Comp. Blochem. Physiol., 1975, Vol. 50A, pp. 131 to 134. Pergamon Press. Printed tn Great Britain
CARBONIC ANHYDRASE IN SOME CORAL REEF FISHES: ADAPTATION TO CARBONATE INGESTION? R. L. SmTH A N D A. C. PAULSON Biology Department, University of Alaska, Fairbanks, Alaska 99701, U.S.A. (Received 1 October 1973)
Abstraet--l. Carbonic anhydrase aetivity was measured in three speeies of coral reef fishes, Acanthurus triostegus, Holocentrus spinlfer and Scarus Jonesl. 2. Significant activity was found in extracts of blood, gill, gut contents, gut mueosa and bile. 3. Intestinal mucosa of the two calcium carbonate ingesting species was twelve times as active as that of the other species, which does not ingest carbonate. 4. In Scarus Jonest, significant differences exist in the intestinal mucosa activities of feeding vs fasted individuals, 5. Our data are consistent with the hypothesis that the ingestion of calcium carbonate may increase the COt load and, therefore, necessitate high levels of gut carbonic anhydrase.
INTRODUCTION A WIDE variety of coral reef fishes ingest moderate to large amounts of calcium carbonate in their diets. Some groups such as the Mullidae (goatfishes), Balistidae (triggerfishes) and Tetraodontidae (puffers) take in rather coarse hits of mollusc shell and fragments of coral skeleton, Others, including the Acanthuridae (surgeonflshes) and Scaridae (parrotfishes) ingest much finer material, usually in the form of finely ground coral rubble or sediments (Hiatt & Strasburg, 1960; Randall, 1967). In the West Indies, Randall (1967) found that up to 75 per cent of the gut contents by weight in most parrotfishes and grazing surgeonfishes consisted of inorganic sediment, presumably calcium carbonate. The anatomy of the alimentary tract of parrotfishes and surgeonfishes is well adapted to this carbonate ingestion. Parrotfishes have massive, parrot-like beaks of fused teeth for cropping the substratum, sets of pharyngeal bones which act as mills for grinding sediment and algae and have no stomachs (AI-Hussaini, 1945; Schultz, 1958; Gohar & Latif, 1963). Surgeons have less massive but welladapted dentition, lack pharyngeal mills and have well-developed stomachs which in grazing species constitutes a thick-walled gizzard (Jones, 1968). The gizzard in grazing acanthurids probably serves a triturating function much like the pharyngeal mill of scarids. There may well be other physiological and biochemical adaptations which suit these fishes for their diet heavy with carbonate particles. This study centers around these latter two groups. We hypothesize that some of the carbonate material ingested by surgeonfishes and parrotfishes is dissolved in the digestive tract and may contribute
to the COt load existing on the COt transport mechanisms of these fishes. Carbonic anhydrase levels in tissues associated with the digestive tract might reflect the magnitude of the COt load generated by carbonate solution. Therefore, we analyzed carbonic anhydrase in blood, gill, gut and gut contents to test this hypothesis. MATERIALS AND METHODS This study was conducted at Eniwetok Atoll, Marshall Islands. Three species contributed data to the study, Acanthurus triostegus, ttolocentrus spinlfer and Scarus jonesL S. ]onesi crops large amounts of CaCOa from the substratum while feeding on algae. A. triostegus ingests only the carbonate particles entangled in the algae it eats. H. spinifer is a predator and takes in very little CaCOa. Thus, differences in enzyme levels might be attributed to the presence of CaCOa in the diet. Fish were obtained by three methods: spear, cast net and hand net. All fish were returned to the laboratory immediately where enzyme analyses were performed on speared fish within 2 hr. Analyses on the digestive tract of A. triostegus were carried out 2 days after capture, as were those on fasted S. Jonesi. Crude enzyme extracts were prepared by macerating the tissues in a glass tissue grinder with 50 vol. of distilled water. Extracts of blood, bile and gut contents were made by shaking vigorously with either 50 or 100 vol. of water depending on the activity of the fluid. These solutions were allowed to extract for 30 rain at 2-4~ before the assays were completed. The assay procedure used was a modification of the indicator method of Maren (1960), Our modifications included using 0.1 ml of an indicator containing 12.5 mg ~o phenol red instead of 0.4 ml of a much weaker dye solution. Rather than 0.1 ml of a buffer consisting of 1 M NatCOa and 1 M NaHCOa, we used 0.4 ml of buffer 131
132
R . L . SMITH AND A. C. PAULSON
made up of 2 M in NaaCOa and NaHCOs, These alter/ttions reduced the sensitivity of the original method approximately eightfold while yielding a typical sere/log calibration curve and uncatalyzed reaction times of about 60see. The calibration curve was constructed wlth known dilutions of a carbonic anhydrase standard (Worthington carbonic anhydrase, 2000 EU/mg). Inhibition experiments were conducted with 0.1 ml of a 0.2 mg diamox (acetazolmide) solution. Analyses of single extracts were performed in triplicate usually yielding values within 5 per cent of each other. Enzyme concentrations are expressed in enzyme units (EU) per ml wet tissue making our data easily comparable with much of the existing literature on carbonic anhydrase reviewed by Maren (1967). Means of data were compared with t-tests. The 95 per cent confidence interval was used in all tests of significance. RESULTS
Table 1 presents the results of five separate lnhlbl. tion experiments utilizing enzyme preparations from blood, small intestine mucosa and large intestine
intestine. There was, however, a significant difference between activities of contents vs mucosa in both small and large intestines. Both A. trlostegus and S. jones/ had much greater carbonic anhydrase levels in the intestinal mucosa than did I-I. splnlfer. S. jones/also had much more enzyme in the gill than did H. splnlfer. Table 3 compares enzyme activities from mucosa S. /ones/ speared during feeding activity with those of individuals caught alive and maintained in a fasted condition for 2 days. These data indicate that the gut mucosa of fasted S. /ones/ had significantly higher enzyme levels than in feeding individuals. The groul~ of fasted S,/onesl is the appropriate one to compare with A. trlostegus and H, splnlfer since A. trlostegus was also fasted and the H, aplnlfer guts were empty. Holocentrus sphtlfer is a nocturnally active species and was speared at dusk, suggesting that no food had been eaten for at least 12hr. Comparison of mueosal activity in H, sphtlfer with A, trlostegus, S, jonesl small intestine and S,/oneM large intestine yields t values of 7.13, 6.39 and 18,9 respectively, all
Table 1, Diamox inhibition of carbonic anhydrase in paired expel'iments utilizing enzyme extracts from small Intestine, large intestine and blood of S. ]tn,r Experiment Small intestine extract 1 Extract 1 + dlamox Small intestine extract 2 Extract 2 + dlamox Large intestine extract 1 Extract 1 +diamox Large intestine extract 2 Extract 2+ diamox Blood extract Blood extract + diamox mucosa of S. /ones/. In all experiments diamox reduced or eliminated observable carbonic anhydrase activity. Carbonic anhydrase activities for the extracts from the three study species are presented in Table 2. Also included are the negative results of enzyme assays performed on sea water, algae and dead coral skeleton. These data indicate that all the preparations from the fishes exhibited carbonic anhydrase activity, while extracts from sea water, dead coral skeleton and algae exhibited no activity, Student's t-tests were performed on the data in Table 2. These tests indicate that the blood of A. trfostegus has a significantly higher enzyme activity than does the blood of S. /onesi, while the gut contents of the two species do not differ in activity. In S. jones/, there is no difference in activities of intestinal contents from small vs large intestine. Neither is there a significant difference between activities from mucosal extracts of small vs large
Enzyme activity (EU/ml wet tissue) 360 126 531 63 522 0 594 81 1098 0 of which are significant when compared to the critical t 0.05 value of 2.92. DISCUSSION
The enzyme assays and inhibition experiments reported above indicate that significant carbonic anhydrase activity exists irt the blood, gill, bile, intestine contents and gut mucosa of the fishes studied. Since there was no demonstrable enzyme activity in the sea water, algae and coral rock normally ingested by A. triostegus and S./onesl (Table 2) we conclude that the carbonic anhydrase in bile, mucosa and gut contents are all endogenous. Comparisons with the existing literature indicate that our gill activities are within the range of reported values for teleostean gills while whole blood activities for S. Jonesl and A. trlostegus equal and exceed, respectively, maximum literature values for teleosts as well as mammals (Maren, 1967). These
Carbonic anhydrase in some coral reef fishes
133
Table 2. Carbonic anhydrase activity from tissues, gut contents and blood of three coral reef fishes. Analyses of algal extracts and sea water are also included EU/ml wet tissue 5:1 S.D.
N
t
A. trtostegus ]. 2. 3. 4.
Wholeblood Stomach contents Intestine contents Intestine mucosa
2486_+662 855:70 1095:30.5 445_+108
11 2 10 2
1, 7 7.53 (+) 2, 3 0.82 ( - ) 3,9 1.78 ( - )
225+80 365:9
4 3
853 + 193 842+6
11 2
1055:55 72-1-52
8 8
9, 10 1.15 ( - ) 10, 12 2,62 (+)
]704-51 1575:75 595:45
12 11 9
9, 11 2.56 (+) 11, 12 0.47 ( - ) 9, 13 1.78 ( - )
H. spinifer 5. Gill 6. Intestine mucosa
5, 8 17.7 (+) 6, II 4.2 (+)
S. jonesi 7. Whole blood 8. Gill Intestine contents 9. Small intestine I0, Large intestine Intestine mucosa 11. Small intestine 12. Large intestine 13. Bile Algae Sea water Dead coral skeleton
0 0 0
3 3 3
Values of t were obtained by comparing the two experiments whose numbers in,unediately precede the t value. Significant differences are indicated by (+). The 95 per cent confidence interval was used on all t-tests. Table 3. Comparison of carbonic anhydrase activity in gut extracts from feeding and fasting S. jonesi Enzyme activity (EU/ml wet tissue) Extract Small intestine Large intestine
Feeding
Fasted for 2 days
t
170+ 51 (N= 12) 157-t-75 ( N = 11)
445 + 120 ( N = 2) 558+51 ( N = 2)
6.83 (+) 5'81 (+)
Values of t indicate fasted fishes had significantly higher enzyme levels than feeding individuals. data apparently conglict with Randall's (1970) statement that carbonic anhydrase activity in fish blood is only about 2 per cent of that in mammals. No significant differences in mucosal enzyme activities from different parts of the intestine are in evidence in the present study. Significant differences have been observed in the rat and dog (Kuriaki & McGee, 1964; Maren, 1967). Carbonic anhydrase values for the intestinal mucosa of S. janesi and A. triostegus exceed reported values for rat colon, chicken small intestine and frog intestine (Maren, 1967; van Goor, 1948). The activity we report in parrotfish bile is in contrast to van Goor's (1948) findings for animals in general. The intestinal mucosa of the two carbonate ingesting species had twelve times the activity present in H. sptntfer while the gill in S. jonesi had only three times that found in H. spinifer. This threefold difference might be attributed to differences in
metabolic activity levels, which are known to be reflected in blood carbonic anhydrase levels and hemoglobin levels (Albers, 1970). However, differences in metabolic rates, if they exist, are insufficient to account for the entire twelvefold difference in mucosa activity. These data are consistent with our hypothesis that calcium carbonate ingestion results in solution of calcium carbonate; and that solution adds to the load on the CO~ transport mechanisms of these fishes. The added load constitutes a selective pressure which lead to higher carbonic anhydrase levels in the intestinal mucosa of carbonate ingesting fishes. We hypothesize a mechanism in which solution of CaCO~ occurs in the presence of weak acids in the lumen of the fish gut. In such a reaction as proposed by Meldrum & Roughton (1933): CaCOa+HA
> CaA2+HaCO8
R. L, SMtTrl AND A, C, PAULSON
134
and ~he rate of solution would be affected by removal of one of the products, HsCOs, by the action of carbonic anhydrase: ~rbonlo
HiCO~
> HaO + C O n
snhy'draao
Department of Biological Sciences and by Dr. Donatd
The products of this second reaction could diffuse into the mucosal ceils and be converted to bicarbonate and hydrogen ions in another carbonic anhydrase facilitated reaction. This hypothesis attaches functional significance to the carbonic anhydrase in intestinal contents and in the mucosa. These findings have suggested the possibility that HCOa-, H~O and CO2 may be involved in electrolyte transfers across the intestinal mucosa. Figure 1 incorporates these reactions into a hypothetical scheme similar to that Lumen
Mueesa
Na*~Na
+
Plasrr ', '
k NHqt"
NH4 I' NH~" 9H~"
CaCO§ HA
~^
c,o~L~ H O+CO --H CO
cL- ct-
deknow/etlgemant.c--Thts research was supported by the U.S. Atomic Energy Commission, Erdwetok Madnt Biological Station through a grant to the Hawaii Institute of Marine Biology. We would like to thank Dr, Philip Helfrich, Director E,M,B.L., for his encouragement and assistance, Material assistance was also provided by the
HOe;--
-- HCO-
3
Fig. 1. Hypothetical scheme of CaCO B solution In the fish intestine and associated electrolyte transfers across the mucosa. Carbonic anhydrase (e.a.) would facilitate the reactions indicated as well as the exchange of HCOafor CI-. Carbonic anhydrase dependent movements of HCOa- and CI- across the mammal intestine have previously been reported (Maren, 1967). proposed for electrolyte transfers across the fresh water teleost gill. (Maetz & Garcia-Romeu, 1964). The gill of marine teleosts cannot transfer electrolytes by exchange diffusion since both Na + and CIinflux would be required in exchange for NI-I4+ and I-ICO8- efflux. However, just such an influx occurs in the gut of marine teleosts (Smith, 1953). The proposed mechanism would have the gut excreting HCO.~- and possibly NH4 +. Carbonic arthydrase is known to facilitate the ion exchanges in the fresh water gill (Maetz & Garcia-Romeu, 1964) and is present in the mucosa of all three marine species tested in this study. The scheme presented in Fig, 1 remains to be tested. We have discussed some other lines of evidence which indicate that calcium carbonate dissolves inside the parrotfish gut (Smith & Paulson, 1974). We mentioned the possibility that the solution of calcium carbonate has nutritional significance, releasing organic material trapped inside the calcium carbonate matrix. Thus, physiologically, carbonate ingestion might well be both an asset and a liability.
Hood, Director, Institute of Marine Sciences, University of Alaska. Special thanks are due to Dr. Ariel Roth, Biology Department, Loma Linda University, for kindly providing the diamox, We also thank R. Rimiticado for helping us catch fish, John Bradbury for constructing the glass reaction chambers and Dr. Russell L. Shoemaker, University of Alaska, for his critical comments on the manuscript, REFERENCES ALilaRs C, (1970) Acid-base balance. In Fish Physiology (Edited by HOAg W, S. & RANDALLD. J.), Vol, IV, pp, 173-208, Academic Press, New York. AL-HUsSAINt A, H, (1945) The anatomy and histologyof the alimentary tract of the coral feeding fish, Status sordldus Klunz. Bull. Inst..E~cyptn 27, 349-:377, OolrAIt H, A, F, & LATIF A, F, A. (1963) Dlgestiv0 proteolytlc enzymes of some searid and labrid fishes (from the Red Sea). Publ. Mar. BIoL Sta. AI.Ghardaqa 12, 4---42, HIAT'r R, W. & STRASaUaO D. W. (1960) Ecological relationships of the fish fauna on coral reefs of the Marshall Islands, EcoL Monogr, 30, 65-127, JONtm R, S, (1968) Ecological relationships In Hawaiian and Johnston Island Acanthuridae (surgeonfishcs), Micron#sign 4, 309-361, KLIR1AKI K. die, MAonlt D. F. (1964) On the carbonic anhydrasr activity of the alimentary canal and pancreas, Life Sc(,3, 137%1382, MA~Z J, & OARCIA-ROM~UF. (1964) The mechanismof sodium and chloride uptake by the gills era freshwater fish, Carasshts auratus~ll. Evidence of NH4+/Na+ and HCOtF/CI- exchanges J, gem Physiol. 47, 12091227. MAR~N T. H. (1960) A simplified micromethod for the detemaination of carbonic nnhydrase and its inhibitors. d. Pharmac. exp. Ther. 730, 26-29. MAREN T. H. (1967) Carbonic anhydrase: chemistry, physiology, and inhibition. PhysloL ReD. 47, 595-781. MSLDROMN. U. & Rovoh"roN F. J. W. (1933) Carbonic anhydrase. Its preparation and properties, J. PhysloL, Lend. 80, 113-142. RANDALLD. J. (1970) Gas exchange in fish. In Fish Physiology (Edited by HoA'~ W. S. & RANDALLD. I.), Vol. IV, pp. 253-292. Academic Press, New York, RANDALLL E. (1967) Food habits of reef fishes of the West Indies. Stud. Trop. Oceanogr. 5, 665-847. SCHUErz L. P. (1958) Review of the parrotflshes, fatally Scaridae. U.S. Nat. Mus. Bull 214, 1-141. SMrrra H, W. (1953) From Fish to Philosopher. Little, Brown, Boston, SMrrrl R. L. & PAUL.SONA, C. (1974) Food transit times and gut pH In two Pacific parrot fishes, Cot~ela, (In press,)
VANGOORI-I. (1948) Carbonic anhydrase, its properties, distribution and significance for carbon dioxide transport. Enzymologla 31, 73-164. Key Word Index--Carbonic anhydrase; coral reef fish', carbonate.
CARBONIC ANHYDRASE IN SOME CORAL REEF FISHES: ADAPTATION TO CARBONATE INGESTION? R. L. SmTH A N D A. C. PAULSON Biology Department, University of Alaska, Fairbanks, Alaska 99701, U.S.A. (Received 1 October 1973)
Abstraet--l. Carbonic anhydrase aetivity was measured in three speeies of coral reef fishes, Acanthurus triostegus, Holocentrus spinlfer and Scarus Jonesl. 2. Significant activity was found in extracts of blood, gill, gut contents, gut mueosa and bile. 3. Intestinal mucosa of the two calcium carbonate ingesting species was twelve times as active as that of the other species, which does not ingest carbonate. 4. In Scarus Jonest, significant differences exist in the intestinal mucosa activities of feeding vs fasted individuals, 5. Our data are consistent with the hypothesis that the ingestion of calcium carbonate may increase the COt load and, therefore, necessitate high levels of gut carbonic anhydrase.
INTRODUCTION A WIDE variety of coral reef fishes ingest moderate to large amounts of calcium carbonate in their diets. Some groups such as the Mullidae (goatfishes), Balistidae (triggerfishes) and Tetraodontidae (puffers) take in rather coarse hits of mollusc shell and fragments of coral skeleton, Others, including the Acanthuridae (surgeonflshes) and Scaridae (parrotfishes) ingest much finer material, usually in the form of finely ground coral rubble or sediments (Hiatt & Strasburg, 1960; Randall, 1967). In the West Indies, Randall (1967) found that up to 75 per cent of the gut contents by weight in most parrotfishes and grazing surgeonfishes consisted of inorganic sediment, presumably calcium carbonate. The anatomy of the alimentary tract of parrotfishes and surgeonfishes is well adapted to this carbonate ingestion. Parrotfishes have massive, parrot-like beaks of fused teeth for cropping the substratum, sets of pharyngeal bones which act as mills for grinding sediment and algae and have no stomachs (AI-Hussaini, 1945; Schultz, 1958; Gohar & Latif, 1963). Surgeons have less massive but welladapted dentition, lack pharyngeal mills and have well-developed stomachs which in grazing species constitutes a thick-walled gizzard (Jones, 1968). The gizzard in grazing acanthurids probably serves a triturating function much like the pharyngeal mill of scarids. There may well be other physiological and biochemical adaptations which suit these fishes for their diet heavy with carbonate particles. This study centers around these latter two groups. We hypothesize that some of the carbonate material ingested by surgeonfishes and parrotfishes is dissolved in the digestive tract and may contribute
to the COt load existing on the COt transport mechanisms of these fishes. Carbonic anhydrase levels in tissues associated with the digestive tract might reflect the magnitude of the COt load generated by carbonate solution. Therefore, we analyzed carbonic anhydrase in blood, gill, gut and gut contents to test this hypothesis. MATERIALS AND METHODS This study was conducted at Eniwetok Atoll, Marshall Islands. Three species contributed data to the study, Acanthurus triostegus, ttolocentrus spinlfer and Scarus jonesL S. ]onesi crops large amounts of CaCOa from the substratum while feeding on algae. A. triostegus ingests only the carbonate particles entangled in the algae it eats. H. spinifer is a predator and takes in very little CaCOa. Thus, differences in enzyme levels might be attributed to the presence of CaCOa in the diet. Fish were obtained by three methods: spear, cast net and hand net. All fish were returned to the laboratory immediately where enzyme analyses were performed on speared fish within 2 hr. Analyses on the digestive tract of A. triostegus were carried out 2 days after capture, as were those on fasted S. Jonesi. Crude enzyme extracts were prepared by macerating the tissues in a glass tissue grinder with 50 vol. of distilled water. Extracts of blood, bile and gut contents were made by shaking vigorously with either 50 or 100 vol. of water depending on the activity of the fluid. These solutions were allowed to extract for 30 rain at 2-4~ before the assays were completed. The assay procedure used was a modification of the indicator method of Maren (1960), Our modifications included using 0.1 ml of an indicator containing 12.5 mg ~o phenol red instead of 0.4 ml of a much weaker dye solution. Rather than 0.1 ml of a buffer consisting of 1 M NatCOa and 1 M NaHCOa, we used 0.4 ml of buffer 131
132
R . L . SMITH AND A. C. PAULSON
made up of 2 M in NaaCOa and NaHCOs, These alter/ttions reduced the sensitivity of the original method approximately eightfold while yielding a typical sere/log calibration curve and uncatalyzed reaction times of about 60see. The calibration curve was constructed wlth known dilutions of a carbonic anhydrase standard (Worthington carbonic anhydrase, 2000 EU/mg). Inhibition experiments were conducted with 0.1 ml of a 0.2 mg diamox (acetazolmide) solution. Analyses of single extracts were performed in triplicate usually yielding values within 5 per cent of each other. Enzyme concentrations are expressed in enzyme units (EU) per ml wet tissue making our data easily comparable with much of the existing literature on carbonic anhydrase reviewed by Maren (1967). Means of data were compared with t-tests. The 95 per cent confidence interval was used in all tests of significance. RESULTS
Table 1 presents the results of five separate lnhlbl. tion experiments utilizing enzyme preparations from blood, small intestine mucosa and large intestine
intestine. There was, however, a significant difference between activities of contents vs mucosa in both small and large intestines. Both A. trlostegus and S. jones/ had much greater carbonic anhydrase levels in the intestinal mucosa than did I-I. splnlfer. S. jones/also had much more enzyme in the gill than did H. splnlfer. Table 3 compares enzyme activities from mucosa S. /ones/ speared during feeding activity with those of individuals caught alive and maintained in a fasted condition for 2 days. These data indicate that the gut mucosa of fasted S. /ones/ had significantly higher enzyme levels than in feeding individuals. The groul~ of fasted S,/onesl is the appropriate one to compare with A. trlostegus and H, splnlfer since A. trlostegus was also fasted and the H, aplnlfer guts were empty. Holocentrus sphtlfer is a nocturnally active species and was speared at dusk, suggesting that no food had been eaten for at least 12hr. Comparison of mueosal activity in H, sphtlfer with A, trlostegus, S, jonesl small intestine and S,/oneM large intestine yields t values of 7.13, 6.39 and 18,9 respectively, all
Table 1, Diamox inhibition of carbonic anhydrase in paired expel'iments utilizing enzyme extracts from small Intestine, large intestine and blood of S. ]tn,r Experiment Small intestine extract 1 Extract 1 + dlamox Small intestine extract 2 Extract 2 + dlamox Large intestine extract 1 Extract 1 +diamox Large intestine extract 2 Extract 2+ diamox Blood extract Blood extract + diamox mucosa of S. /ones/. In all experiments diamox reduced or eliminated observable carbonic anhydrase activity. Carbonic anhydrase activities for the extracts from the three study species are presented in Table 2. Also included are the negative results of enzyme assays performed on sea water, algae and dead coral skeleton. These data indicate that all the preparations from the fishes exhibited carbonic anhydrase activity, while extracts from sea water, dead coral skeleton and algae exhibited no activity, Student's t-tests were performed on the data in Table 2. These tests indicate that the blood of A. trfostegus has a significantly higher enzyme activity than does the blood of S. /onesi, while the gut contents of the two species do not differ in activity. In S. jones/, there is no difference in activities of intestinal contents from small vs large intestine. Neither is there a significant difference between activities from mucosal extracts of small vs large
Enzyme activity (EU/ml wet tissue) 360 126 531 63 522 0 594 81 1098 0 of which are significant when compared to the critical t 0.05 value of 2.92. DISCUSSION
The enzyme assays and inhibition experiments reported above indicate that significant carbonic anhydrase activity exists irt the blood, gill, bile, intestine contents and gut mucosa of the fishes studied. Since there was no demonstrable enzyme activity in the sea water, algae and coral rock normally ingested by A. triostegus and S./onesl (Table 2) we conclude that the carbonic anhydrase in bile, mucosa and gut contents are all endogenous. Comparisons with the existing literature indicate that our gill activities are within the range of reported values for teleostean gills while whole blood activities for S. Jonesl and A. trlostegus equal and exceed, respectively, maximum literature values for teleosts as well as mammals (Maren, 1967). These
Carbonic anhydrase in some coral reef fishes
133
Table 2. Carbonic anhydrase activity from tissues, gut contents and blood of three coral reef fishes. Analyses of algal extracts and sea water are also included EU/ml wet tissue 5:1 S.D.
N
t
A. trtostegus ]. 2. 3. 4.
Wholeblood Stomach contents Intestine contents Intestine mucosa
2486_+662 855:70 1095:30.5 445_+108
11 2 10 2
1, 7 7.53 (+) 2, 3 0.82 ( - ) 3,9 1.78 ( - )
225+80 365:9
4 3
853 + 193 842+6
11 2
1055:55 72-1-52
8 8
9, 10 1.15 ( - ) 10, 12 2,62 (+)
]704-51 1575:75 595:45
12 11 9
9, 11 2.56 (+) 11, 12 0.47 ( - ) 9, 13 1.78 ( - )
H. spinifer 5. Gill 6. Intestine mucosa
5, 8 17.7 (+) 6, II 4.2 (+)
S. jonesi 7. Whole blood 8. Gill Intestine contents 9. Small intestine I0, Large intestine Intestine mucosa 11. Small intestine 12. Large intestine 13. Bile Algae Sea water Dead coral skeleton
0 0 0
3 3 3
Values of t were obtained by comparing the two experiments whose numbers in,unediately precede the t value. Significant differences are indicated by (+). The 95 per cent confidence interval was used on all t-tests. Table 3. Comparison of carbonic anhydrase activity in gut extracts from feeding and fasting S. jonesi Enzyme activity (EU/ml wet tissue) Extract Small intestine Large intestine
Feeding
Fasted for 2 days
t
170+ 51 (N= 12) 157-t-75 ( N = 11)
445 + 120 ( N = 2) 558+51 ( N = 2)
6.83 (+) 5'81 (+)
Values of t indicate fasted fishes had significantly higher enzyme levels than feeding individuals. data apparently conglict with Randall's (1970) statement that carbonic anhydrase activity in fish blood is only about 2 per cent of that in mammals. No significant differences in mucosal enzyme activities from different parts of the intestine are in evidence in the present study. Significant differences have been observed in the rat and dog (Kuriaki & McGee, 1964; Maren, 1967). Carbonic anhydrase values for the intestinal mucosa of S. janesi and A. triostegus exceed reported values for rat colon, chicken small intestine and frog intestine (Maren, 1967; van Goor, 1948). The activity we report in parrotfish bile is in contrast to van Goor's (1948) findings for animals in general. The intestinal mucosa of the two carbonate ingesting species had twelve times the activity present in H. sptntfer while the gill in S. jonesi had only three times that found in H. spinifer. This threefold difference might be attributed to differences in
metabolic activity levels, which are known to be reflected in blood carbonic anhydrase levels and hemoglobin levels (Albers, 1970). However, differences in metabolic rates, if they exist, are insufficient to account for the entire twelvefold difference in mucosa activity. These data are consistent with our hypothesis that calcium carbonate ingestion results in solution of calcium carbonate; and that solution adds to the load on the CO~ transport mechanisms of these fishes. The added load constitutes a selective pressure which lead to higher carbonic anhydrase levels in the intestinal mucosa of carbonate ingesting fishes. We hypothesize a mechanism in which solution of CaCO~ occurs in the presence of weak acids in the lumen of the fish gut. In such a reaction as proposed by Meldrum & Roughton (1933): CaCOa+HA
> CaA2+HaCO8
R. L, SMtTrl AND A, C, PAULSON
134
and ~he rate of solution would be affected by removal of one of the products, HsCOs, by the action of carbonic anhydrase: ~rbonlo
HiCO~
> HaO + C O n
snhy'draao
Department of Biological Sciences and by Dr. Donatd
The products of this second reaction could diffuse into the mucosal ceils and be converted to bicarbonate and hydrogen ions in another carbonic anhydrase facilitated reaction. This hypothesis attaches functional significance to the carbonic anhydrase in intestinal contents and in the mucosa. These findings have suggested the possibility that HCOa-, H~O and CO2 may be involved in electrolyte transfers across the intestinal mucosa. Figure 1 incorporates these reactions into a hypothetical scheme similar to that Lumen
Mueesa
Na*~Na
+
Plasrr ', '
k NHqt"
NH4 I' NH~" 9H~"
CaCO§ HA
~^
c,o~L~ H O+CO --H CO
cL- ct-
deknow/etlgemant.c--Thts research was supported by the U.S. Atomic Energy Commission, Erdwetok Madnt Biological Station through a grant to the Hawaii Institute of Marine Biology. We would like to thank Dr, Philip Helfrich, Director E,M,B.L., for his encouragement and assistance, Material assistance was also provided by the
HOe;--
-- HCO-
3
Fig. 1. Hypothetical scheme of CaCO B solution In the fish intestine and associated electrolyte transfers across the mucosa. Carbonic anhydrase (e.a.) would facilitate the reactions indicated as well as the exchange of HCOafor CI-. Carbonic anhydrase dependent movements of HCOa- and CI- across the mammal intestine have previously been reported (Maren, 1967). proposed for electrolyte transfers across the fresh water teleost gill. (Maetz & Garcia-Romeu, 1964). The gill of marine teleosts cannot transfer electrolytes by exchange diffusion since both Na + and CIinflux would be required in exchange for NI-I4+ and I-ICO8- efflux. However, just such an influx occurs in the gut of marine teleosts (Smith, 1953). The proposed mechanism would have the gut excreting HCO.~- and possibly NH4 +. Carbonic arthydrase is known to facilitate the ion exchanges in the fresh water gill (Maetz & Garcia-Romeu, 1964) and is present in the mucosa of all three marine species tested in this study. The scheme presented in Fig, 1 remains to be tested. We have discussed some other lines of evidence which indicate that calcium carbonate dissolves inside the parrotfish gut (Smith & Paulson, 1974). We mentioned the possibility that the solution of calcium carbonate has nutritional significance, releasing organic material trapped inside the calcium carbonate matrix. Thus, physiologically, carbonate ingestion might well be both an asset and a liability.
Hood, Director, Institute of Marine Sciences, University of Alaska. Special thanks are due to Dr. Ariel Roth, Biology Department, Loma Linda University, for kindly providing the diamox, We also thank R. Rimiticado for helping us catch fish, John Bradbury for constructing the glass reaction chambers and Dr. Russell L. Shoemaker, University of Alaska, for his critical comments on the manuscript, REFERENCES ALilaRs C, (1970) Acid-base balance. In Fish Physiology (Edited by HOAg W, S. & RANDALLD. J.), Vol, IV, pp, 173-208, Academic Press, New York. AL-HUsSAINt A, H, (1945) The anatomy and histologyof the alimentary tract of the coral feeding fish, Status sordldus Klunz. Bull. Inst..E~cyptn 27, 349-:377, OolrAIt H, A, F, & LATIF A, F, A. (1963) Dlgestiv0 proteolytlc enzymes of some searid and labrid fishes (from the Red Sea). Publ. Mar. BIoL Sta. AI.Ghardaqa 12, 4---42, HIAT'r R, W. & STRASaUaO D. W. (1960) Ecological relationships of the fish fauna on coral reefs of the Marshall Islands, EcoL Monogr, 30, 65-127, JONtm R, S, (1968) Ecological relationships In Hawaiian and Johnston Island Acanthuridae (surgeonfishcs), Micron#sign 4, 309-361, KLIR1AKI K. die, MAonlt D. F. (1964) On the carbonic anhydrasr activity of the alimentary canal and pancreas, Life Sc(,3, 137%1382, MA~Z J, & OARCIA-ROM~UF. (1964) The mechanismof sodium and chloride uptake by the gills era freshwater fish, Carasshts auratus~ll. Evidence of NH4+/Na+ and HCOtF/CI- exchanges J, gem Physiol. 47, 12091227. MAR~N T. H. (1960) A simplified micromethod for the detemaination of carbonic nnhydrase and its inhibitors. d. Pharmac. exp. Ther. 730, 26-29. MAREN T. H. (1967) Carbonic anhydrase: chemistry, physiology, and inhibition. PhysloL ReD. 47, 595-781. MSLDROMN. U. & Rovoh"roN F. J. W. (1933) Carbonic anhydrase. Its preparation and properties, J. PhysloL, Lend. 80, 113-142. RANDALLD. J. (1970) Gas exchange in fish. In Fish Physiology (Edited by HoA'~ W. S. & RANDALLD. I.), Vol. IV, pp. 253-292. Academic Press, New York, RANDALLL E. (1967) Food habits of reef fishes of the West Indies. Stud. Trop. Oceanogr. 5, 665-847. SCHUErz L. P. (1958) Review of the parrotflshes, fatally Scaridae. U.S. Nat. Mus. Bull 214, 1-141. SMrrra H, W. (1953) From Fish to Philosopher. Little, Brown, Boston, SMrrrl R. L. & PAUL.SONA, C. (1974) Food transit times and gut pH In two Pacific parrot fishes, Cot~ela, (In press,)
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