Changes in respiratory functions during metamorphosis of the bullfrog, Rana catesbeiana

Changes in respiratory functions during metamorphosis of the bullfrog, Rana catesbeiana

Respiration Physiology (1973) 17, 276282; North-Holland Publishing Company, Amstedm CHANGES IN RESPIRATORY FUNCTIONS DURING METAMORPHOSIS OF TH...

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Respiration

Physiology

(1973) 17, 276282;

North-Holland

Publishing

Company,

Amstedm

CHANGES IN RESPIRATORY FUNCTIONS DURING METAMORPHOSIS OF THE BULLFROG, Rana catesbeiana’

JOHN J. JUST, RANDALL School qf Biological Sciences,

N. GATZ and EUGENE

and in adults.

All animals

Bicarbonate

concentrations

appropriate

corrections

about

of the blood were calculated Carbon

increase

using the Henderson-Hasselbalch thereafter.

from 7.82 in stage XX tadpoles

unchanged

to about

XXV

were taken for analysis. equation

with

from about 5 torr at stage XX to

Bicarbonate

concentration

from a low of 8.4 mM/L at stage XX to a high of 27.6 mM/L in adults.

decreased

essentially

of R. catesheiuna from stages XVIII through

dioxide tension increased

15 torr at stage XXIII with no significant

progressively blood

in tadpoles

were kept at 23 “C for at least 2 weeks before samples

for constants.

JR.

of Kentucky,Lexington, Kentucky 40506, U.S.A.

University

Abstract. Blood pH and Pc,,, were measured

C. CRAWFORD,

7.65 at stage XXI and thereafter

even in the adult (pH = 7.68). The critical phase of respiratory

increased

In uivo pH of cardiac metamorphosis

remained occurs

during stages XXI and XXII and takes place in about 5 days. Acid base balance

Metamorphosis

Blood HCOj

Ontogeny

Blood pH

R. catesbeiana

Blood Paz

Tadpoles

Bullfrogs

Although many physiological and biochemical changes occurring during anuran metamorphosis have been extensively investigated (review Frieden and Just, 1970) the respiratory system has received little attention. Adult aquatic vertebrates have a lower blood CO, tension and bicarbonate concentration than do terrestrial vertebrates (Howell et al., 1970). These physiological differences in adults may have an evolutionary significance in the transition from water to air breathing and are apparently due to a reduction in ventilation which is possible because of the high oxygen concentration in air, relative to water (Rahn, 1966). Since anuran amphibian larvae occupy an aquatic environment, changes in respiratory function may be expected to occur during the transition from water to air

Accepted for publication

I5 December

1972.

’ Supported Grants

by Biomedical Science Grant 5-SO5RR07114-04 GB-7288 (ECC), and GB-19981 (ECC).

Thanks

are expressed

to Jeanne

Just and Dahlia

Moore 276

(JJJ), NIH ROl AM 16126-01 (JJJ), NSF

for help in preparing

this manuscript.

RESPIRATORY

METAMORPHOSIS

IN BULLFROG

TADPOLES

277

Erasmus, Howell and Rahn (1970~71) have shown that young Ram concentration in the blood than do adult bullfrogs. The purpose of this study is to determine at what point in metamorphic development of the bullfrog the shift in CO2 tension and bicarbonate concentration occurs and to relate this information to changes occurring in the morpholo~ of the respiratory system. breathing.

catesbeiana tadpoles have a lower CO, and bicarbonate

Materials and methods Rana catesbeiana tadpoles weighing between 1.5 and 30 g were collected from local ponds during July and August. The animals were maintained in aerated tap water (PO2 135-150 torr) at 23 “C. They were fed boiled spinach and the water was changed every other day. No attempt was made to prevent the animals from reaching the surface and gulping air, a phenomenon often observed in nature (Savage, 1952). After the animals had acclimated for at least two weeks they were staged according to the morphological criteria of Taylor and Kollros (1946). The investigation was limited to metamorphic stages XVIII to XXV and adults. Briefly, tadpoles in stage XVIII have fully developed hind limbs but show no sign of forelimb eruption which occurs at stage XX. Tail regression begins at stage XXI and by stage XXII the tail is approximately one half its original length. Stage XXV is the last metamorphic stage, the tail has completely disappeared and the animal resembles a miniature bullfrog. Adult R. catesbeiana were purchased in February and maintained at 23 “C for two weeks before being used for experiments. Both tadpoles and adults were pithed and quickly opened to expose heart and great vessels. The conus arteriosis of tadpoles was severed and the cardiac end inserted into a cold heparinized capillary tube. Blood was collected by the pumping action of the heart, which displaced a small oil droplet in the capillary tube. Blood was drawn from the ventricle of adult frogs using a heparinized needle and syringe. All blood samples were obtained in fess than two minutes and analysis carried out immediately. Blood pH and Pcol were determined with Radiometer microelectrodes. All measurements were carried out at 23 “C. Carbon dioxide tensions were also determined by the Astrup (1956) technique. There was essentially no difference in P co2 determined by the Astrup method and the Radiometer Pco2 microelectrode when the values were above 7 torr, therefore only the Astrup values are reported. Bicarbonate concentrations were calculated from the Henderson-Hasselbalch equation utilizing temperature and pH corrections for the solubility of CO, and pK’, of carbonic acid (Severinghaus, Stupfel and Bradley, 1956a, b). Buffer slopes were determined with tonometered blood. All data were statistically analyzed using the Duncan (1955) multiple analysis of variance.

Results The partial pressure of CO, is plotted separately rather than on the conventional Davenport diagram, to better illustrate the in viva CO, tensions in the blood of tadpoles at various stages of development (fig. I). The values for Pco, at stages XVIII,

278

J. J. JUST,

R. N. GATZ

E. C. CRAWFORD,

JR.

/’

5- I-f, (6)

AND

(4)

16)

XVIII

xx

XXII

XXIV

ADULT

STAGES

Fig. 1. Carbon

dioxide

tension

in the blood

mean P,.,, at various

stages of development.

number

is the number

in parenthesis

of metamorphosing

R. catesbeiuna.

Verticle bars indicate

of animals

Closed circles represent

one standard

tested at that particular

error of the mean. The

stage. The average

number

of days

: Stage XIX : 1.4 days; stage XX : 3.3 days; stage XXI; 2.9 days; stage XXII: 2.5 days; stage XXIII : 1.5 days; stage XXIV: 9.5 days.

R. catesbeiana

tadpoles

require

to complete

various

stages are

40 .

30 . GROUP 5 Ilo. 6FmUP 4 (11.7)

%

GROW 3 113.9) 1

b” 20 . 2

GROUP 2 (15.01

IO

I

.

GROUP I (9.6) 1

. * . . . .

I. 6.7 Fig. 2. Plasma HCO;

7.1

buffer curves for metamorphosing

values and closed symbols

the standard

6.9

errors

represent

of the means. Group

7.3

7.5 7.7 PH R. catesbeiana.

in vitro tonometered

1 consists

of 16 animals

1 I

7.9 Open symbols

represent

in ho

values. Verticle and horizontal

pH and bars are

in stages XVIII, XIX and XX; group

consists of 18 animals in stages XXI and XXII; group 3 consists of 9 animals in stages XXIII and XXIV; group 4 consists of 4 animals in stage XXV and group 5 consists of 6 adults. The number in parenthesis represents

the mean buffer slope, AHCO;/ApH.

2

RESPIRATORY

METAMORPHOSIS

IN BULLFROG

TADPOLES

279

XIX, and XX approach the theoretical maximum for gill respiration (Rahn, 1966) of about 5 torr. At stage XXI the blood Pco, rose to 8.9 torr. By stage XXII the P,, increased to 12.5 torr. This value is significantly different from stages XVIII, XIX and XX at the 1% level, and from the adult values at the 5% level. The average Pcoz from stage XXIII to adults were all above 15 torr, and were not significantly different from each other. When the buffer curves of different stages were plotted (fig. 2), they feil into five differences between the mean bicarbonate concentration of group 4 (24.4 mM HCO; / group. Group 1 consists of stages XVIII, XIX and XX ; group 2 of stages XXI and XXII ; group 3 of stages XXIII and XXIV ; group 4 of stage XXV ; and group 5 of the adult. The in nivo bicarbonate concentration of the blood increases steadily during normal anuran metamorphosis, from a low of 8.4 mM/L in group 1 to a high of 27.6 mM/ L in group 5. The differences between the mean of HCO; concentration (14.9 mM/L) in group 2 and the mean bicarbonate concentration of all other groups is statistically significant at the i% level. The difference between the mean of group 3 (22.1 mM I-ICO;/L) and the means of group 1,2 and 5 are significant. There is no signi~cant difference between the mean bicarbonate concentration of group 4 (24.4 mM HCO;/ L) and group 5 (27.6 mM HCO,/L). In addition to the bicarbonate changes during metamorphosis, changes were also observed in the in vim pH of the blood. A tendency towards acidosis was observed as the animals matured. The in t!ivo pH of group 1 animals was 7.82 f0.02 while that of group 5 animals was 7.68 F0.03. The differences between the mean of group 1 and the mean of all other groups is statistically significant at the 5% level. The mean buffer slopes also change during development as is seen in fig. 2. The buffer slope of group 1 is 9.6, the slope increases to a high of 15.0 in group 2 and begins to decline to 13.8 in group 3. Groups 4 and 5 have buffer slopes of 11.7 and 10.2 respectively. Discussion The Pco, values for stages XVIII, XIX and XX approach the theoretical maximum for aquatic respiration of approximately 5 torr (fig. l), in contrast to the 2 torr reported for tadpoles of unknot stages of development (Erasmus et al., 1970/71~. The difference in Pco2 between that recorded in fig. 1 and the earlier work couid be due to sampling techniques, slow gill ventilation, or gulping of air. However, when four stage XVIII animals were denied access to the surface they had a mean Pco2 of 2.3 torr. Although the physiological role of air gulping observed by us and others (Savage, 1952) is unclear, these data suggest that air gulping probably a~ompanied by decreased gills and/or cutaneous blood flow can contribute to relatively high COz tensions in tadpoles. The physiological data (fig. 1) suggest that gills are the major site of gas exchange in tadpoles up to stage XX, when animals are in an environment containing high O2 concentrations. The gross morphology suggests that tadpoles up to stage XX have

280

J. J. JUST, R. N. GATZ AND E. C. CRAWFORD,

JR.

STAGE XVIII DA

DC

STAGE

XXII

DA

i/-;;

DC

VA

STAGE

XXIV

vc

Fig. 3. A schematic representation of the blood supply to the respiratory organs of R. cutesbeiana at various stages of development. (Schematized from Witschi, 1956.) The following abbreviations are used in this figure: DA dorsal aorta, VA - ventral aorta, DC - dorsal carotid, VC - ventral carotid H ~ heart, CA cutaneous artery, PA-pulmonary artery. The arabic numerals indicate the aortic arches and associated gills. The roman numerals indicate the Taylor-Kollros stages of development.

three potential respiratory surfaces : gills, skin, and lungs. Pulmonary gas exchange seems possible because of the observed air gulping, presence of gas in the lungs even in very young tadpoles, and because there is essentially no change in lung weight relative to body weight through stage XX (Atkinson and Just, unpublished observation). The lungs and skin could act as gas exchange organs under certain conditions because pulmonary and cutaneous arteries branch from the dorsal root of the sixth aortic arch, one of the 4 aortic arches which supply blood to gills {fig. 3, XVIII). Lungs and skin thereby receive blood which has passed through a gill where at least some gas exchange should occur prior to entrance of the blood into the pulmonary and cutaneous circuits. During times of reduced gill ventilation or in water with a low 0, concentration, the lungs and skin may play a more important role in gas exchange. The physiological significance of this exchange, however, would depend upon the portion of the cardiac output passing through the sixth aortic arch relative that passing through the other three arches. Based on physiological and morphological data, the most active phase in the transition from water breathing to air breathing occurs during stages XXI and XXII. During this period (about 5 days) the CO, tension increases from 4.5 to 12.5 torr (fig. 1). S~ultaneousIy, there occurs a significant involution of gills, and a 3Oo/;r increase in the ratio of Iung to body weight (Atkinson and Just, unpublished observation). In addition to the biochemical changes which occur in the gills and lungs

RESPIRATORY

METAMORPHOSIS

IN BULLFROG

TADPOLES

281

during stages XXI and XXII, there are shifts in the respiratory circulatory pattern. The dorsal vascular connection between the 3rd and 4th aortic arches is greatly diminished. The 5th aortic arch is in an advanced stage of regression and the dorsal anatomical connection between the 6th aortic arch and the dorsal aorta disappears (fig. 3, XXII). These morphological changes result in increased circulation through the pulmonary and cutaneous circuits which arise from the 6th aortic arch and essentially result in the establishment of the adult circulatory pattern by stage XXIV (fig. 3, XXIV). It would appear during these stages @XI and XXII) that the animals possess three respiratory organs: gills, lungs, and skin, with lungs assuming an increasing importance in gas exchange. The net result of the physiological and morphological changes is that by stage XXIII the blood has attained Pco2 values comparable to the adult where both lungs and skin but not gills participate in gas exchange. By stage XXIII gas exchange across the lungs is apparently essential for survival since our observations (and probably others ; Etkin, 1934) show that no animal denied free access to air survived beyond stage XXIII. As a result of the increase in Pco3 due to aerial breathing the pH of the blood should decrease (Rahn, 1966). However, Erasmus et al. (1970/71) have shown that there is a substantial increase in bicarbonate concentration concomitant with the increase in CO, tension, and they found little difference in blood pH between tadpoles and adult frogs. There is essentially no difference between the values we obtained for pH and HCO; in tadpoles at stage XVIII, XIX and XX, nor do these values differ signi~~ntly from those reported by Erasmus et al_ (1970/71~ for tadpoles acclimated to 20 “C. From stage XXI to XXV there is a progressive accumulation of HCO; and a decrease in pH from 7.82 to about 7.68. Helff (1932) observed a decrease in blood pH from 7.5 to 7.2 during the development of R. clamitans. Helffs experiments were conducted at room temperature with no clarification of what the temperature was or whether it was controlled. More recently, Girard (1971) has shown that the pH of chick embryo blood decreases from 7.52 at 5 days to 7.31 at 19 days of incubation. This suggests an apparent acidosis during respiratory development of some vertebrates which is not completely compensated for by an increase in HCO;. It should be pointed out that these stages of bullfrog development are further compli~ted by other biochemical changes which may alter blood pH. It is during this time that the conversion from ammonotelic to ureotelic nitrogen excretion takes place (for review see Cohen, 1970). Also, the concentration of plasma proteins increases, particularly albumin (Feldhoff, 1971), and adult hemoglobin first appears in the circulation (Just and Atkinson 1972). The relative contribution of these various factors to the buffer characteristics of the blood during metamorphosis of R. cafesbeiana have not been evaluated. Our data suggest that during the critical stages of respiratory metamorphosis the pH of the blood decreases. The resulting “acidosis” could be due to a lag between morphological changes related to breathing air, and biochemical changes related to the buffering characteristics of the blood. If this were the case the blood pH should

282

J. J. JUST,

R. N. GATZ

AND

E. C. CRAWFORD,

JR.

eventually attain adult values upon completion of metamorphosis. However, the pH of adult frogs in these experiments was not different from values obtained during the critical phases of development, and was lower than the pH of young tadpoles. Though our values for pH of adult frog blood (7.68) is within the usual range for poikilotherms acclimated to 23 ‘C, they are somewhat lower than the 7.87 reported by Howell et al. (1970) for frogs at 20 “C. This difference could be due to population differences or handling techniques. Also, their measurements were made on sciatic arterial blood ; our samples were taken by cardiac puncture, essentially the same technique we used to obtain blood from tadpoles. Our results suggest, however, that there is a slight decrease in blood pH as the water breathing tadpole develops into an air breathing frog. References Astrup,

P. (1956). A simple electrometric

blood and plasma, plasma

total content

at a fixed carbon

technique

of carbon

dioxide

for the determination

dioxide in plasma,

tension

P. P. (1970). Biochemical

differentiation

Duncan,

D. B. (1955). Multiple

Erasmus,

B. Dew., B. J. Howell and H. Rahn (1970/71). Ontogeny

range and multiple

chicken. Respir. Physiol. 11: 46-53. Etkin, W. (1934). The phenomena of anuran metamorphosis. Feldhoff, Frieden, Girard,

Physiol. 40B Action,

changes

ed. by G. Litwack.

H. (1971). Respiratory

normal

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F tests. Biometrics

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in the bullfrog during

and

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albumin

during

bullfrog

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H. (1966). Aquatic

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Camp.

in amphibian

metabolic

metamorphosis.

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In: Mechanisms

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Press, 52 p.

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13 : 343- 35 I. metamorphosis

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X. Hydrogen-ion

concentration

of the blood of

balance in cold-b!ooded

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during

vertespon-

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Savage, R. M. (1952). Ecological, physiological and anatomical tadpoles. Proc. Zool. Sot. London 122: 467-514. Severinghaus,

balance

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anuran larvaesduring involution. Biol. Bull. 63: 405-418. Howell, R. J., R. W. Baumgardner, K. Bondi and H. Rahn (1970). Acid-base

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acid pK’ with pH

J. Appl. Physiol. 9: 189-196. Severinghaus,

J. W., M. Stupfel and A. F. Bradley (1956b). Variations

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(1946). Stages in the normal

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