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Sea turtle blood serum protein concentrations correlated with carapace lengths
03~~-9h29:X2~110337-03m00:0 Q 19x? ~rrgsmon Press Ltd
Corny. Biochem. Phyud. Vol. 72A. No. 3. pp. 337 to 339, 1982 Printed in Great Britain.
SEA TURTLE CONCENTRATIONS
BLOOD SERUM PROTEIN CORRELATED WITH CARAPACE LENGTHS
WAYNE
‘Department
FRAIR’ and B. K. SHAH’ The King’s College, Briarcliff Manor, NY 10510, U.S.A. and ‘Rockland Research Institute, Orangeburg. NY 10962, U.S.A.
of Biology,
(Recriwl 3 Murclr 1982) Abstract,-I.
Sea turtles with longer lengths of upper shells have higher concentrations
protein over a wide range of animal sizes. 2. In very large sea turtles the concentration
of serum protein
INTRODUCTION
appears
of total serum
to drop.
there were more females than males included in this investigation, no difference based on sex has been obDuring ontogeny of sea turtles their erythrocytes tend served. to increase in size and decrease in number (Frair, Most blood samples were obtained in May. July. 1977; see also Bachere. 1980). A similar size difference August. October, November and December. and plots had been reported for Javanese toad (Bufo) erythroof all specimens by month show the type of regression cytes by Church (1961). who also pondered possible indicated in Fig. I and Table I. However. the monthly changes in proteins of the hemomatrix. Thus, the data are inadequate as a basis for conclusions about present project was set up to explore the possibility geographical or seasonal differences. that there is a concomitant alteration of erythrocytes A PC not included was for a small specimen (3.71. and the quantity of serum protein. and this hypothesis for a 4.9cm Chdonia). this having been taken was confirmed. (uniyuely in this study) before the animal had an opportunity to eat. Blood was removed 8 hr after this hatchling was dug from a nest in the sand 56 days MATERIAL Ai’iD METHODS after the eggs were laid, Plasma was more golden than Blood was taken from sea turtles bj cardiocentesis, and most others and did not clot. the serum uhtained after clotting was utilized either fresh Also omitted from the calculations were 22 values or after freezer storage. Biuret reagent was employed to obtained From captive-reared animals (8 for 3 Cnrertu; determine total serum protein concentration (PC). The ani8 for 3 Chekmia; 6 for 1 Errtmochelys) obtained over mals were mostly aquarium specimens, but others included time spans as long as 45 months and for individual some commercial captures and feral females on their nestturtles with CL ranging from 5.0 to 48.5 cm. For the ing beaches (see Frair, 1977). Measurement was straightline carapace length (CL). Experimental phases of the work single Eretmoche/ys specimen the correlation of PC were performed during various seasons of the years with CL was not significant. However when the 1968~1980. values for the 3 Curetta specimens were combined there resulted an R2 of 0.55 (P < O.OS),and for Chelonia an R2 of 0.78 (P < 0.01). Thus, these ontogenetic RESULTS studies, even though quite provisional, offer some support for the conclusions that as sea turtles increase There is a significant positive correlation between in CL their PC is augmented. carapace length and PC as showed in Fig. I and There was a question as to whether to include PC Table 1. Cuwtta and Eretrrwchrlys regression curves from C‘lzr/orzilcfemales on their nesting beach because were not statistically different and so one plot is the PC values were so low and their CL the longest shown and a single set of parameters given in Table I. The Fig. 1 curves for Drrnmhc~lys and ~~pi~~~~lz~~~.~for the species. Interestingly, the specimens with highest PC (4.08) and the lowest (1.24) (this was omitted ~~i[.u~~Lr are the most provisional. because of incongruity with all other values). as well Although there are a number of possible sources of as one intermediate (3.91). were bled before nesting, error for the values obtained, including undetected ill whereas the other three values (3.18-3.91) were from health of specimens. possible hemodilution of blood turtles bled shortly after completion of egg laying. by pericardial fluid and in some cases incomplete In the case of Derrnochdys, the laying females (also clotting of the blood, an effort was made to include largest, except for the single mature male Drrrrraonly values not deemed questionable on the basis of chelys. in these studies) had lower PC. The two values available information. In some cases, especially among for sniall Dwmochrly were from unsexed specimens the larger specimens. the serum was very cloudy, reared by Paul Hirschmann for 42 days in captivity probably from digestive products. Centrifugation (eggs incubated 63 days). They were flown From Flor(about 7500 x .y) was used to clarify the serum, otherida to New York (19-20 August 1969) and were dead wise biuret values were considerably higher. Although C.BP 73:3n--a
337
WAYNF FRAN and B. K. SHAH
CARAPACE LENGTH IN CENTIMETERS Fig. t. Graph
of weight compared with CL of green turtles. theioniu m~dus. (both sexes) from Hirth (1971).
regression calculations. Because of these values, we incline to believe that the lower PC for nesting Chelor~iu and DWUNXM,KS are associated with larger CL rather than egg production or laying. DISCUSSION
6
upon arrival. Their bodies were opened and blood removed from the atriums. probably within 12 hr from time of death. With ~epi~oc~e~~s niiaacra, there was no significant difference between PC for nesters (Surinam) and nonnesters (5 males and 6 females from Mazatlan. Mexico). Therefore PC for nesters were included in Table
1. Parameters
and statistics
The PC values previously reported for sea turtles (Frair, 1964) must now be reconsidered with regard for animal size. Interestingly, the PC appears to increase concomitantly with erythrocyte enlargement in the same animals (Frair, 1977). Kuramoto (1981) reported a relationship between size of genome and erythrocytes in amphibians. Regarding reptiles, this matter needs further investigation. In turtles. Factors expected to have a significant effect upon serum proteins would include nutrition. season and other environmental factors, such as lead pollutants. Bonnet (1979) reported virtually no change in serum protein between a group of small green sea turtles fasted for 5 days and another group which was fed (wt. difference between groups 26 g). Frogs fasted for 28 days showed a drop in serum protein (Singh. 1976). Bashtar (1979) has discovered, in C~~~/~~}I~~~ rn~&s. lower totai serum protein, hemoglobin, erythrocyte counts and hematocrits in winter than in summer, but his values do not include consideration of turtle size. for PC = (I i- h(CL) + c(CLY
Organisms curerru (37)-Evermochrl~~25) Ch&& D~rl?l0&lj*.~ Lepidochelys kempi Lepidochelys oliwcea * Square of multiple correlation coefficient is defined as a square of correlation assayed serum proteins and calculated serum proteins from the regression model. i P < 0.01.
between
Sea turtle serum protein
-
339
concentrations
In order that results of this investigation and that of Frair (1977) may be compared more readily with other allometric studies based on weights (such as that of Prothero, 1980) Figs. 2 and 3 are included to show the general correspondence between weight and CL of sea turtles (see Hirth, 1982; and for Lepidochelys oliuacea Marquez, 1976). Ackno~~ledyenlenrs-Laboratory aid has been given by Judv Kunz and Lora Sullivan. Statistical help and other suggestions have come from Robert J. Frank and Stanley L. Arlton. Adam J. Keseleski prepared graphs. Joan MillsPont assisted with translation and Ruth Pro1 the manuscript. Partial support for preparation of this paper was received from the Faculty Research Fund of The King’s College Alumni Associatibn. REFERENCES
-I Fig. 3. Graph of weight (mass) compared with CL of hawksbill turtles, Errt/~tochel)‘s iwhricura. (unsexed) from Hughes (1974).
Seidel (19741, in considering some freshwater turtles. found lower winter protein values, but any differences in protein concentration related to size were not indicated. Rozhaja et al. (1980) found PC to be lower in land turtles (Test& hermanni) living in polluted regions. However, in the light of this and all other considerations, we, at the present time, still do not recognize any evidence suggesting that the increase in PC of sea turtles is associated with anything except the normal physiology accompanying size increase. However all of the above factors need further study. Sturbaum & Bergman (1981) measured what appeared to be an insignificant increase in PC after box turtles were subjected to 2 hr of heat exposure. In considering sea turtles we would expect larger animals to retain metabolic heat better, but it remains to be determined whether temperaturk correlates with PC in sea turtles. Further research suggested by the results reported in this present paper would include quantification of specific proteins as well as physiological tests involving oncotic pressures and thermal considerations with turtles of various sizes. Presumably, homeostatic hemodynamic conditions at various ages would maintain appropriate osmotic pressures across membranes in contact with the plasma (see Burggren, 1982).
BACHERE E. (1980) Recherches hematologiques chez la tortue marine, Chelonia my&s (L.) en elevage. Dissertation, Centre Universitaire de la Reunion, France, 179 pp. BASHTAR A.-R. M. E.-S. (1979) Some aspects of the hematology of the tortoise Testudo kleinmanni Lortet. and the turtle Chelonia mydas Linnaeus. Thesis, Cairo University, Egypt, 157 pp. BONNET B. (1979) Irifluence of the nutritional conditions on the organic composition of blood and urine in the juvenile sea turtle Cheloniu r~~ydas L. Aqr~ac~rl~~rre 16. 253-260. BURGGREN W. W. (1982) Pulmonary blood plasma filtration in reptiles: a “wet” vertebrate lung? Science 215, 77-78. CHURCH G. (1961) Auxetic growth in the Javanese toad. Bufo m2lunostictus. Science 133, 2012-2014. FRAIR W. (1964) Turtle family relationships as determined by serological tests. In Tuxonornic Biochemistry und Serology (Edited by LLXINEC. A.). pp. 5355544. Ronald Press. New York. FRAIR W. (1977) Sea turtle red blood cell parameters correlated with carapace lengths. Conlp. Biochenl. Physiol. XiA, 467-472. HIRTH H. F. (1971) Synopsis of biological data on the green turtle Chelonia mydas (Linnaeus) 1758. F.A.O. Fish. Biol. Synopsis 85, 71 pp. HIR~H H. F. (1982) Weight and length relationships of some adult marine turtles. Bull. Mar. Sci. 32, 336-341. HUGHES G. R. (1974) The Seu Turf/es of Sour&Eusf Africa. I. Stufus. Morphology und Distrihurions. Oceanographic Research Institute Investigations Report 35, pp. I-144. KURAMOTO M. (1981) Relationships between nhmber, size and shape of red blood cells in amphibians. Camp. Biothem. Physiol. 69A, 771-775. MARQUEZ R. M. (1976) Sinopsis de datos biologicos sobre la tortuga golfina Lepidochelps olicacea (Eschscholtz, 1829). INP Sin. sohre Pesca 2, 1-61. PROTHFRO J. W. (1980) Scaling of blood parameters in mammals. Co~lp. Biockern. Physiol. 67A. 649-657. ROZHAJA D. A., D~R,MAI
Corny. Biochem. Phyud. Vol. 72A. No. 3. pp. 337 to 339, 1982 Printed in Great Britain.
SEA TURTLE CONCENTRATIONS
BLOOD SERUM PROTEIN CORRELATED WITH CARAPACE LENGTHS
WAYNE
‘Department
FRAIR’ and B. K. SHAH’ The King’s College, Briarcliff Manor, NY 10510, U.S.A. and ‘Rockland Research Institute, Orangeburg. NY 10962, U.S.A.
of Biology,
(Recriwl 3 Murclr 1982) Abstract,-I.
Sea turtles with longer lengths of upper shells have higher concentrations
protein over a wide range of animal sizes. 2. In very large sea turtles the concentration
of serum protein
INTRODUCTION
appears
of total serum
to drop.
there were more females than males included in this investigation, no difference based on sex has been obDuring ontogeny of sea turtles their erythrocytes tend served. to increase in size and decrease in number (Frair, Most blood samples were obtained in May. July. 1977; see also Bachere. 1980). A similar size difference August. October, November and December. and plots had been reported for Javanese toad (Bufo) erythroof all specimens by month show the type of regression cytes by Church (1961). who also pondered possible indicated in Fig. I and Table I. However. the monthly changes in proteins of the hemomatrix. Thus, the data are inadequate as a basis for conclusions about present project was set up to explore the possibility geographical or seasonal differences. that there is a concomitant alteration of erythrocytes A PC not included was for a small specimen (3.71. and the quantity of serum protein. and this hypothesis for a 4.9cm Chdonia). this having been taken was confirmed. (uniyuely in this study) before the animal had an opportunity to eat. Blood was removed 8 hr after this hatchling was dug from a nest in the sand 56 days MATERIAL Ai’iD METHODS after the eggs were laid, Plasma was more golden than Blood was taken from sea turtles bj cardiocentesis, and most others and did not clot. the serum uhtained after clotting was utilized either fresh Also omitted from the calculations were 22 values or after freezer storage. Biuret reagent was employed to obtained From captive-reared animals (8 for 3 Cnrertu; determine total serum protein concentration (PC). The ani8 for 3 Chekmia; 6 for 1 Errtmochelys) obtained over mals were mostly aquarium specimens, but others included time spans as long as 45 months and for individual some commercial captures and feral females on their nestturtles with CL ranging from 5.0 to 48.5 cm. For the ing beaches (see Frair, 1977). Measurement was straightline carapace length (CL). Experimental phases of the work single Eretmoche/ys specimen the correlation of PC were performed during various seasons of the years with CL was not significant. However when the 1968~1980. values for the 3 Curetta specimens were combined there resulted an R2 of 0.55 (P < O.OS),and for Chelonia an R2 of 0.78 (P < 0.01). Thus, these ontogenetic RESULTS studies, even though quite provisional, offer some support for the conclusions that as sea turtles increase There is a significant positive correlation between in CL their PC is augmented. carapace length and PC as showed in Fig. I and There was a question as to whether to include PC Table 1. Cuwtta and Eretrrwchrlys regression curves from C‘lzr/orzilcfemales on their nesting beach because were not statistically different and so one plot is the PC values were so low and their CL the longest shown and a single set of parameters given in Table I. The Fig. 1 curves for Drrnmhc~lys and ~~pi~~~~lz~~~.~for the species. Interestingly, the specimens with highest PC (4.08) and the lowest (1.24) (this was omitted ~~i[.u~~Lr are the most provisional. because of incongruity with all other values). as well Although there are a number of possible sources of as one intermediate (3.91). were bled before nesting, error for the values obtained, including undetected ill whereas the other three values (3.18-3.91) were from health of specimens. possible hemodilution of blood turtles bled shortly after completion of egg laying. by pericardial fluid and in some cases incomplete In the case of Derrnochdys, the laying females (also clotting of the blood, an effort was made to include largest, except for the single mature male Drrrrraonly values not deemed questionable on the basis of chelys. in these studies) had lower PC. The two values available information. In some cases, especially among for sniall Dwmochrly were from unsexed specimens the larger specimens. the serum was very cloudy, reared by Paul Hirschmann for 42 days in captivity probably from digestive products. Centrifugation (eggs incubated 63 days). They were flown From Flor(about 7500 x .y) was used to clarify the serum, otherida to New York (19-20 August 1969) and were dead wise biuret values were considerably higher. Although C.BP 73:3n--a
337
WAYNF FRAN and B. K. SHAH
CARAPACE LENGTH IN CENTIMETERS Fig. t. Graph
of weight compared with CL of green turtles. theioniu m~dus. (both sexes) from Hirth (1971).
regression calculations. Because of these values, we incline to believe that the lower PC for nesting Chelor~iu and DWUNXM,KS are associated with larger CL rather than egg production or laying. DISCUSSION
6
upon arrival. Their bodies were opened and blood removed from the atriums. probably within 12 hr from time of death. With ~epi~oc~e~~s niiaacra, there was no significant difference between PC for nesters (Surinam) and nonnesters (5 males and 6 females from Mazatlan. Mexico). Therefore PC for nesters were included in Table
1. Parameters
and statistics
The PC values previously reported for sea turtles (Frair, 1964) must now be reconsidered with regard for animal size. Interestingly, the PC appears to increase concomitantly with erythrocyte enlargement in the same animals (Frair, 1977). Kuramoto (1981) reported a relationship between size of genome and erythrocytes in amphibians. Regarding reptiles, this matter needs further investigation. In turtles. Factors expected to have a significant effect upon serum proteins would include nutrition. season and other environmental factors, such as lead pollutants. Bonnet (1979) reported virtually no change in serum protein between a group of small green sea turtles fasted for 5 days and another group which was fed (wt. difference between groups 26 g). Frogs fasted for 28 days showed a drop in serum protein (Singh. 1976). Bashtar (1979) has discovered, in C~~~/~~}I~~~ rn~&s. lower totai serum protein, hemoglobin, erythrocyte counts and hematocrits in winter than in summer, but his values do not include consideration of turtle size. for PC = (I i- h(CL) + c(CLY
Organisms curerru (37)-Evermochrl~~25) Ch&& D~rl?l0&lj*.~ Lepidochelys kempi Lepidochelys oliwcea * Square of multiple correlation coefficient is defined as a square of correlation assayed serum proteins and calculated serum proteins from the regression model. i P < 0.01.
between
Sea turtle serum protein
-
339
concentrations
In order that results of this investigation and that of Frair (1977) may be compared more readily with other allometric studies based on weights (such as that of Prothero, 1980) Figs. 2 and 3 are included to show the general correspondence between weight and CL of sea turtles (see Hirth, 1982; and for Lepidochelys oliuacea Marquez, 1976). Ackno~~ledyenlenrs-Laboratory aid has been given by Judv Kunz and Lora Sullivan. Statistical help and other suggestions have come from Robert J. Frank and Stanley L. Arlton. Adam J. Keseleski prepared graphs. Joan MillsPont assisted with translation and Ruth Pro1 the manuscript. Partial support for preparation of this paper was received from the Faculty Research Fund of The King’s College Alumni Associatibn. REFERENCES
-I Fig. 3. Graph of weight (mass) compared with CL of hawksbill turtles, Errt/~tochel)‘s iwhricura. (unsexed) from Hughes (1974).
Seidel (19741, in considering some freshwater turtles. found lower winter protein values, but any differences in protein concentration related to size were not indicated. Rozhaja et al. (1980) found PC to be lower in land turtles (Test& hermanni) living in polluted regions. However, in the light of this and all other considerations, we, at the present time, still do not recognize any evidence suggesting that the increase in PC of sea turtles is associated with anything except the normal physiology accompanying size increase. However all of the above factors need further study. Sturbaum & Bergman (1981) measured what appeared to be an insignificant increase in PC after box turtles were subjected to 2 hr of heat exposure. In considering sea turtles we would expect larger animals to retain metabolic heat better, but it remains to be determined whether temperaturk correlates with PC in sea turtles. Further research suggested by the results reported in this present paper would include quantification of specific proteins as well as physiological tests involving oncotic pressures and thermal considerations with turtles of various sizes. Presumably, homeostatic hemodynamic conditions at various ages would maintain appropriate osmotic pressures across membranes in contact with the plasma (see Burggren, 1982).
BACHERE E. (1980) Recherches hematologiques chez la tortue marine, Chelonia my&s (L.) en elevage. Dissertation, Centre Universitaire de la Reunion, France, 179 pp. BASHTAR A.-R. M. E.-S. (1979) Some aspects of the hematology of the tortoise Testudo kleinmanni Lortet. and the turtle Chelonia mydas Linnaeus. Thesis, Cairo University, Egypt, 157 pp. BONNET B. (1979) Irifluence of the nutritional conditions on the organic composition of blood and urine in the juvenile sea turtle Cheloniu r~~ydas L. Aqr~ac~rl~~rre 16. 253-260. BURGGREN W. W. (1982) Pulmonary blood plasma filtration in reptiles: a “wet” vertebrate lung? Science 215, 77-78. CHURCH G. (1961) Auxetic growth in the Javanese toad. Bufo m2lunostictus. Science 133, 2012-2014. FRAIR W. (1964) Turtle family relationships as determined by serological tests. In Tuxonornic Biochemistry und Serology (Edited by LLXINEC. A.). pp. 5355544. Ronald Press. New York. FRAIR W. (1977) Sea turtle red blood cell parameters correlated with carapace lengths. Conlp. Biochenl. Physiol. XiA, 467-472. HIRTH H. F. (1971) Synopsis of biological data on the green turtle Chelonia mydas (Linnaeus) 1758. F.A.O. Fish. Biol. Synopsis 85, 71 pp. HIR~H H. F. (1982) Weight and length relationships of some adult marine turtles. Bull. Mar. Sci. 32, 336-341. HUGHES G. R. (1974) The Seu Turf/es of Sour&Eusf Africa. I. Stufus. Morphology und Distrihurions. Oceanographic Research Institute Investigations Report 35, pp. I-144. KURAMOTO M. (1981) Relationships between nhmber, size and shape of red blood cells in amphibians. Camp. Biothem. Physiol. 69A, 771-775. MARQUEZ R. M. (1976) Sinopsis de datos biologicos sobre la tortuga golfina Lepidochelps olicacea (Eschscholtz, 1829). INP Sin. sohre Pesca 2, 1-61. PROTHFRO J. W. (1980) Scaling of blood parameters in mammals. Co~lp. Biockern. Physiol. 67A. 649-657. ROZHAJA D. A., D~R,MAI