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Research Plan – LFRG
The primary focus of research in my lab has been to reveal the physiological mechanisms for maintenance of salt balance in fish native to the extremely ion-poor, acidic waters of the Rio Negro, a major tributary of the Amazon River. The waters of the Rio Negro drain nutrient-poor jungle soils (Furch 1984), have extremely low salt concentrations ([Na+] and [Cl-] < 50 μmol L-^1 , [Ca2+] < 10 μmol L-^1 ), and are very acidic (pH ≤ 4.5). This sort of water chemistry poses significant challenges for freshwater fish that regulate internal salt levels (primarily Na+^ and Cl-) at concentrations far above the surrounding water. If a typical North American or European fish, such as a minnow or trout, were placed in Rio Negro water internal Na+^ and Cl-^ levels would fall rapidly, initiating a cascade of physiological disturbances (Milligan & Wood 1982) that would culminate in death within just a few hours. Yet despite these challenges the Rio Negro is an amazing species rich rivers system, with more species inhabiting its waters than in all the waters of North America combined (Val & Almeida-Val 1995). The question we have been addressing is, how are fish native to the Rio Negro able to maintain ion balance in such extreme conditions? To begin to answer that question we must first briefly review how freshwater fish regulate internal salt concentrations. The gills are the primary organs of salt regulation in freshwater fish and they separate concentrated body fluids from dilute freshwater by a single cell layer (Fig. 1). Consequently salts diffuse out of the blood into the water across the large surface area of the gills. To maintain internal levels above the surrounding water fish must actively transport Na+^ and Cl- into their bodies and this also occurs at the gills. The problem with Rio Negro water is that the dilute and acidic nature of the water tends to interfere with active salts transport and greatly stimulate diffusive losses. To maintain ion balance in ion- poor, acidic waters fish must maintain uptake and/or reduce diffusive loss. Over the last several years our work have identified a suite of specializations in Rio Negro species, including generally reduced stimulatory effects of low pH on diffusive salt loss and reduced inhibitory effects of low pH on active uptake (Gonzalez et al. 1997). However, the most interesting adaptation we have uncovered is that the ion transport mechanisms in two closely related species, neon and cardinal tetras ( Paracheirodon innesi and Paracheirodon axelrodi , respectively), are completely insensitive to pH (Gonzalez & Preest 1999, Gonzalez & Wilson 2001, Preest et al. 2005). Before our work, studies of Na+^ and Cl-^ transport in all other species found that both they are sensitive to low pH and are completely inhibited at pH 4.5 and below (McDonald 1983). In contrast, we found that both Na
and Cl
- uptake in tetras is unaffected down to pH 3.25, the lowest pH we tested. The question we are now pursuing is how this pH insensitivity is achieved at the cellular/molecular level? This question is particularly ATP Blood 140 mM Na+ Water < 1 mM Na+ Na+ 3Na+ H+ Na+ Na+ H+ 2K+
- (^) - Gill Epithelium ATP a b Figure 1. Mechanisms of sodium transport across the gills of freshwater fish.
timely given the current ongoing debate concerning the mechanisms responsible for Na
transport in freshwater fish. Currently there are two competing models for exactly how Na+^ is taken up from the surrounding water into the gill cell. In the older model (option a in Fig. 1 on previous page) Na
is moved into the gill epithelial cell across the water-facing, or apical, membrane, in direct exchange for H+^ by a so-called antiport protein (NHE; Maetz & Garcia-Romeau 1964). The driving force for this exchange is thought to be a low Na
concentration inside the cell produced by the action of Na
/K
- ATPase (NaK), a transport protein that moves 3 Na
- out of the cell in exchange for 2 K+, working on the basolateral (blood facing side) membrane. More recently (option b in Fig. 1) it has been proposed that H
are pumped out of the cells, across the apical membrane, into the water via a H
- ATPase and this creates an electrical potential that draws Na
in through a Na
- specific protein channel (ENaC; Lin & Randall, 1991, 1993, Sullivan et al. 1995). Once inside the gill cell, NaK transports Na+^ out of the cell into the blood as in model a. A notable feature of both models is the necessity for H
excretion into the water, which may explain the sensitivity to pH (water with low pH means it has a high H+^ concentration which makes it more difficult to move H+^ out of a cell into the water) that is commonly observed in non-Rio Negro species. It also raises the intriguing possibility that, given their insensitivity to pH, tetras are employing a novel mechanism that does not require H
excretion. I have applied for sabbatical for next Fall ’09 and if this LFRG is funded I will expand my research focus beyond that described in my regular FRG application to include a research trip to the Rio Negro where I will examine a variety of species collected direction from the river. In the first part of the research (as described in the regular FRG) I propose to continue my work focusing on neon and cardinal tetras with an integrative approach that utilizes physiological, biochemical, immunohistochemical, and molecular biology techniques to identify, localize, and functionally characterize the transport proteins present on the tetra gills. We have already begun our investigation by measuring rates of Na+^ transport in blackskirt tetras ( Gymocorhymbus ternetzi ), a relative of neon and cardinal tetras, exposed to pharmacological agents that are highly specific inhibitors of various components of the transport models described above. That work has yielded surprising results, suggesting that while EnaC is involved in Na+^ transport, H+- ATPase is not. We must now repeat these experiments with neon and cardinal tetras. Our next step will then be to survey the gills of the tetras with antibodies that have been raised for NaK, ENaC, NHE, and H+-ATPase to visualize whether these transporters are present, and if so where. To do this work, gills will be excised from fish and after fixation they will be incubated with antibodies that have been raised specifically for the various protein transporters. A second antibody, specific for the first and with a visible color tag, is then applied allowing us to localize the presence of the different transporters with a microscope. We have already begun this work as well with cardinal tetras and it appears that ENaC and NAK are present, but not H
- ATPase or NHE. The absence of H
- ATPase has been supported with western blotting techniques in which antibodies are applied to proteins separated on an electrophoresis gel. Still we need to do further work to confirm these findings as well as to support our other results. Ultimately we hope to clone the genes for the transporters and compare their nitrogenous base sequence to other transporter genes in the database. Then we will be able to look at aspects of expression of the genes in low pH waters. The second goal will be to assess whether the transporters we describe in neon and cardinal tetras are found in other species of fish native to the Rio Negro. To do this I will travel
Preest, M., R.J. Gonzalez & R.W. Wilson. 2005. A pharmacological examination of the Na
and Cl
- transport mechanisms in freshwater fish. Physiological and Biochemical Zoology 78: 259 - 272. Val A.L. and V.M.F. de Almeida-Val. 1995. Fishes of the Amazon and Their Environment. Springer-Verlag, Berlin.
Budget Explanation
I am requesting money to purchase fish and supplies for the work described above. Tissue fixation supplies include supplies and chemicals for fixing, embedding, sectioning, and mounting samples for analysis. Several different antibodies that are specific for different transporter proteins will be purchased along with several staining kits. Molecular supplies include pre-cast gels, membranes and chemicals required for the Western blotting procedure. I have increased the amounts relative to the regular FRG budget to purchase additional supplies to take to Brazil. I also request 4 days rental of the Amanai II research vessel at $2,000 per day. Others will pay for additional days.
Budget
Item
Cardinal tetras, 70 @ $5.00 ea. $350. Neon tetras, 70 @ $2.00 ea 140. Tissue Fixation Supplies 1250. Antibodies 1625. Localization Kits 1455. Molecular Supplies 1750. Rental of Research Vessel 8000. TOTAL $14,
CURRICULUM VITAE
RICHARD J. GONZALEZ
Address Personal Information
Department of Biology Date of birth: August 27, 1959 University of San Diego Place of birth: Chicago, Illinois 5998 Alcalá Park Marital Status: Married, one child San Diego, CA 92110 U.S.A. Phone#: (619) 260- 4077 Fax#: (619) 260- 6804 e-mail: gonzalez@sandiego.edu
Education/Honors
University Professor. 1999. University of San Diego. Ph D. in Biology (1989). The Pennsylvania State University. Thesis Advisor-William A. Dunson. Minority Student Fellowship from the Graduate School, The Pennsylvania State University, 1983/1984. B.S. in Organismal Biology, (1981). The University of Kansas, with departmental honors and distinction.
Professional Experience
2007 – Present: Chair, Department of Biology, University of San Diego.. 2005 – Present: Professor, Department of Biology, University of San Diego. 1998 – 2005: Associate Professor, Department of Biology, University of San Diego. 1992 – 1 998: Assistant Professor, Department of Biology, University of San Diego. 1991 – 1992: Postdoctoral Researcher/Lecturer. University of Chicago, Department of Organismal Biology and Anatomy. 1990 – 1991: Postdoctoral fellow in the lab of Gordon McDonald, Department of Biology, McMaster University. 1988: Research Assistant, Department of Biology, The Pennsylvania State University. 1984: Research Assistant, Rutgers University and New Jersey Department of Fish and Game, Endangered Species Program. 1981: Research Assistant, Department of Systematics and Ecology, The University of Kansas.
Teaching Experience
The University of San Diego Biology 478 – Vertebrate Physiology, Lecture and Lab Biology 190 – Evolution, Genetics, and Ecology, Lecture Biology 225L – Principles of Cell Biology Laboratory
Preest, M., R.J. Gonzalez & R.W. Wilson. 2005. A pharmacological examination of the Na
and Cl
- transport mechanisms in freshwater fish. Physiological and Biochemical Zoology 78: 259 - 272. Brauner, C., T. Wang, Y. Wang, J.G. Richards, R.J. Gonzalez , N.J. Bernier, W. Xi, M. Patrick & A.L. Val. 2004. Limited extracellular but complete intracellular acid-base regulation during short-term environmental hypercapnia in the armored catfish, Liposarcus pardalis. Journal of Experimental Biology 207: 3381 – 3390. Sardella, B., J. Cooper, R. Gonzalez & C.J. Brauner. 2004. The effect of temperature on juvenile Mozambique tilapia hybrids ( Oreochromis mossambicus x O. urolepis hornorum ) exposed to full-strength and hypersaline seawater. Comparative Biochemistry and Physiology Part A, 137: 621 – 629. Sardella, B., V. Matey, J. Cooper, R. Gonzalez & C.J. Brauner. 2004. Physiological, biochemical, and morphological indicators of osmoregulatory stress in ‘California’ Mozambique tilapia ( Oreochromis mossambicus x O. urolepis hornorum ) exposed to hypersaline water. Journal of Experimental Biology 207: 1399 - 1413. Wood, C.M., A.Y.O. Matsuo, R.W. Wilson, R.J. Gonzalez , M.L. Patrick, R.C. Playle & A.L. Val. 2003. Protection by natural blackwater against disturbances in ion fluxes caused by low pH exposure in freshwater stingrays endemic to the Rio Negro. Physiological and Biochemical Zoology 76: 12 – 27. Wang, Y., R.J. Gonzalez , M.L. Patrick, M. Grosell, C.G. Zhang, Q. Feng, J.Z. Du, C.M. Wood, & P.J. Walsh. 2003. Unusual physiology of scaleless carp, Gymnocypris przewalskii , in Lake Qinghai: A high altitude alkaline saline lake. Comparative Biochemistry and Physiology Part A 134: 409 – 421. Wood, C.M., A.Y.O. Matsuo, R.J. Gonzalez , R.W. Wilson, M.L. Patrick & A.L. Val. 2002. Mechanisms of ion transport in Potamotrygon , a stenohaline freshwater elasmobranch native to the ion-poor blackwaters of the Rio Negro. Journal of Experimental Biology 205: 3039 - 3054. Patrick, M.L., R.J. Gonzalez , C.M. Wood, R.W. Wilson, T.J. Bradley & A.L. Val. 2002. The characterization of ion regulation in Amazonian mosquito larvae: evidence of phenotypic plasticity, population-based disparity and novel mechanisms of ion uptake. Physiological and Biochemical Zoology 205: 223 - 236. Patrick, M.L., R.L. Ferreira, R.J. Gonzalez , C.M. Wood, R.W. Wilson, T.J. Bradley & A.L. Val.
- Ion regulation patterns of mosquito larvae collected from breeding sites in the Amazon rainforest. Physiological and Biochemical Zoology 75: 215 - 222. Gonzalez , R.J., C.M. Wood, R.W. Wilson, M.L. Patrick, & A.L. Val. 2002. Diverse strategies of ion regulation in fish collected from the Rio Negro. Physiological and Biochemical Zoology 75: 37 - 47. Patrick, M.L., R.J. Gonzalez & T.J. Bradley. 2001. Sodium and chloride regulation in freshwater and osmoconforming larvae of Culex mosquitoes. Journal of Experimental Biology 204: 3345 - 3354. Gonzalez , R.J., L. Milligan, A. Pagnotta* & D. G. McDonald. 2001. Effect of air breathing on acid-base and ion regulation after exhaustive exercise and during low pH exposure in the bowfin, Amia calva. Physiological and Biochemical Zoology 74: 502 - 509. Gonzalez R.J & R.W. Wilson. 2001. Patterns of ion regulation in acidophilic fish native to the ion-poor, acidic Rio Negro. Journal of Fish Biology 58: 1680 - 1690. Gonzalez , R.J. & D.G. McDonald. 2000. Ionoregulatory responses to temperature change in two species of freshwater fish. Fish Physiology and Biochemistry 22: 311 – 317.
Wilson, R.W., C.M. Wood, R.J. Gonzalez , M.L. Patrick, H. Bergman, A. Narahara & A.L. Val.
- Net ion fluxes during gradual acidification of extremely soft water in three species of amazonian fish. Physiological and Biochemical Zoology 72: 277 - 285. Gonzalez , R.J. & M. Preest. 1999. Mechanisms for exceptional tolerance of ion-poor, acidic waters in the neon tetra ( Paracheirodon innesi ). Physiological and Biochemical Zoology 72: 156 - 163. Wood, C.M., R.W. Wilson, R.J. Gonzalez , M.L. Patrick, H. Bergman, A. Narahara & A.L. Val.
- Responses of an Amazonian teleost, the tambaqui ( Colossoma macropomum ) to low pH in extremely soft water. Physiological Zoology 71: 658 - 670. Gonzalez , R.J., C.M. Wood, R.W. Wilson, M. Patrick, H. Bergman, A. Narahara & A.L. Val.
- Effects of water pH and Ca 2+ concentration on ion balance in fish of the Rio Negro, Amazon. Physiological Zoology 71: 15 - 22. Gonzalez , R.J., V. M. Dalton* & M.L. Patrick. 1997. Ion regulation in ion-poor, acidic water by the blackskirt tetra ( Gymnocorymbus ternetzi ), a fish native to the Amazon River. Physiological Zoology 70: 428 - 435. Gonzalez , R.J., J. Drazen, S. Hathaway, B. Bauer* & M. Simovich. 1996. Physiological correlates of water chemistry requirements in fairy shrimps (anostraca) from southern California. Journal of Crustacean Biology 16: 286 - 293. Gonzalez , R.J. & D.G McDonald. 1994. The relationship between oxygen uptake and ion loss among fish from diverse habitats. Journal of Experimental Biology 190: 95 - 108. Feder, M.E., R.J. Gonzalez , T. Robbins* & C.R. Talbot. 1993. Bulk flow of the medium and cutaneous sodium uptake in frogs: Potential significance of sodium and oxygen boundary layers. Journal of Experimental Biology 174: 235 - 246. Gonzalez , R.J. & D.G. McDonald. 1992. The relationship between oxygen consumption and ion loss in a freshwater fish. Journal of Experimental Biology 163: 317 - 332. Gonzalez , R.J. & W.A. Dunson. 1991. Does water pH control habitat segregation of sibling species of sunfish ( Enneacanthus )? Wetlands 11: 313 - 323. McDonald, D.G., J. Freda, V. Cavdek, R.J. Gonzalez & S. Zia. 1991. Interspecific differences in gill morphology of fresh water fish in relation to tolerance of low pH environments. Physiological Zoology 64: 124 - 144. Gonzalez , R.J., R.S. Grippo & W.A. Dunson. 1990. The disruption of sodium balance in brook trout by manganese and iron. The Journal of Fish Biology 37: 765 - 774. Gonzalez , R.J. & W.A. Dunson. 1989. Mechanisms for tolerance of sodium loss during exposure to low pH of the acid-tolerant sunfish Enneacanthus obesus. Physiological Zoology 62: 1219 - 1231. Gonzalez , R.J. & W.A. Dunson. 1989. Differences in low pH tolerance among closely related sunfish of the genus Enneacanthus. Environmental Biology of Fishes 26: 303 - 310. Gonzalez , R.J., C.H. Mason* & W.A. Dunson. 1989. Anomalous tolerance of low pH in a euryhaline killifish, Fundulus heteroclitus. Comparative Biochemistry and Physiology 94C: 169 - 172. Gonzalez , R.J. & W.A. Dunson. 1989. Acclimation of sodium regulation to low pH and the role of calcium in the acid-tolerant sunfish Enneacanthus obesus. Physiological Zoology 62: 977 - 992. Gonzalez , R.J. & W.A. Dunson. 1987. Adaptations of sodium balance to low pH in a sunfish ( Enneacanthus obesus ) from naturally acidic waters. Journal of Comparative Physiology 157B: 555 - 566. Freda, J. & R.J. Gonzalez. 1986. Daily movements of the treefrog Hyla andersoni. Journal of Herpetology 20: 469 - 471.
