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Permanent link (DOI): https://doi.org/10.7939/R3BR8MQ2D

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Respiration, Acid/Base, Ammonia and Ionoregulatory Strategies in the Pacific hagfish (Eptatretus stoutii) Open Access

Descriptions

Other title
Subject/Keyword
Hagfish
Agnathan
Acid
Base
Ammonia
Respiration
Ionoregulation
Regulation
Homeostasis
Ammonium
Carbonic Anhydrase
Skin
Branchial
Gill
Cutaneous
Oxygen
Mitochondrial Rich Cell
MRC
Proton
Nitrogen
Urea
Rh glycoprotein
Nernst Potential
Active transport
Osmoconform
Cyclostome
Bicarbonate
Recovery
Compensation
Evolution
Transport
Physiology
Excretion
Exercise
EPOC
Ion Transport
Hypercapnia
Hypercarbia
Blood gas
Metabolism
NHE
Sodium Proton Exchanger
Chloride Bicarbonate Exchanger
Sodium
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Clifford, Alexander M
Supervisor and department
Goss, Greg (Biological Sciences)
Examining committee member and department
Gallin, Warren (Biological Sciences)
Young, James (Physiology)
Allison, Ted (Biological Sciences)
Wright, Patrica (University of Guelph, Integrative Biology)
Department
Department of Biological Sciences
Specialization
Physiology, Cell and Developmental Biology
Date accepted
2016-09-12T15:20:55Z
Graduation date
2016-06:Fall 2016
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Hagfish feed on putrefied carrion, which poses several environmental challenges to the scavenger including hypoxia (Low PO2), hypercapnia (high PCO2) and high environmental ammonia (HEA). To any other organism, these conditions would be physiologically challenging; however, hagfish seem to have adapted to survive and thrive in this type of environment. While feeding, hagfish immerse their head and gills in the decaying flesh leaving their trunk exterior to the carcass, allowing for potential cutaneous exchange of acid/base (A/B) equivalents, ammonia and O2. Hagfish are osmoconformers and do not regulate plasma [Na+] or [Cl-] but do regulate the divalent ions SO42-, Mg2+ and Ca2+; however, the hormone(s) controlling this regulation have remained elusive. In this thesis, I describe some of the physiological strategies that Pacific hagfish (Eptatretus stoutii) employ to withstand and recover from stresses in A/B status, and exposure to ammonia and hypercapnia. Furthermore I identify the relative branchial and cutaneous contributions to overall maintenance of A/B status, ammonia excretion, and O2 acquisition through the use of custom designed separated-flux chambers. Finally I characterize the glucocorticoid and mineralocorticoid responses of the hagfish and identify the role of the corticosteroids previously shown to act on hagfish corticosteroid receptor. An environment with a high PCO2 will initially cause acidification of the blood followed by compensatory metabolic plasma [HCO3-] elevations. I simulated these perturbations in hagfish through injections of either HCl or NaHCO3 (total H+/HCO3- load: 6000 µmolkg-1). Hagfish excreted the acid load via excretion of acid equivalents in the gill region while the resultant base load was excreted in the skin-only posterior region, indicating that the gills may not be involved in recovery from acute metabolic hypercarbia. However, following chronic hypercapnia exposure (72 h; 0.6% CO2) to induce hypercarbia, hagfish utilized both gill and skin mechanisms to similar degrees for HCO3- excretion, indicating that the gills are capable of excreting base equivalents. Hagfish exposed to chronic hypercapnia (48 h; 4% CO2) compensated for blood acidosis by mounting an ~70 mmol L-1 plasma [HCO3-] response. Upon reintroduction into normocapnic seawater, plasma [HCO3-] was rapidly corrected within 8 h. This correction occurred with impressive rates of plasma HCO3- loss (at peak: ~10 mmol kg-1 h-1) occurring primarily via carbonic anhydrase mediated CO2 offloading with only minor contributions from direct HCO3-/Cl- flux and insignificant contributions from increased glomerular filtration. During 48 h HEA (20 mmol L-1 [TAmm]; [total ammonia]) exposure, plasma [TAmm] increased 100-fold to over 5000 μmol L-1 while ammonia excretion (JAmm) was transiently inhibited. Plasma [TAmm] stabilized after 24–48 h exposure, possibly through lowering NH3 influx by reducing body NH3 permeability. Ammonia balance was subsequently maintained through the reestablishment of JAmm against inwardly directed ΔPNH3 and NH4+ electrochemical gradients. Restoration of JAmm by the hagfish during ammonia exposure likely involves secondary active transport of NH4+, possibly mediated by Na+/NH4+ (H+) exchange. Recovery from HEA in ammonia-free water was characterized by considerable ammonia washout, and restoration of plasma [TAmm] within 24 h. During recovery, JAmm occurred via branchial (70-80%) and cutaneous (20-30%) mechanisms. Excised hagfish skin fluxes revealed concentration-dependent (0.05 – 5 mmol L-1) JAmm with 8-fold greater JAmm across skin excised from HEA-exposed hagfish. Using immunohistochemical staining with a hagfish-specific Rhcg (ammonia/ammonium transporter) antibody, I demonstrated that Rhcg is present in cutaneous epidermal layers consistent with physiological data. Cutaneous O2 uptake by the hagfish has been previously suggested as the major site of systemic O2 acquisition. However, I show that hagfish rely primarily (85%) on branchial O2 uptake. When the branchial region was locally immersed in hypoxic conditions (4.6 kPa O2), hagfish did not utilize cutaneous mechanisms to acquire available O2 in the normoxic anterior chamber, suggesting that even when presented with restricted O2 availability, hagfish do not use cutaneous mechanisms to supplement metabolic O2 requirements. Using handling stress and mineral challenges (SO42- injections), I show that hagfish are capable of eliciting glucocorticoid and mineralocorticoid responses. Furthermore I show that these responses are not mediated by alterations in plasma cortisol, corticosterone, 11-deoxycorticosterone or 11-deoxycortisol. Overall in this thesis, I show that hagfish have developed novel cutaneous A/B and ammonia handling mechanisms along with extraordinary tolerances and effective recovery strategies to cope with exposure to extreme perturbations (e.g. hypercapnia, hypoxia, HEA). It is these strategies and mechanisms that allow this unique organism to survive and thrive in their demersal environment.
Language
English
DOI
doi:10.7939/R3BR8MQ2D
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
Clifford, A.M., Goss, G.G., Roa, J.N., Tresguerres, M., 2015. Acid/base and ionic regulation in hagfish, in: Edwards, S.L., Goss, G.G. (Eds.), Hagfish Biology. CRC Press, Boca Raton, Fl, pp. 277–298. doi:10.1201/b18935-12Clifford, A.M., Guffey, S.C., Goss, G.G., 2014. Extrabranchial mechanisms of systemic pH recovery in hagfish (Eptatretus stoutii). Comp. Biochem. Physiol. A 168, 82–89. doi: 10.1016/j.cbpa.2013.11.009.Clifford, A.M., Goss, G.G., Wilkie, M.P., 2015b. Adaptations of a deep sea scavenger: High ammonia tolerance and active NH4+ excretion by the Pacific hagfish (Eptatretus stoutii). Comp. Biochem. Physiol. A 182C, 64–74. doi:10.1016/j.cbpa.2014.12.010Clifford, A.C., Zimmer, A.M., Wood, C.M., Goss, G.G. 2016. It’s all in the gills: Evaluation of O2 uptake in Pacific hagfish refutes a major respiratory role for the skin. Journal of Experimental Biology. In Press, doi: 10.1242/jeb.141598

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