquatic animals such as fish these are called gills. Another fundamental process that is essential for life is maintaining a balance between the amounts of acid and base that our bodies make, and the amount we excrete. To maintain a healthy acid-base balance (a constant pH in our cells is essential for normal function) the removal of acids and bases from our various excretory organs (especially kidneys) must be well integrated. We have discovered that fish living in extremely salty (hypersaline) conditions (e.g. flatfish called flounders in drying up marine lagoons, and a small killifish in ponds near the salty Dead Sea in Israel) have to overcome difficulties with dehydration. In doing so they break the normal biological rules regarding the above 2 processes (gas exchange and acid-base balance). This is because they have a great thirst for drinking the salty water they live in which contains large amounts of calcium. As this calcium-rich fluid passes through the intestine, the fish actively secrete a form of carbon dioxide (called bicarbonate ions). This in turn causes the calcium they have drunk to precipitate in a solid form known as calcium carbonate (a mineral also found in limestone rocks). The phenomenon of precipitation is very valuable to the fish as it prevents them absorbing the unwanted calcium into their blood, but also makes it easier for them to absorb the valuable water they have swallowed, and overcome the dehydration effects of living in hypersaline waters. UNUSUAL GAS EXCHANGE: However, the volume of fluid these fish need to drink, and the quantity of calcium present in this fluid, are so great that they need to excrete massive amounts of bicarbonate ions to make sure all the calcium gets precipitated in the gut. Now, the bicarbonate ions (and the precipitated carbonate)are a form of carbon dioxide (CO2), so when this mixture is excreted by the intestine, it breaks the normal rule that fish excrete all their CO2 from their respiratory organ (the gills). This is highly unusual and probably requires an evolutionary adaptation in the way their red blood cells carry respiratory gases such as O2 and CO2 around the body. We aim to study how the molecules that carry these gases in red blood cells (called haemoglobin) have evolved to cope with this anatomical separation of O2 uptake (by the gills) and CO2 excretion (by the intestine). UNUSUAL ACID-BASE BALANCE: As well as being a form of CO2, the bicarbonate ions (and the precipitated carbonate) are bases (opposite of acids), so when this mixture is excreted by the intestine, it also breaks the normal rule that the kidneys do most of the work regarding acid-base balance. We have also discovered that, because the intestine is excreting massive amounts of base, the gills are obliged to balance this by excreting massive amounts of acid at the same time. Normally the gills of fish only excrete acid during very short periods when they are suffering from an acute change in the pH of their body cells (e.g. after intense exercise which produces lactic acid). However, these fish living in hypersaline waters appear to be doing this (excreting acid) all the time, and at extremely high rates. This makes them an ideal species to study the mechanisms by which the gills (and similar organs in humans like our kidney) are able to excrete acid from the body. In this study we aim to examine what genes are important in transporting the acid out of cells, which may lead to the discovery of new genes that we were not previously aware were involved in this process. In the long term this could be useful for biomedical research, if we can understand the human diseases in which people are unable to excrete acid sufficiently and suffer from kidney failure.

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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