Citation
Comparison of osmoregulation in two species of the genus Fundulus

Material Information

Title:
Comparison of osmoregulation in two species of the genus Fundulus
Creator:
Burnside, Dale Frederick, 1943-
Publication Date:
Copyright Date:
1969
Language:
English
Physical Description:
ix, 114 leaves. : illus. ; 28 cm.

Subjects

Subjects / Keywords:
Animals ( jstor )
Body weight ( jstor )
Fish ( jstor )
Fresh water ( jstor )
Gills ( jstor )
Oxygen ( jstor )
Oxygen consumption ( jstor )
Salinity ( jstor )
Sea water ( jstor )
Surface areas ( jstor )
Atheriniformes ( lcsh )
Dissertations, Academic -- Zoology -- UF
Osmoregulation ( lcsh )
Zoology thesis Ph. D
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis -- University of Florida.
Bibliography:
Bibliography: leaves 109-113.
General Note:
Manuscript copy.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
022558875 ( AlephBibNum )
13842057 ( OCLC )
ADA1595 ( NOTIS )

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COMPARISON OF OSMOREGULATION IN

TWO SPECIES OF THE GENUS Fundulus













By
DALE FREDERICK BURNSIDE


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY














UNIVERSITY OF FLORIDA
1969
























For

MY PARENTS
who gave mo life

and for

LAURA
who shares my life












ACKNOWLEDGEMffNTS


I would like to express my sincere appreciation to Dr. Frank G.

Nordlie. Without his academic and personal counseling, I could not

have completed this research.

Dr. Robert M. DeWitt has liberally provided equipment and has

dealt with the many problems of organizing my committee.

I would like to acknowledge Dr. Brian K. McNab and Dr. Daniel

Belkin for giving generously of their time in serving on my committee

and evaluating many of my ideas.

My thanks go to all my fellow graduate students for providing the

academic atmosphere in which I have carried out my research. I would

especially like to thank Mr. Charles G. Yarbrough and Mr. Carl M. Colson

for many stimulating conversations. I would like to thank Mr. Colson,

Mr. Richard S. Fox III, and Mr. Paul Maslin for helping with the

collection of animals.

I would like to thank my darling wife, Laura, for her patience and

understanding and for helping type the final manuscript.

Finally thanks are due to Mr. and Mrs. Gerrald Gantt for providing

a typewriter for final processing of the manuscript.












TABLE OF CONTENTS

Page

ACKNOWLEDGENENTS. . . . . . . . . . . . iii

LIST OF TABLES . . . . . . . . . . v

LIST OF FIGURES . . . . . . . . . . vii

INTRODUCTION. . . . . . . . . 1

MATERIALS AND METHODS . . . . . . . . . . 4

Collection, Maintenance, and Acclimation of Fish . 4
Plasma Samples . . . . . . . . . . . 5
,e-termination of Blood Plasma Concentration. * * 5
Activity . . . . a * * * *. * 7
Intact Animal Oxygen Consumption ,, . . . . 8
Oxygen Consumption of Excised Gills, . . .* . 13
Gill Surface Area. . . . . . . . 15
Permeability . .. .. . . . . . 16
Statistical Methods . ............... 17

RESULTS . . . . . . . . . . . . . 18

Activity . . . . . . . . . . ...* 18
Concentration of Blood Plasma. .. . ... . . 18
Intact Animal Oxygen Consumption . . . . . 25
Oxygen Consumption of Excised Gills. ... . . . 51
Comparison of Variations in Oxygen Consumption of
Intact Animals and Excised Gills ... . . . 76
Apparent Permeability . . . . . ....... 78
Anatomical Relationships . . . . 83

DISCUSSION. . . . . . . . . . . . . 95

LITERATURE CITED. . . . . . . . . . . . 109

BIOGRAPHICAL SKETCH . . . . . . . . . . . 114












LIST OF TABLES


Table Page

1 THE DOUBLE LOGARIT~IIC RELATIONSHIP BETWEEN OXYGEN
CONSUITTION AND WEIGHT OF INTACTr F. similis AT
FOUR SALINITIES. . . ... . . . . * 34

2 STATISTICAL COI PARISONS OF SLOPES AND INTERCEPTS OF
THE DOUBLE LOGARIThICPRLATIOI1SHIP BET-EEN OXYGEN
CONSUEP2TIOhN AND WEIGHT OF INTACT F. similis AT FOUR
SALINITIES . . ... .. * . . . . 35

3 THE DOUBLELOGARITHI -C RELATIONSHIP BETWEEN OXYGEN
CONSUI'PTION AN'D ViTTGHT OF INTACT F. chrsotus AT
FOUR SALIIIITIES. .. . . . . . . . *

4 STATISTICAL COi PARISONS OF SLOPES AND INTERCEPTS OF
THE DOUBLE LOGARITHIJC RELATIONSHIP BETWEEN OXYGEN
CONSUNiPTION AND WEIGH. OF INTACT F. chrysotus AT FOUR
S LINITIES . . . . . . . . 46

5 INTERSPECIFIC STATISTICAL COIALRISOIS OF SLOPES AND
INTERCEPTS OF THE DOUBLE LOGA.ITHITC RELATIONSHIP
BETWEEN OXYGEN CONISUITION AND WEIGHT OF INTACT
ANIMALS AT FOUR SXLINITIES . . . . . . . 47

6 OXYGEN CONSUl-PTION OF INTACT F. similis AS A FUNCTIION
OF BODY SIZE ANDA SALINITY. . . .. . . . . 48

7 OXYGEN CONSUTI'.ION OF I-TACT F. chrysots AS A FUNCTION
OF BODY SIZE AND SALINITY. . . . . . . . 49

8 THE DOUBLE LOGARITHPIC RELATIONSHIP BETWEEN OXYGEN
CONSUlJPTION ANID DRY -WIGHT OF EXCISED GILLS OF
F. sinilis AT FOUR SALINITIES. . . . . . 60

9 STATISTICAL COj-ARISONS OF SLOPES AND II!TERCEPTS OF
THE DOUBLE LOGLRIThi-C RELATION:S!IP E TiEI' OXYGEN
CONSUJPTION AND DRY EIGHT OF EXCISED GILLS OF
F. similis AT FOUR SALTIITIES. . . . . . . 61

10 THE DOUBLE LOGARITIi-C RTI.ATICNSHIP BETWEEN OXYGEN
CONSUMPTION AND DRY 'WEIGHT OF EXCISED GILLS OF
F. c sotus AT FOUR SAJINITIES. .. . . ... . 70





Table Page

11 STATISTICAL COMPARISONS OF SLOPES AND INTERCEPTS OF
THE DOUBLE LOGARITHMIC RELATIONSHIP BETWEEN OXYGEN
CONSUMPTION AND D Y WEIGHT OF EXCISED GILLS OF
F. chrSsoJus AT FOUR SALINITIES.. ...... ... 72

12 INTERSPECIFIC STATISTICAL COMPARISONS OF SLOPES AND
INTERCEPTS OF THE DOUBLE LOGARITHMIC RELATIONSHIP
BETWEEN OXYGEN CONSUMPTION AND DRY WEIGHT OF EXCISED
GILLS AT FOUR SALINITIES . . . . . . . . 73

13 OXYGEN CONSUMPTION OF EXCISED GILLS OF F. sim.lis
AS A FUNCTION OF BODY SIZE AND SALINITY. . . . . 74

14 OXYGEN CONSUMPTION OF EXCISED GILLS OF F. chrysotus
AS A FUNCTION OF BODY SIZE AND SALINITY. . . . . 75

15 COMPARISON OF PERCENTAGE CHANGE IN OXYGEN CONSUMPTION
OF INTACT ANIMALS AND EXCISED GILLS AS A FUNCTION
OF SALINITY. . . . . . . . . . . . 77

16 APPARENT PERMEABILITY OF F. similis AS A FUNCTION OF
BODY SIZE AND SALINITY . . . . ..... ... 79

17 APPARENT PERMEABILITY OF F. chrysotus AS A FUNCTION OF
BODY SIZE AND SALINITY .. . . . . . . . 81

18 ANATOMICAL RELIONSHIPS . . . . . . . 88

19 INTERSPECIFIC CO PARISON OF SLOPES AND INTERCEPTS OF
ANATOMICAL RELATIONSHIPS . . . . . . . . 89

20 ENERGY EXPENDITURE FOR OSMOREGULATION (POTTS, 195). . 102

21 ENERGY EXPENDITURE OF F. similis FOR OSEOREGULATION. . 103

22 ENERGY EXPENDITURE OF F. chrysotus FOR OSOREGPULATION. 104











LIST OF FIGURES

Figure Page

1 System for measuring the oxygen consumption of
intact animals. (a) cooling coil; (b) filtration
flask; (c) airstone; (d) circulation pump; (e)
oxygen probe; (f) rubber stopper; (g) 100-ml sample
bottle; (h) magnet; (i) air hose; (j) magnetic
stirrer. . . * * * . * . . 10

2 Activity record of F. sirmlis after placement in res-
piration chambers. Data are the totals from records
of five fish. The period of minimal activity is
indicated, . . . * . * . 20

3 Activity record of F. chrysotus after placement in
respiration chambers. Data are the totals from records
of five fish. Tho period of animal activity is
indicated, , @ e ,* , @ e 22

4 Concentration of the blood plasma of both species as
a function of the concentration of the medium. Two
standard deviations are indicated around each mean.. . 24

5 Oxygen consumption of intact F. similis in fresh
water as a function of body weight. Slope = 0.6896 +
0.0755. Intercept = -1.0655 + 0.0436. Correlation
coefficient = 0.91.. . . . . . . . . . 27

6 Oxygen consumption of intact F. similis in 1/3 sea
water as a function of body weight. Slope = 0.4916 +
0,0772. Intercept = -0.9508 + 0.0274. Correlation
coefficient = 0.81.. . . . . . . . . . 29

7 Oxygen consumption of intact F. similis in 2/3 sea
water as a function of body weight. Slope = 0.5432 +
0.0927. Intercept = -0.9281 + 0,0294. Correlation
coefficient = 0.77.. , . . .. . a 31

8 Oxygen consumption of intact F. similis in full sea
water as a function of body weight. Slope = 0.5801 +
0.0625. Intercept = -0.8239 + 0.0469. Correlation
coefficient = 0.89.. . . . . . . . . . 33


vii







9 Oxygen consumption of intact F. chrysotus in fresh
water as a function of body weight. Slope = 0.3578 -
0.0348. Intercept = -0.9208 + 0.0118. Correlation
coefficient = 0.91. . . .. . . .. 37

10 Oxygen consumption of intact F. chrysotus in 1/3 sea
water as a function of body weight. Slope = 0.3430 +
0.0518. Intercept = -0.9066 + 0.0148. Correlation
coefficient = 0.81. . ..... . . . . . 39

11 Oxygen consumption of intact F. chrysotus in 2/3 sea
water as a function of body weight. Slope = 0.4119 +
0.0511. Intercept =-0.9066 +; 0.0215. Correlation
coefficient = 0.86. . . . . . . . . 41

12 Oxygen consumption of intact F. chrysotus in full sea
water as a function of body weight. Slope = 0.5077 +
0.0424. Intercept = -1.0177 + 0.0126. Correlation
coefficient = 0.93. . . . . . .. . . 43

13 Oxygen consumption of excised gills of F. similis
in fresh water as a function of dry weight of gills.
Slope = 0.3151 0.1311. Intercept = 0.7121 + 0.1325.
Correlation coefficient = 0.47. . . . . . 53

S14 Oxygen consumption of excised gills of F. similis
in 1/3 sea water as a function of dry weight of gills.
Slope = 0.5817 + 0.0673. Intercept = 0.4751 + 0.0685.
Correlation coefficient = 0.87. . . . 55

15 Oxygen consumption of excised gills of F. similis
in 2/3 sea water as a function of dry weight of gills.
Slope = 0.4144 + 0.0764. Intercept = 0.7526 + 0.0888.
Correlation coefficient = 0.77. . . . . . . 57

16 Oxygen consumption of excised gills of F. similis
in full sea water as a function of dry weight of gills,
Slope = 0.2453 + 0.0592. Intercept = 0.9531 + 0.0685.
Correlation coefficient = 0.66. . . . . . 59

17 Oxygen consumption of excised gills of F. chrysotus
in fresh water as a function of dry weight of gills.
Slope = 0.3436 + 0.1216. Intercept = 0.6022 + 0.0801.
Correlation coefficient = 0.60. . . . . . . 63

18 Oxygen consumption of excised gills of F. chrysotus
in 1/3 sea water as a function of dry weight of gills.
Slope = 0.1383 + 0.3402. Intercept= 0.7599 + 0.1991.
Correlation coefficient = 0.11. . . . .. . . 65


viii


Figure


Page








19 Oxygon consumption of excised gills of F, chrysotus
in 2/3 sea water as a function of dry weight of gills.
Slope = 0.5173 + 0.1845. Intercept = 0.5533 + 0.0979.
Correlation coefficient = 0.59. . . . . 67

20 Oxygen consumption of excised gills of F. chrysotus
in full sea water as a function of dry weight of gills.
Slope = 0.4584 + 0.1315. Intercept = 0,6302 + 0.0937,
Correlation coefficient = 0.67. , . . . . 69

21 Surface area of gills of F. similis as a function of
body weight. Slope = 0,8457 + 0.0307. Intercept
2.4330 0.0204. Correlation coefficient = 0.99, 85

22 Surface area of gills of F. chrysotus as a function of
body weight. Slope = 1.1753 0,0294. Intercept
2.3623 + 0.0133. Correlation coefficient = 0.99. . 87

23 Dry weight of gills of F. similis as a function of
body weight. Slope = 0.9874 j 0.0545. Intercept =
0.8940 + 0,0201. Correlation coefficient = 0.89. . 92

24 Dry weight of gills of F. chrysotus as a function of
body weight. Slope = 0.9671 t 0,1022. Intercept =
0.7021 + 0.0284. Correlation coefficient = 0.65. . 94


Figure


Page










INTRODUCTION


The integrity of any organism is maintained by the functioning of

homeostatic systems. Various parameters are physiologically controlled

at levels differing from environmental levels. Because an organism is

an open system, energy is required not only to establish these internal

levels but also to maintain them. The energetic cost of regulating ary

parameter is a function of:

1. the difference between the maintained level and

the environmental level

2. the degree to which the animal is open to the

parameter

3. the efficiencies of the controlling mechanisms.

The osmoregulatory system is one by which body fluid concentration

can be maintained at a relatively constant level irrespective of varia-

tions in the concentration of the external median. General aspects of

osmoregulation have been reviewed by Krogh (1939), Hober et. al. (1945),

Baldwin (1948), and Potts and Parry (1964). Osmoregulation in teleost

fishes has been reviewed by Smith (1932), Black (1951, 1957), Gordon

(1964), and Parry (1966).

To study the problem of teleost osmoregulation, I have chosen

two congenoric Cyprinodont species which inhabit areas of different sa-

linity conditions. The ouryhaline Fndultus similis (Baird and Girard)

lives in salt marshes where salinity conditions are highly variable,







The comparatively stenohaline Fundulus chrysotus (Holbrook) has been

found occasionally in waters of up to 24.7 ppt (Kilby, 1955), but

large populations are restricted to fresh water.

The energetic cost of osmoregulation in fishes is the sum of the

transport work performed by the kidneys, gills, and intestinal tract.

The gills account for the majority of the energetic expenditure

(Margaria, 1931; Potts, 1954). In order to estimate the energetic

cost of osmoregulation, I have measured the oxygen consumption of intact

animals and excised gills as a function of the concentration difference

between the animal and the environment.

The degree to which the osmoregulatory system of an animal is open

to the environment is termed permeability. Because the energetic effi-

ciencies of the controlling mechanisms are not considered in this study,

they have been combined with permeability to yield a compound factor

which I call apparent permeability. If the efficiencies are high or

the permeability is low, the apparent permeability is low. If the

efficiencies are .low or the permeability is high, then the apparent

permeabiltiy is high. Apparent permeability varies directly with permc-

ability and inversely with the efficiencies of the controlling

mechanisms.

Parry (1958) has suggested that body size is an important factor

affecting the ability to osmoregulate. This factor has been almost

completely ignored in osmoregulatory studios. In the present study,

body size is considered by making plots of log oxygen consumption-log

body weight at different salinities. This type of analysis has been

carried out by Hickman (1959) for fishes and by Gilchrist (1956), Rao

(1958), and Lumbye (1958) for various invertebrates. For tissue res-







piration studies, the dry gill tissue weight is correlated with oxygen

consumption and with body weight in order to consider the influence of

body size.


Purposes

The purposes of this study areas

1. to determine the precision of osmoregulation of two

closely related species of fish which are from habi-

tats of different salinity conditions

2. to consider the energetic cost of osmoregulation for

these two species by measuring the oxygen consumption

of intact animals and excised gills as a function of

salinity

3. to calculate apparent permeability as a function of

body size and salinity

4, to compare the magnitude and direction of changes in

the oxygen consumption of intact animals and excised

gills as a function of body size and salinity.












MATERIALS AND METHODS


Collection, Maintenance, and Acclimation of Fish


Seines were used to collect F. similis from tidal creeks near Cedar

Key, Florida. The salinity at times of collection varied from 8 to

34 ppt. Specimens of F. chrysotus were collected in the Gainesville,

Florida,area from Lake McCloud and River Styx with dipnots, seines, and

small dredges.

Fish were maintained in 10-gallon aquaria. Water was aerated and

filtered. Temperature was regulated between 20 and 250 C. During the

studies with intact animals, a 12-hour light and 12-hour dark cycle was

maintained. For the blood plasma studies and tissue metabolism experi-

ments, light was not controlled. Fish were fed commercial shrimp

pellets.
4
F. si-milis was acclimated to the different salinities in a step-

wise fashion. Usually the salinity at collection was near that of full

sea water. Therefore, the fish were first kept in full sea water from

Marinelanc of Florida (35.5 ppt). Two days later, 3/4 of the fish

were transferred to a dilution of 2/3 sea water to 1/3 fresh spring

water. In another two days, 2/3 of these fish were transferred from

2/3 sea water into a 1/3 sea water mixture. Because F. similis could

not survive 100 spring water, 1/2 of the fish in 1/3 sea water were

transferred into a mixture equaling ca. 1 ppt salinity. After all

transfers had been completed, a period of two weeks was allowed for







full acclimation. Parry (1966) has suggested that only a few hours

are required for fish to become acclimated to salinity.

F. chrysotus was also acclimated in a stepwise fashion. The

process began in fresh water and proceeded into 1/3, 2/3, and full sea

water. A period of two days was allowed between transfers and a period

of two weeks was allowed for full acclimation.



Plasma Samples


Plasma samples were obtained using heparinized capillary tubes.

Fish were removed from the aquaria and excess moisture in the gill re-

gion was absorbed with tissue paper. A hoparinized capillary tube

(1.4-1.6 mm in diameter) was placed adjacent to the isthmus and gently

twisted and pushed into the heart. When the capillary tube met re-

duced resistance and the heart had been pierced, the tube was slightly

retracted. WJhole blood entered the tube.

Samples were centrifuged at 10,000 rev/min for 15 minutes to separ-

ate the plasma from the blood cells. For large samples (all F. similis

and some F. chrysotus), the plasma was separated from the cells by

breaking the tube at the plasma-cell interface. Then the plasma was

transferred to a new capillary tube. Such a separation was too diffi-

cult for small samples and the plasma was frozen in a vertical posi-

tion with the column of cells under it.



Determination of Blood Plasma Concentration


The determination of blood plasma concentration was modeled after

Gross (1954), Hoar (1960), and Pavlovskii (1964). This method is per-







forced by comparing the time of melting of frozen solutions of unknown

osmolar concentration with solutions of known osmolar concentration

when allowed to warm very slowly and linearly in a cold brine (satu-

rated NaC1) bath. Because only 0.001-0.01 rl of sample is needed to

fill a small portion of a capillary tube, this method is desirable for

small quantities.

A 1-gallon battery jar was suspended in a 2.5-gallon bucket which

had a 12.5 cm diameter hole in the bottom. StyrofoaQ. insulation was

placed between the bucket and battery jar. The bucket was elevated by

three legs. Reflected light was passed through the battery jar con-

taining cooled brine. One polaroid plate, 0.030 inches thick and 30%

light transmission, was placed immediately below the battery jar and

another was placed above the battery jar in the "crossed" position.

This allowed 1% light transmission and caused crystals to appear to

glow. Gentle circulation of the brine was provided by an air-driven

stirrer. For suspending samples in the battery jar, a capillary tube

holder was made from a plastic soap dish.

Standard solutions were mixed to known concentrations as follows

according to Harned and Owen (1958, page 492)

Molarity Osmotic Coefficient Osmolarity

0.1 NaCl 1.86 .186

0.2 NaCl 1.84 .368

0.3 NaC1 1.83 .549

0.5 Iacl 1.81 .905

An experimental run consisted of these four standards plus two to

four unknowns. All volumes were approximately equal. The standards

and unlncwns were placed in the holder and frozen with dry ice. The







brine was cooled to between -8 and -100 C with dry ice. The capillary

tube holder was suspended in the cooled bath and the polaroid plates

were positioned. The bath was allowed to warm at the rate of ca. 1 C

every half hour during the critical temperature range from -2 to 00 C.

The thawing of ice crystals was observed and time was recorded as the

last crystal melted.

Then the concentrations of the unknowns could be determined from

a graph of osmolar concentration and time of thing of standards.

The accuracy of this method is + 5-10 mosm/liter.





The system for measuring the effects of experimental handling-ono

the activity of fish consisted of a 6.3 volt light source with an infra-

red filter and a Hunter photorelay connected to an Esterline Anxgu

event recorder.

The experimental fish was placed in a flask (either 250 or 500 ml,

depending upon the size of the fish) between the infrared light source

and the photorelay. Whenever the fish broke the beam, the event was

recorded. During an experiment, water was kept aerated with an air-

stone and temperature was maintained constant at 200 C. All experi-

ments were continued for 24 hours in the dark.

Five individuals of each species were used. The number of events

on the recorder paper was- grouped into 1-hour intervals. Data from

each of five fish of the sane species were added together for each

hour interval. The total number of times that the beam was broken per

hour was graphed against time. The period of minimal activity was

thus determined.







Intact Animial OQ:yen Consunption


Apparatus

The system for measuring the oxygen consumption of intact animals

was modified from that of Fry and Hart (1947).

The respiration chamber consisted of a one-arm suction filtration

flask fitted with a one-hole rubber stopper through which a glass tube

was passed. The end of the glass tube inside the flask was positioned

as far away from the arm as possible (see Figure 1). Two sizes of

flask were used (250 and 500 ml),depending upon the size of the fish.

A 19-liter aquarium served as both the temperature bath and the

resevoir for the circulating medium. One end of the aquarium was re-

moved and replaced by plexiglass through which a hole was drilled. A

glass tube, 5 cm long, was passed through this hole around which aquar-

ium cement was applied to prevent leakage. Both ends of the glass

tube were connected to tygon tubing. The longer tube on the outside

of the aquarium passed through a circulation pump and back into the

aquarium. The shorter tube on the inside was connected to the glass

tube of the respiration flask. When the circulation pump was turned on,

water left the flask via the glass tubing and was replaced by water

entering through the arm of the flask. See Figure 1 for flow direc-

tions.

Water circulated through the cooling coil from a constant tempera-

ture bath held at 200 C. An air pump vented through airstones main-

tained a high oxygen concentration in the aquarium water.

To provide insulation and darkness, the aquarium was placed in a

large Styrofoaz cooler which was painted flat black on the inside.








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Determination of Dissolved Oxygen

The Precision galvanic cell oxygen analyzer reads linearly in

milliamperes of electricity flowing between cathode and anode, It meas-

ures oxygen concentration to within 0.1 ppm at constant temperature.

The analyzer's flow requirement of 1.0 ft/sec was met with an electro-

magnetic stirrer (see Figure 1).

Approximately 200 ml of water at the samo temperature and salinity

as the water in the 19-liter aquarium was vigorously aerated overnight.

A Winkler determination for dissolved oxygen indicated that this water

was consistently 99-100l saturated. This water was used as a standard

of reference for the oxygen analyzer readings.

Readings in milliamperes were taken for 100 ml of the standard

water and for 100 ml of an unknown sample, The following calculation

yielded dissolved oxygen in the unknown samples


milliamperes-sample X amount dissolved at saturation
milliacperes-standard for given temperature and salinity


Saturation values were read from a nomogram which was supplied with

the oxygen analyzer.


Experimental Procedure and Analysis

The experimental animals were fasted for 24 hours prior to their

use. Each fish was placed in a full respirometer flask which was

stoppered and connected to the tygon tubing inside the aquarium. The

circulation pump was turned on and adjusted to give a flow rate of

100-150 ml/min. This rate had no visible effects cn the fish and was

fast enough so that the water in the flask would stay as vell aerated

as the water in the aquarium. The fish was allowed to remain in this

condition until the period of minimal activity had been reached.






At the beginning of an experiment, a 100 ml sample of water was

removed through the outside tygon tubing and oxygen concentration was

determined. The system was then stopped from flowing for 30 minutes

and a second sample was then taken from inside the flask. The dead

space (tygon and glass tubing) was cleared and a 100 ml water sample

was collected through the outside tube. The oxygen concentration was

determined and subtracted from the first determination. The system was

then allowed to flow for 30 minutes and the entire procedure was re-

peated from the beginning. Five such determinations were made during

a 4.5-hour period. Fish were superficially dried with paper towels and

weighed after experiments on a TorsioQ balance to a precision of 0,01 g.

Experiments with blue dye demonstrated that no mixing of water

entering and leaving the flask occurred while drawing samples.

The average of five readings was considered to be an estimate of

routine oxygen consumption as defined by Beamish and Mookherjii (1964).

The log of oxygen consumption (ml/hr) was correlated with the log of

body weight (g) for each species at four different salinities.

Log oxygen consumption = A log body weight + log B

where

A = increase in log oxygen consumption relative to

increase in log body weight

B = amount of oxygen consumed by a 1.0 g fish.

Special Considerations

Fry (1957) has suggested that the following variables may affect

the oxygen consumption of fish:

1. experimental temperature and thermal history

2. oxygen and carbon dioxide tensions







3. seasonal influences

4, diurnal variations

5. activity

6. nutritional state

7. sex

8. body weight

9. environmental salinity.

In this study, an attempt was made to consider each of those fac-

tors and to allow only body size and environmental salinity to vary.

Diurnal variations, sex, and seasonal influences are not discussed else-

where in this paper. Experiments were performed between 0800 and 1200

(Eastern Standard Time) and between 2000 and 2400 (Eastern Standard

Time). No difference was apparent in the results between these two

groups. Sex of individuals was determined, but was found not to sig-

nificantly affect oxygen consumption. The duration of these studies

did not permit the observation of possible seasonal influences on

oxygen consumption.



Oxygen Consumption of Excised Gills


Eperimental Setup

The oxygen consumption of excised gills was determined using a

Gilson differential respirometer. This is a closed system in which

the reaction vessels are separated from a compensation chamber by a

manometer (Umbreit et al., 1956). Fifteen ml capacity flasks were

used. The center well of the flask contained a piece" of folded filter

paper which had been saturated with 10% KOH. The main compartment of

the flask contained the gills and 2.0 ml of water of the desired

salinity.






Experimental Procedure

The gill apparatus was excised as a unit. A single cut was made at

the caudal end of the isthmus. The isthmus was pulled cephalad exposing

the gill apparatus. After the gill apparatus had been teased loose

using a scapel and dissecting needles, it was separated from the fish

with a forceps and divided into right and left halves. This division

allowed the tissue to lie flat in the manometer vessels and be completely

covered by the medium.

.Oxygen consumption was corrected to standard conditions. Meas-

urements were made at 200 C and barometric pressure was recorded at the

beginning and end of each experiment.

A period of thirty minutes was allowed for equilibration and then

readings were taken at 15-minute intervals for a period of 2.0 hours.

At the end of each experiment, the gills were removed from the manom-

eter vessels and the surplus liquid was absorbed from the tissue with

paper towels. The gills were placed in small unstoppered vials and

dried for one week at 1100 C in a drying oven. At which time, the dry

tissue weight was determined with a Mettle )analytical balance to a

precision of 0.1 mg.


Analysis

For each species in each medium, a plot of log dry gill weight (mg)

against log oxygen consumption (,ul/hr) was made.

Log oxygen consumption = A log dry gill weight + log B.

The wet body weight of each fish was also recorded and log dry

gill weight (mrg) was plotted as a function of wet body weight (g).

Log dry gill weight = A log wet body weight + log B.







Special Considerations

Krebs (1950) has discussed two important aspects of tissue

metabolism
1. the choice of medium

2. treatment of tissues.

In the present study, the medium was an artificially prepared sea

water (Svedrup et al., 1942) and dilutions were made with distilled

water to produce salinities which would be consistent with those of

the blood plasma studies. The gills were excised as an intact unit

and tissue damage was minimal.


Gill Surface Area


The method of determining the surface area of the gills was

according to Gray (1954) and Hughes (1966). Intact fish were weighed

and their gills removed. The number of filaments on each gill arch

was counted for the gill apparatus on one side of the fish. The average

length of these filaments was determined by measuring every fifth fila-

ment on each gill arch. The average number of lamellae per mm was

determined by making five counts on the filaments of each gill arch.

The average area of the lamellae was determined by sampling ten lamellae

per fish. These were drawn by means of a ZeichentubuiP and a planimeter

was used to determine surface area. The lamellae are triangular in

cross section. Because these drawings are two dimensional, their areas

were multiplied by a factor of three to get total surface area. It is

believed that this is an underestimate of the true surface area be-

cause the triangular cross sections have curved sides. The formula for

gill surface area is:







SA = 8 X F L X 1 X sa

where:

SA = total gill surface area in mm2

8 = total nunbor of gill arches

-F = average number of filaments per gill arch

L = average length of filaments in mm

1 = average number of laiellae per mm

sa = average surface area of lamellao in mm2.

For each species, log surface area (nm2) was correlated with log

wet body eight (g).

Log surface area = A log wet weight + log B.

Fish and gills were stored in 10% formalin during these measure-

ments and correction factors for shrinkage and weight change Twre

applied according to Parker (1963).



Permeability


Gill permeabilities were inferred from other data by using the

equation of Potts (1954):

W = KA (B-M) R T In B/M

whe re

W = work in cal/hr

K = permeability in moles/mm2-molar differonco-hr

A = surface area in mm2

B = blood concentration in moles/liter

M = medium concentration in moles/liter

R = 1.987 cal/C-mole

T = absolute temperature in oK.







This equation may be rewritten
W
K = A(B-II) R T In B/,

Work was derived from gill oxygen consumption readings by multi-

plying data by 4.8 cal/ml 02. It was decided better not to subtract

a small amount of energy used for maintenance of the gill cells, because

Harvey et al. (1967) have show that this portion of the metabolism is

also instrumental in ion transport. Surface area of the gills was known

from the relationships in Table 18. Molar concentrations of the medium

and blood were calculated from osmolar concentration data.

Assumptions upon which these calculations are based are considered

in the discussion.



Statistical Methods


Regression lines were calculated according to the least squares

method for curvilinear regression (Miller and Freund, 1965).

Regression coefficients are given with one standard error of the

estimate and are compared by the method given in Bailey (1959).

Intercepts are given with one standard deviation according to

Snedecor (1965). Both the variation around the sample mean and the

variation of the regression coefficient are taken into account.

Comparison of intercepts involved the use of "pooled" variances from

the regression and the test statistic for intercepts according to Miller

and Freund (1965).

Sample standard deviations from regressions were calculated for

each regression line according to Snedecor (1965).

Means of the blood plasma samples were compared using a standard

"t" test statistic for hypotheses concerning two means after Miller and

Freund (1965).












RESULTS


Activity


Both speciesare most active immediately after being placed in

the respiration flasks. Activity then decreases to a minimal level

and eventually increases until the end of the experiment (see Figures

2 and 3). Between 11 and 15 hours after the beginning of the experi-

ments, F. similis exhibits minimal activity which is approximately 40%

of the original level of activity. The period of minimal activity for

F. chrysotus occurs between 7 and 18 hours after being placed in res-

piration flasks, and is loss than 1% of the original activity. Meas-

urements of routine oxygen consumption were made during these periods

of minimal activity.



Concentration of Blood Plasma


As the concentration of the medium is raised from 25 mosm/litor

to 1,000 mosm/liter, the concentration of the blood plasma of

F. similis increases from 339. 15.9 mosm/litor to 397 + 7.1 mosm/liter

(Figure 4). At CM] = 370 mosm/liter, the mean fish plasma concen-

tration equals that of the medium. All means are significantly dif-

ferent from each other except those of fish in 1/3 and 2/3 sea water.

The concentration of the blood plasma of F. chrysotus increases

from 246 + 22.8 mosm/liter to 459 26.3 mosm/liter as the medium.



























Figure 2.


Activity record of F. similis after placement in respiration
chambers. Data are the totals from records of five fish.
The period of minimal activity is indicated.
















































Tim~ hours


400-


3501-


3001-


2501-


2001-


1501
(]Q!


I- I


I I


2U





























Figure 3. Activity record of F. chysotus after placement in respira-
tion chambers. Data are the totals from records of five
fish. The period of minimal activity is indicated.

















251



201


Timo-hours

















C
0


0

cs






0






aS
0
o














4-1;
0








0








okd






00







o
P0






OQ
0
^1
C
^! T

























































Ci
AZ!,vulouis-ems-uld P0018


ar



I,,
0
U)

'0r
Qa







is increased from 15 nosm/liter to 660 mosm/liter. At [M] = 980

miosm/liter, the blood plasma concentration drops to 297 25.3 mosm/

liter (Figure 4). The point of osmoneutrality is at [M- = 405 mosm/

liter = B] All means are significantly different from one another

(P<.001).


Intact Ainmal Oyen Consmaption


There are significant variations in the oxygen consumption of

intact F. sidilis in response to variations in salinity. The results

are presented graphically in Figures 5 8 and are summarized in

Table 1. The slopes of the log oxygen consumption-log body weight

regressions are lowest in 1/3 sea water, 0.4916, and increase as

IB M increases. In fresh water, the slope is 0.6896, an increase

of 40%. In 2/3 sea water, the slope increases 10% to 0.5432 and in

sea water it is 0.5801, an increase of 30%. There are no significant

difference among these slopes (T.able 2).

The intercepts of these regression lines increase from -1.0655

in fresh water (0.086 ml/hr) to -0,8239 in full sea water (0.150

nl/hr), All intercepts are statistically different from each other,

except those in 1/3 and 2/3 sea water (Table 2).

The slopes of the log oxygen consunption-log body weight re-

gressions for F. chrysotus follow the same pattern as for F. similis.

See Figures 9 12 and Table 3. The slopes are minimal in 1/3 sea
water, 0.3430, and increase as (B MI increases. In fresh water,

the slope- is 0.3578, a 10% increase over the slope in 1/3 sea water.

In 2/3 sea water, it increases 12% to 0.4119 and it is 0.5077, a 15%

increase, in full soa water. Statistically, the slope in sea water

























Figure 5. Oxygen consumption of intact F. similis in fresh water as
a function of body weight. Slope = 0.6896 + 0.0755.
Intercept = -1.0655 + 0.0436. Correlation coefficient =
0.91.

































fresh water


0.02


Body WVight -g


























Figure 6.


Oxygen consumption of intact F. similis in 1/3 sea water as
a function of body weight. Slope = 0.4916 + 0.0772.
Intercept = -0.9508 + 0.0274. Correlation coefficient =
0.81.

























* S


113 sea water


Body Woight-g


N020

i


0.021


0.01

























Figure 7. Oxygon consumption of intact F. similis in 2/3 sea water as
a function of body weight. Slope = 0.5432 + 0.0927,
Intercept = -0.9281 + 0.0294. Correlation coefficient =
0.77.





















0.40
*
0.30



**
bo
S0.20-




0.10- e




0.0

0
0.05 -
02/3 a2 water


x
0

0.02 -




0.O 1 I._,,.. ,. .|....i.11 *
0.1 0W2 0.5 1 2 3 4


Mody Weight-g


























Figure 8. Oxygen consumption of intact F. similis in full sea water as
a function of body weight. Slope = 0.5801 + 0.0625.
Intercept = -0.8239 + 0.0469. Correlation coefficient
0.89.


























c .0





0.02
0 01 I s I I










0.1 0.2 0.5 1 2 5 10

Body Weight-g











0 o 0 0
0 0 0 0
n Hl l






O O O O
* *



S o o0 0



O O O O
-o c- O
o0 CO C \ L0



U) +1 +1 +1 +1
0 o
'.rO O O 0

F 0 0 O 0 0
0H r- 0 0 0
N I I I I



o r


0 o



H O O O O


V, C9- c C0
U) *+1 +1 +1 +1








E-4 0 0

4)9 4 0
SH N n







EOi

9 r;


O

0
O0Rr


0
0\ N l U N












TABLE 2

STATISTICAL COI-PARISONS OF SLOPES AND INTERCEPTS OF
THE DOUBLE LCGARITIMIC RELATIONSHIP BETWEEN OXYGEN CONSUI.iPTION AND
WEIGHT OF INTACT F. similis AT FOUR SALINITIES




Comparison Slopes Intercepts



f-1/3 P>.05 *P<.05


f-2/3 P>.05 *P<.01


f-3/3 P>.05 *P<,O01

1/3-2/3 P>.05 P>.05


1/3-3/3 P>.05 *P<.01

2/3-3/3 P>.05 *P<.05
.V


*Considered to be statistically significant.




























Figure 9,


Oxygen consumption of intact F. chrysotus in fresh water as
a function of body weight. Slope = 0.3578 + 0.0348.
Intercept = -0.9208 + 0.0118. Correlation coefficient =
0.91.



































fresh water


2 34


Body Weight-g


0.01 L
0.1


0.5


























Figure 10. Oxygen consumption of intact F. chrysotus in 1/3 sea water
as a function of body weight. Slope = 0.3430 + 0.0518.
Intercept = -0.9066 + 0.0148. Ccrrelation coefficient =
0.81.










































0.01 I I I I I I
0.1 0.2 0.5 1 2 3 4


Body Weight -g




























Figure 11. Oxygen consumption of intact F. chrysotus in 2/3 sea water
as a function of body weight. Slope = 0.4119 + 0.0511.
Intercept = -0.9066 + 0.0215. Correlation coefficient =
0.86.











































Body Weaght-g


























Figure 12.


Oxygen consumption of intact F. chrysotus in full sea water
as a function of body weight. Slope = 0.5077 + 0.0424.
Intercept = -1.0177 + 0.0126. Correlation coefficient =
0.93.




























O

0-40


EH



oB











S-


+1


o














P4
0


















4,
0
k4H



















U
0


4 g
Ot
CO
0


o
o









O




O
0
rI


0
o



I







o0
o

CO-



o
+1












0
O 4
























N
+1

CO

































N


,N N N/


cH (1N <^
H- CN (r%


O









0
C-'


0'


0
+1
N
N

.0
H







O
0
+1
N
0


go
CrI \0







differs from both the slope in fresh water (P<.01) and the slope in

1/3 sea water (P<.05). The hypothesis that any other differences in

slopes exist is rejected (P>.05). See Table 4.

The intercepts of the regression lines for F. chrysotus do not

show as much variation as those for F. similis. They are -0.9208

(0.120 ml/hr) in fresh water and -0.9066 (0.124 ml/hr) in 1/3 and 2/3

sea water. However, the intercept decreases in full sea water to

-1.0177 (0.096 rml/hr), a result which is significantly different from

all other intercepts (P<.001). See Table 4,

An interspecific comparison of slopes at each salinity shows

that they differ in fresh water (P<.001). In all other salinities, a

difference in slopes does not exist (P>.05). The intercepts differ in

fresh and full sea water (P<.001), but do not differ in 1/3 and 2/3

sea water (P>.05). See Table 5.

Tables 6 and 7 show oxygen consumption of F. similis and'

F. chrysotus of five sizes at four different salinities. These values

are calculated from the relationships given inTables 1 and 3.

Changes are calculated relative to values in 1/3 sea water which is

nearest to being osmotically equivalent to the blood plasma.

Oxygen consumption is lowest in fresh water for similis

weighing from 0.5 to 4.0 g. A 4.0 g fish has approximately the same

oxygen consumption in fresh and 1/3 sea water. A 10.0 g fish shows

a 21,1% increase in oxygen consumption in fresh water. In 2/3 sea

water, fish of all sizes show increases in oxygen consumption (1.6%

to 18.6%). In full sea water, increases range from 25.0% for a 0.5 g

fish to 63.8% for a 10.0 g fish. All fish in a hyperosmotic medium

show increases in oxygen consumption relative to 1/3 sea water. Small












TABLE 4


STATISTICAL COMPARISONS OF SLOPES AND INTERCEPTS OF
TIE DOUBLE LOGARITIHflC RELATIONSHIP BETTVEEN OXYGEN CDNSUIPTION AND
WEIGHT OF INTACT F. chrysotus AT FOUR SALINITIES




Comparison Slopes Intercepts


f-1/3 P>.05 P>.05

f-2/3 P>.05 P>.05


f-3/3 *P<.oi *P<,ool

1/3-2/3 P>.05 P>.05


1/3-3/3 *P<.05 *P<.O01

2/3-3/3 P>.05 *P<.001


*Considerod to be statistically significant.












TABLE 5

INTERSPECIFIC STATISTICAL COMPARISONS OF SLOPES AND INTERCEPTS OF
THE DOUBLE LOGARTIINIC RELATIONSHIP BETWEEN OXYGEN CONSUMIFTION AND
WEIGICT OF INTACT ANhIMALS AT FOUR SALINITIES




Medium Slopes Intercepts


f *P<,001 *P<.001


1/3 P>.05 P>.05


2/3 P>.05 P>.05


3/3 P>.05 *P<.001


*Considered to be statistically significant.












* *
C? u- \ "

N n+

C; (3


0

I o




















+0
ccA












I 0
"

















+ o


0

0













0


0 0 0 0









* *0


0 H CM l
Cn M \ N C\ H







0 0 0 0 0
* 0 0 0 0
0 r- CM 0
H


* * *


+ + + H




0 0 0 0 0
1 1
0000


cs





I o

CO)


-t


*da
-H
H

* 0





CQ H







0 *
0O




4rx


0
0





*H




0 (
o
-.0












'I 0
C


0:
0,

a
2










to
\0 13
I o



o







0
to






II
1 0
I| -
0
( 1











II








+0
* -


to-
" ^









'-A


I I I I


0

O 2








0
H 0
I 1





S0
CO


o0


CO NM -- ,I. n C-
03 r r r
0 0 0 0 0






I I I



O H H
CO 0 0C 0
o& o \ 1 0
0 r-! r-i r'l N -I




0 0 0 0 0








O 0 0 04 0


o 4o o o
S
O 0 H H N


I I I I I

c0 Cm \0 Co c-
0 Co 0\ r-
0 0 0 0 0





e7. -C - 'A
00 0 CO0



.& H 0 N in
I I + +


o 0 0 0 0


Id
0
0

0


0a
o H

*rl





S*r
go







pc

OQ
g







fish (loss than 4.0 g) in a hypoosmotic medium show decreases in

oxygen consumption,while large fish (more than 4.0 g) in the same

salinity show increases in oxygen consumption.

The oxygen consumption of F. chrysotus is relatively stable from

fresh to 2/3 sea water and then drops in full sea water. In fresh

water, all fish show slight decreases in oxygen consumption ranging

from 4.2% for a 0.5 g fish to 2.Z2 for a 2.0 g fish. In 2/3 sea water,

fish below 1.0 g show a slight decrease in oxygen consumption,and fish

above 1,0 g show a slight increase in oxygen consumption. In full sea

water, all fish show decreased oxygen consumption with the greatest

depressions occurring in the smallest fish.

The slopes obtained for the double logaritmic relationship be-

tween oxygen consumption and body weight are lower for both species

in all sclinities than those reported for other fishes. Job (1955)

found slopes of 0.802 0.856 for Salvelinus fontinalis. Hickman (1959)

found a slope of 0.86 to represent the effect of weight on the oxygen

consumption of the starry flounder, Platichthys stellatus. He also

found slope values of 0.84 for lemon sole, ?arohys vetulus, and 0,90

for sanddab, Citharichthys stigLmaus. O"Hara (1968) found slopes of

0.717 and 0.710 for Loomis machrochirus and Lepomis fgbbosus re-

spectively. Beamish aeid Mookherjii (1964) found an average slope of

0.85 for Carassius auratus. Only the value inferred from data of

Wells (1935) for Fndulus parvipinnis approaches the values in this

study. His data imply a slope of 0.50. All other studies concerning

the dependence of oxygen consumption on weight in fishes have been

conducted using larger fish than those in this study and only data of

Wells are for fish of a comparable size range.






Oxygen Consurption of Excised Gills

The slopes of the regression lines of log oxygen consumption-log

dry gill weight for F. sinilis are maximal (0.5817) in 1/3 sea water

and decrease as JB MI increases (Figures 13 16 and Table 8).

In fresh water, the slope decreases 46s to 0.3151. In 2/3 sea water,

it is 0.4144, a 29% decrease. In full sea water, the slope is minimal

at 0.2453, a 57Z decrease. Statistically, the slope in full sea

water differs from both the slope in 1/3 sea water (P<.001) and the

slope in 2/3 sea water (P<.05). See Table 9.

The intercepts of these regression lines for F. similis are

minimal in 1/3 sea water (0.4751) and increase as IB Mi increases.

In fresh water, the intercept increases 50% to 0.7121. Intercepts

are 0.7526 and 0.9531 in 2/3 and 3/3 sea water respectively. These

correspond to increases of 57p and 101% over the intercept in 1/3

sea water. The intercept in 1/3 sea water differs from the intercept

in 2/3 sea water (P<.02) and from the intercept in full sea water

(P<,001). See Table 9.

The gills of F. chrysotus were more difficult to handle because

they were smaller (1 20 mg dry weight) than gills of F. similis

(1 157 nig dry weight). Only 55% (66 of 120) of the excised gills
survived the operation and consumed oxygen when placed in the res-

pironmeters. Therefore, the following results are of questionable

value.

The slopes of the regression of log oxygen consumption-log dry

gill weight for F. chrysotus vary neither directly nor indirectly

with IB MI (Figures 17 20 and Table 10). The slope is minimal in



























Figure 13. Oxygen consumption of excised gills of F. similis in fresh
water as a function of dry weight of gills. Slope =
0.3151 + 0.1311. Intercept = 0.7121 + 0.1325. Correlation
coefficient 0.47,
























20



0.0

c
. 10 -
E

c

uo 5r
~f re~
x
0

2-





12 5 10


Dry Gill vJfrit~ht mng


ish water


a



























Figure 14. Oxygen consumption of excised gills of F. sim7lis in 1/3
sea water as a function of dry weight of gills. Slope =
0.5817 + 0.0673. Intercept = 0.4751 + 0.0685. Correlation
coefficient = 0.87.
































1/3 sea water


200


Dry Gill Weight -mg


2001


a00



I20


8 10


1 5


* &


4o


1 2


5 10


























Figure 15.


Oxygen consumption of excised gills of F. similis in 2/3
sea water as a function of dry weight of gills. Slope =
0.4144 + 0.0764. Intercept = 0.7526 + 0.0888. Correlation
coefficient = 0.77.






























0
g


a


E


C
c


0


Dry Gill Wcight-mg



























Figure 16. Oxygen consumption of excised gills of F. similis in full
sea water as a function of dry weight of gills. Slope
0.2453 + 0.0592. Intercept = 0.9531 + 0.0685. Correlation
coefficient = 0.66.




















2001-


50



20 sva


10

sea water
5-


2


I I


5 10


I I I


I I I-


Dry Cill Wight-mg


1 2


50 uu 0a0v








I10


0 0
oo


R H
H




CO
W CO







co




0 3
H


0



O



0


C1









+4
Q






c-










+1


4
0












C)
*H


+1






0






U
o<
C-


0^ ClV (n


0 C-
n \O
C'6 \o


C\('4N t


c\




O-


+1
*
0









+1





o-l


*
0
+1



0


0o







Co
'o

0







0
o

+1



c0






O0
0
o


+1



0












TABLE 9
STATISTICAL COIMPARSONS OF SLOPES AND INTERCEPTS OF
THE DOUBLE LOGARITHI-C RELATIONSHIP BETWEEN OXYGEN CONSU1IPTION AND
DRY WEIGHT OF EXCISED GILLS OF F. similis AT FOUR SALINITIES




Comparison Slopes Intercepts


f-1/3 P>.05 P>.05

f-2/3 P>.05 P>.05

f-3/3 P>.05 P>.05

1/3-2/3 P>.05 *P<.02

1/3-3/3 *P<.ooI *P<.ooi

2/3-3/3 *P<.05 P>.05

*Considered to be statistically significant.



























Figure 17. Oxygen consumption of excised gills of F. chlysotus in
fresh water as a function of dry weight of gills.
Slope = 0.3436 + 0,1216. Intercept = 0.6022 + 0.0801.
Correlation coefficient = 0.60.
































9*

fresh wat


frGsh E wat


Dry Gill Weight-mg


201-


10


a'







0
0
"^
a:
P


12 5 2_~;__e~~__ dV_


5

























Figure 18. Oxygen consumption of excised gills of F. chysotus in 1/3
sea water as a function of dry weight of gills.
Slope = 0.1383 + 0.3402. Intercept = 0.7599 + 0.1991.
Correlation coefficient = 0.11.
























20-




o
0.



0
E



a 5-
0


c
0 %

1/3 sea water

o 2




1 10 2I
1 2 5 10 20


Dry Gill Weight-mg

























Figure 19. Oxygen consumption of excised gills of F. chrysotus in 2/3
sea water as a function of dry weight of gills.
Slope = 0.5173 + 0.1845. Intercept = 0.5533 + 0.0979.
Correlation coefficient = 0.59.
































2/3 sea wator


Dry Gill Weight mg



























Figure 20.


Oxygen consumption of excised gills of F. crSysotus in
full sea water as a function of dry weight of gills.
Slope = 0.458- + 0.1315. Intercept = 0.6302 + 0.0937.
Correlation coefficient = 0.67.











































Dry Gill Weight -mg


























H



4~-3


0
0






oa


F-I




0H"I
o




gr


+)















0
+1

o

o

H







W

+1















vl
0






0O
o





O o
S E


fk% R ((:
H C'J CC)









C11 10 0
H- R^ \0 C
C'r^ '^0


0 1o C- C
-l r- r- r-


0%G
U-N
r-
O




O
C0



O
Co








o
O


+1
CNI


\0








H
0
+1



0


o 0

+1 +1



o o
* *

0 0







1/3 sea water, 0.1383. In fresh water, it increases 148% to 0.3436.

In 2/3 sea water, the slope is 0.5173, a 2714 increase. However, in

full sea water the slope drops to 0.4584, a 231% increase over the

slope in 1/3 sea water. There are no statistical differences among

these slopes (Table 11).

The intercept of this relationship for F. chrysotus is minimal in

2/3 sea water, 0.5533. It increases 9% to 0.6022 in fresh water. In

1/3 sea water the slope is nis.dmal at 0.7599, an increase of 385 over

the slope in 2/3 sea water. The intercept for the relationship in

full soa water is 0.6302, 5% greater than the intercept for the re-

lationship in 2/3 sea water. Statistically, none of these intercepts

differs from each other (Table 11).

Coparing slopes and intercepts at each salinity for the two

species shows that the intercepts differ in sea water (P<.01). See

Table 12.

Tables 13 and 14 show the amount of oxygen consumed by excised

gills of various sizes of fish in four different salinities. Gill

weight is calculated for each size fish based on the relationships in

Table 18. Oxygen consumption is derived from the relationships in

Tables 8 and 10. For a small F. stmilis (0.5 g), oxygen consumption

of the gills increases as JB MI increases. A 1.0 g fish also shows

increases relative to 1/3 sea water when it is in a hyperosmotic

medium, but there is no rise in gill oxygen consumption when it is in

a hypoosmotic medium. Fish above 1.0 g show decreases *' in

gill oxygen consumption in fresh water. Fish up to ca. 4.0 g show

increases in gill oxygen consumption during hypoosmotic regulation.

Above this weight, decreases in gill oxygen consumption occur and












TABLE 11

STATISTICAL COMPARISONS OF SLOPES AND INTERCEPTS OF
THE DOUBLE LOGARITHMIC RELATIONSHIP BETWEEN OXYGEN CONSUMPTION AND
DRY WEIGHT OF EXCISED GILLS OF F. chrysotus AT FOUR SALINITIES




Comparison Slopes Intercepts


f-1/3 P>.05 P>.05

f-2/3 P>.05 P>.05


f-3/3 P>.05 P>.05

1/3-2/3 P>.05 P>.05


1/3-3/3 P>.05 P>.05

2/3-3/3 P>.05 P>.05


*Considered to bo statistically significant.




73







TABLE 12

INTERSPECIFIC STATISTICAL COMPARISONS OF SLOPES AIND INTERCEPTS OF
THE DOUBLE LOGARITHMIC RELATIONSHIP BETWEEN OXYGEN CONSUMPTION AND
DRY WEIGHT OF EXCISED GILLS AT FOUR SALINITIES


I edium Slopos Intercepts


f ?>.05 P>.05


1/3 P>.05 P>.05


2/3 P>.05 P>.05


3/3 P>.05 *P<,.O


*Considered to be statistically significant.















N 3






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become greater as (B MI increases. The results for F. clrysotus

show increases in gill oxygen consumption relative to 1/3 sea water

for all fish in full sea water and for fish larger than 0.5 g in 2/3

sea water. A 0.5 g fish shows a decreased gill oxygen consumption in

2/3 sea water. Fish weighing 1.0 g or less have depressed oxygen

consumption in fresh water. Fish weighing 1.5 g or 2.0 g show in-

creases in gill oxygen consumption in fresh water.

Oxygen consumption of excised gills increases less than propor-

tionately as dry gill weight increases. Slopes range from 0.2453 to

0.5817 for F. si2ailis and from 0.1383 to 0.5173 for F. chrysotus.

Studies of Weymouth et al. (1944), Bertalanffy and Perozynski (1951,

1953) and Vernberg (1954, 1956) state that, at least in some tissues,

weight specific metabolic rate decreases as body size increases.

More specifically, studios with gill tissue by Dehnel and McCanghran

(1964) and Holmes and Stott (1960) have indicated slopes of 0.33 and

0.86 respectively. These are considerably greater than the slopes

found in this study.



Comarison of Variations in Oxygen Consumption of
Intact Animals and Excised Gills


Changes in oxygen consumption of intact animals do not parallel

changes in oxygen consumption of excised gills as a function of

salinity (Table 15). In fresh water, intact F. similis shows reduc-

tions in oxygen consumption of animals weighing loss than 4.0 g.

Animals below 1.0 g show increased gill oxygen consumptions, but

animals above 1.0 g show decreased gill oxygen consumption. In 2/3

sea water, increases in the oxygen consumption of intact animals are







TABLE 15

COIPTARISON OF PERCENTAGE CHANGE IN OXYGEN CONSUiSPTION OF
INTACT ANI,-ALS AND EXCISED GILLS AS A FUNCTION OF SALINITY


Fundulus similis Fundulus chrysotus

Wt Intact Excisgd Wt Intact Excised
(g) animalgill (g) animal gill


Lresh water

-33.9%

-23.2%

-12.6%

+0.9%

+21.1%

2/3 sea water
+1.6%

+5.4-

+9.2%

+13.1%

+18.6%

.3.2 sea water
+25.0%

+33.9%

+41.7%

+50.6%

+63.8-


+19.5%

0.0%

-17.0%

-31.0%

-46.0%



+50.0%

+34.0%

+19.0%

+6.6%

-8.4%



+89.0%

+51.0%

+19.0%

-5.2e

-30.1%


0.50

0.75

1.00

1.50

2.00



0.50

0.75

1.00

1.50

2.00



0.50

0.75

1.00

1.50

2,00


fresh water

-4.2%

-3.6%

-3.3%

-2.8%

-2.2%

2/3 sea water

-4.0

-1.9%

0.0%

+2.8%

+5.0%

3/3 sea water

-30.6%

-26.0%

-22,6%

-17.5%

-12.7%


0.50

1.00

2.00

4.00

10.00



0.50

1.00

2,00

4.00

10.00



0.50

1.00

2.00

4.00

10.00


-15.7-
-8.4%

-3.1%

+5.0%

+11.2%



-11.2%

+3.3%

+14.6%

+32.9%

+47.9%



+0.5%

+14.0%

+24.5%

+41.1%

+54.1%


*Data are given as a percentage change in oxygen
consumption relative to oxygen consumption in 1/3 sea water.







present in all sizes of fish and the percentage increase becomes

larger as body size increases. Oxygen consumption of excised gills

also shows increases, but the percentage decreases with increasing

body size, until at 10.0 g there is an 8.4% reduction in gill oxygen

consumption. In 3/3 sea water, all F. similis show increased oxygen

consumption by intact animals and the percentage change relative to

1/3 sea water increases with increasing body size. Fish weighing

2.0 g or less show increased gill oxygen consumption, but 4.0 and

10.0 g fish show decreased gill oxygen consumption.

All sizes of F. chrysotus show decreased oxygen consumption of

intact animals in fresh water. Animals weighing 1.0 g or less also

show decreased gill oxygen ccnr.omption. However, animals weighing

either 1.5 or 2.0 g show increases in gill oxygen consumption. In

2/3 sea water, oxygen consumption of intact animals decreases for

animals of less than 1.0 g and increases for animals weighing more

than 1.0 g. Oxygen consumption of gills of animals of all sizes

except 0.50 g shows increases. In 3/3 sea water, oxygen consumption

of intact animals of all sizes is reduced,while gill oxygen consumption

of all animals is elevated.

There is no apparent correlation between changes in oxygen con-

sumption of intact animals and changes in oxygen consumption of

excised gills for either of the species.



Apparent Pormo ability


Apparent permeabilities which have been calculated with the

equation of Potts (1954) are given in Tables 16 and 17. In all












TAhLE 16

APPARENT PERPJEABILITY OF F. sinjiis AS A
FUNCTION OF BODY SIZE AND SALINITY



B-M = +314-. B-M = +25'

Wt Surface area Permeability change Permeability
(g) (mm2) (,/m2M-.r) % (M/mm-M-hr)

0.50 150 4.11 X 10-6 -99.0% 3.84 X 10-4

1.00 251 2.82 X 10"6 -99.15 3.17 X 10-4

2.00 490 1.93 x 10o6 -99.3% 2.61 X 10-

4.00 887 1.32 X 10-6 -99.4% 2.51 X 10-4

10.00 1,941 0.80 X 10-6 -99.5% 1.66 X 10"4

#Difference between the medium concentration and
blood plasma concentration in milliosmoles/liter.

Moles per square millimeter per molar concentration
difference per hour.












TABLE 16 (Extended)


B-M = -31f7

Perrmeability
(1/mmn -M-hr)


5.06 x 10"6


3.72 X 10-6


2.73 X 10-6


2.00 X 10-6


1.33 X 10-6


change



-98.7%


-98.8%


-99.o0%


-99.1%


-99.25


B-M = -6o30

Perme ability*
(M/mm2-IM-hr)


2.23 X 10-0


1.46 X 10-6


0.95 X 10-6


0.62 X 10-6


0.35 X 10-6


change
-


change
%




-99.5%


-99.6%


-99.7%

-99.8%


_ __I__ __ __ __ __


~CII~_ _














TABLE 17

APPARP NT PERPEABILITY OF F. chr2otus AS A
FUNCTION OF BODY SIZE AND SALINITY


B-M = +231# B-M = +66#

Wt Surface area Permeability* change Permeability*
(g) (rm2) (1,1/p2-M-hr) % (./2ra.M-br

0.50 102 4.36 X 10-6 94.5 7.36 x 10-5


0.75 164 3.11 x 10-6 94.0% 5.14 X 10-5

1.00 230 2.45 X 10-6 93.6% 3.82 X 10-5


1.50 371 1,73 x 10-6 93.1% 2.51 X 10~5

2.00 520 1.35 X 10-6 92.7% 1.85 X 10-5


TDifference between the rIndium
blood plasma concentration in


concentration and
milliosmoles/liter.


*Moles per square millimeter per molar concentration
difference per hour.















TABLE 17 (Extended)


B-M = -201#

Permebility*
(/M/ma -K-hr)


1.16 x 10-5


8.79 X 10-6


7.25 x 10-6


5.51 X 10-6


4.53 x 10-6


change



35.3%


82.9%


81.0%





75.5%


B-M = -683

Permeability*



1.17 X 10-6


8.73 X 10-7


7,05 x 10-7


5.26 X 10-7


4.25 X 10-7


change
-


change



98.5%


98.3%


98.2%


98.0%


97.7%


-- -- ------- ---


--------


---


- -- -- -- -- -- ----







salinities for both species, the smallest fish have the greatest

permeabilities, Percentage decreases relative to permeabilities in

1/3 sea water are also given in Tables 16 and 17. The percentage

decrease from 1/3 sea water increases with body size in all salinities

for F. similis. For F. chrysotus, this factor decreases with body

size in all salinities.


Anatomical Relationships


The log gill surface area-log body weight relationship for

F, similis indicates that gill surface area increases less than

proportionately as body weight increases (Figure 21 and Table 18).

The slope equals 0.8457 and the intercept is 2,4330. A similar

analysis for F. chrysotus shows that gill surface area increases

more than proportionately as body weight increases. The slope equals

1.1753 and the intercept is 2.3623 (Figure 22 and Table 18). The

slopes differ statistically from each other, as do the intercepts

with P<,01 in both cases (Table 24).

The most notable studies concerning gill surface area of fishes

are by Gray (1954), Steen and Kruysse (1964), and Hughes (1966).

Gray (1954) found surface areas ranging from 188 mm2/g for Lophopsetta

maculatus to 1773 mm2/g for Brevoortia tyrannus, Steen and Kruysse

(1964) found that a 550 g eel had 530 cm2 of gill surface area or

1,075 v 2/g. Hughes (1966) found surface areas ranging from 168 mm2/g
for a 24 g Callio ymlus lyra to 845 nm2/g for an 85 g Clupea harengas.

The surface area of F. similis ranges from i54.3 mm2/g for a 9.25 g

fish to 313.4 nm2/g for a 1.46 g fish. The surface area of F, chry-

sotus ranges from 148.6 mm2/g for a 0.36 g fish to 279.7 rm2/g for a

3.88 g fish.



























Figure 21. Surface
weight.
0.0204.


area of gills of F. similis as a function of body
Slope = 0.8457 + 0.0307. Intercept = 2.4330 +
Correlation coefficient = 0.99.












































Body vWoiht-0



























Figure 22. Surface area of gills of F. chrysotus as a function of
body weight. Slope = 1.1753 + 0.0294. Intercept
2.3623 + 0.0133. Correlation coefficient = 0.99.















































Cody Weibht -









o o" c
O0 0\ Co
o 0 0
0 C)^ CL- CO








O O O\ o
+ *








0 0 0



C N H N
o o o o
+1 0i CS Ct `0
o o 0 0
4 +1 +1 +1 +1

p0 C' \0 C 0 .

S*
&. H Co0 O 0

HH

0

.0 0 0 O

1 +1 +1 +1 +1


no o C
00 H 0







S0 0 0 N 0
HN N 01 '10
) rl C\ CD 0















c4 C

0 C -)



S * *
r 0r 0 -H1



ao 0 M
0 0




89








TABLE 19

INTERSPECIFIC COMPARISON OF SLOPES AND
INTERCEPTS OF ANATOMICAL RELATIONSHIPS


Relationship Slopes Intercepts



Surface area-Body wt # *P<.01 *P<.01


Dry gill uv-Body t# \ P>.05 *P<.001


SDouble logarithmic relationship.


* Considered to be statistically significant.






Only Price (1931) has previously studied the gill surface area

of a fish as a function of body weight and has found a slope of 0.78

for Micropterns dolomiou. This is lower than the slopes of 0.846 for

F. similis and 1.175 for F. chrysotus.

Dry gill weight increases proportionately with body weight in

both F. similis and F. chrysotus (Figures 23 and 24). The slopes are

0.9874 for F. similis and 0.9671 for F. clhrsotus. These are not

significantly different from each other (P>.05). The intercepts of

this relationship are 0.8940 for F. similis and 0.7021 for F. chrysotus.

The intercepts are significantly different from each other (P<.001).

These anatomical relationships and statistical comparisons are

reviewed in Tables 18 and 19.




Full Text

PAGE 1

COMPARISON OF OSMOREGULATION IN TWO SPECIES OF THE GENUS Fundulus By DALE FREDERICK BURNSIDE A DISSERTATION PRESENTED TO THE GRADUATE COUNOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1969

PAGE 2

UNIVERSITY OF FLORIDA 3 1262 08552 2695

PAGE 3

For I'll PAREHTS who gave rno life and for LAURA vjho shares my life

PAGE 4

ACKNO^CEDGEISOTS I iTOuld like to egress rny sincere appreciation to Dr, Frank G, Nordlie, V/ithout his academic and p3rsonal coui-isoling, I could not have completed this research, Dr, Robert M, DeWitt has liberally provided equlpraont and has dealt with the many problems of organizing rny committee, I would like to acknowledge Dr, Brian K, McNab and Br, Daniel Belkin for giAring generously of their time in serving on my cominittee and evaluating many of tny ideas, Ky thaiTks go to all ny fellow graduate students for providing the academic atmosphere in which I have carried out my research, I would especially like to thank Mr, Charles G, larbrovigh and Mr, Carl M, Colson for many stiroulating conversations, I would like to thank Ifr, Colson, Mr, Richard S. Fox III, and Kr, Paul Maslin for helping vdth the collection of animals, I would like to thank my darling wife, Laura, for her patience and understanding and for helping type the final manuscript. Finally thanks are duo to Mr, and Mrs, Gerrald Gantt for providing a typeiwiter for final processing of the manuscript. iii

PAGE 5

TABLE OF CONTENTS Page ACeJOVJLEDGEJiENTS. iii LIST OF TABLES v LIST OF FIGURES vii INTRODUCTION 1 MATERIALS Mm METHODS ^^ Collection, Maintenance, and Accliraation of Fish ..... h Plasma Samples .» «.......•••.• 5 Detorraination of Blood Plasma Concentration, ....... 5 Activity . . ...•..«..••.•••.. 7 Intact Aniraal Oxygen Constmption •......•••... 8 Oxygen Consxunption of Excised Gills. •..•••.•••• 13 Gill Surface Area .....••••.•••.» 15 Permeability •....•.... ..• l6 Statistical Methods. .•.......*....••... 17 RESULTS 18 Activity ........... 18 Concentration of Blood Plasna. .............. 18 Intact Animal Oxygen ConsuEiption .*•....*..•.• 25 Oxygen Consumption of Excised Gills. ...« 51 CoiTr):)3j'iscn of Variations in Oxygen Consxxmption of Intact Animals and Excised Gills .••.......•*• 76 Apparent Perneability, .................. 78 Anatomical Relationships ••.•.....••...«.. 83 DISCUSSION. 95 LITERATURE CITED. 109 BIOGRAPHICAL SKETCH Hk iv

PAGE 6

LIST OF T/.5LE3 Table Page 1 THE DODELELOGARITHl-IC RELATION SHIP BBTVffiEN OXYGEN CONSUIT-TIOh' M^D WEIGHT OF IlfTACT F, sirtilis AT FOUR SALIKITIES 3^ 2 STATISTIC/iL COM^AHISOIJS OF SLOPES Al^ID irJTERCEPTS OF THE DOUBLE LOGARITir-TC RELATIOrSIUP ESTVEEi: OXYGEN COKSul-PTIO!: AICD INSIGHT OF ICTACT F. si;rdlis K£ FOUR S.'ILIIHTIES T 35 3 THE DOUBLELOGA^JTeTC REL.ATI0M3RIF BSTI'JEEN OXTGEI^ CGNSUi:PTIOi; AI^ID VrEIGKT OF lOTACT F. chrysottts AT FOUR SALIIIITIES T Wjk ^ATISTIC;L COl-TARISOKS OF SLOPES AlID INTERCEPTS OF THE DC'U33I.E LOGAFvITin-ZC RELATIONSHIP EETIJEEN OXYGEN CONSUi'IPTIOK mH V.'EIGHj? CF intact F. ehrysottis /iT FOUR S/LINITIES T ^6 5 HCTSRSPECIFIC STAriS"JIC>\L CO'T.ARISONS OF SLOPES AND INTSRCBFrS OF THE DOiJELE LCG/.RITHiTC RLLATIONSHJP BBTvSSN OXYGEN COI.'SUITTION I'llD VJSIGHT OF INTACT Aiai-IALS AT FOUR SAJ.INITIES ^7 6 OXYGEiJ COKSUIK-ION OF li'TTACT F. simUis AS A FUICCTION OF BODY SIZE AND S/iLINITI. ^S 7 OXYGHI CONSUl'PTION CF P'TACT F. c^r}rsotv^ AS A FUNCTION OF BODY SIZE MvT) S/ilNITY. .7 ^9 8 THE D0L15LE LOGARTTHIZC RELATIONSHIP BSTVSBN OXYGEl^' CONSUNPTION A13 DRY V.^GKT OF EXCISED GILI.S OF F, s.imlis AT FOUR S.'ILINITIES . . 60 9 ST/iTISTIGAL C01-?/J?IS0NS OF SLOPES AND INTERCEPTS OF THE MUBLS LOG/JlIThiaC REJ^ATIONSi-ZP BET'VffiEN OXYGEN CCNSUl-PTION AI'3D DKY VrEIGHT OF EXCISED GIXLS OF F. sinalis AT FOUR 3/LINITIES 6l 10 THE DOUBLE LOGARITIU-ZCR-J.ATICNSHIP BSTV.i:SN OXYGEN CONSU^'TTICN /JjD DRY '.EIG.HT OF EXCISED GILLS OF F. cbrysotus KT FOUR SAI.INITIES 70

PAGE 7

Table Page 11 STATISTIG/i COI'IP/J^JSONS OF SL.OPES AIJD IKrTERCEPTS OF THE DOUEy-: LOGARITHMIC RELATICKSHIP BETI'/EEN OXYGEN COKSUI^ffTIOi; AiJD Din vaiGHT OF EXCISED GHiS OF !• clEZSotus AT FOUR SALINIT'IES 72 12 INTERSPICCIFIC STM'I,STICi\L C0I'5PMIS0NS OF ,SI-.OPES AND INTERCEPTS OF THE DOUBLE LOGARITHKEC RJiLATIONSHIP BETV.'ESN OXYGEN COIJSUi>lPTION /U'iD DtUi lEIGhT OF EXCISED GILLS AT FOUR SALINITIES 73 13 OXYGEl'! CONSUMPTION OF EXCISED GILLS OF F. siallis AS A FUIICTION OF BODY SIZE AKD SALIiaTY 7^ 14 OXYGEN CONSUI-PTION OF EXCISED G3iL3 OF F. chrysotus AS A FUNCTION OF BODY SIZE MID S/^LINITY 75 15 COirPARISON OF PERCENTAGE CH/JIGS IN OXYGEN CONSUI-rTION OF INTACT ANIM/iLS AI'ID EXCISED GILLS AS A FUN'CTION OF SALINITY. 77 16 APPAREOT PERI!EABILITY OF F. simJlis AS A FUNCTION OF BODY SIZE Mm SALINITY 79 17 APPARSOT PERI-EABILITY OF F. ctoysot us A3 A FUl^'CTION OF BODY SIZE AITO SALINITY .7 81 18 AIUTOIUCAL RELAl'IOHSHIPS 88 19 H'JTSRSPBCIFIC COITARISON OF SLOPES .\ND INTEEGSPT3 OF Al^ATOMICAL RELATIONSHIPS 89 20 EI^iERGY EXPENDITUiS FOR OSKORSGULATION (POTTS, 195^^) ... 102 21 ENERGY EXPEIIDITURE OF F. similis FOR OSMOREGULATION. . . 103 22 ENERGY EXPEIIDITURE OF F. chrysotns FOR OSI^OPJiXiUL.ATION . , 104 vi

PAGE 8

LIST OF FIGURES Figirre Pago 1 System for rneasuring the oxygen consvimption of intact animals, (a) cooling coil; (b) filtration flask; (c) airstone; (d) circulation pump; (e) 03!ygen probe; (f) rubber stopper; (g) lOO-nl saiaple bottle; (h) magnet; (i) air hose; (j) magnetic stirrer. •.••••••••••••••••tt»i*t 10 2 Activity record of F, s ird.l is after placement in respiration chambers. Data ai'e the totals from records of five fish. The period of mininal activity is indicated, .,,,,,,,,,,,,•*,,,,,,,, 20 3 Activity record of F, chrysotus after placement in respiration chambers. Data are the totals from records of five fish. The period of Bdninal activity is indicated, ,.,,•,,,*••,*•,, ,**,**t 22 if Concentration of the blood plasina of both species as a fionction of the concentration of the medium. Two standard deviations are indicated around each Kean,, , , 2k 5 0:
PAGE 9

Figxire Page U 9 0:
PAGE 10

Figxire Page 19 Oxygen consunption of excised gills of F, clir ysotu s in 2/3 sea water as a function of dry iraight of gills. Slope = 0.5173+ 0,1045. Intercept = 0.5533 + O.O979. Correlation coefficient = 0,59t . t ••••••••• • 6? 20 Oxygen consuinption of excised gills of F, chirsottis in full sea water as a function of dry veigl:t of gills. Slope = 0.455^ + 0.1315. Intercept = O.6302 + 0.0937. Correlation coefficient =0,67. •..••..••••• ^9 21 Surface area of gills of F, simllis as a function of body weight. Slope = 0,8457 + O.O307. Intercept = 2,4330 + 0.0204. Correlation coefficient ~ 0.99, . , , 85 22 Surface area of gJJLls of F, clirysotus as a function of body weight. Slope = 1,1753+ 0,0294, Intercept = 2,3623+ 0,0133, Correlation coefficient = 0,99, ... 8? 23 Dry weight of gills of F, sipiilis as a function of body freight. Slope = 0,98W± O.O545, Intercept = 0,8940 + 0,0201. Correlation coefficient = 0,89. ... 92 24 Dry wreight of gills of F. chrysotus as a function of body vroight. Slope = 0,9^ + 0.1022. Intercept = 0,7021 + 0,028/+. Correlation coefficient = 0,65. . , , 9^*-

PAGE 11

nTOlODUCTION The integrity of any organism is maintained by the functioning of homoostatic systems. Varioiis paraiaeters aro physiologically controlled at levels differir^ from environmental levels. Becatiso an organism is an open system, energy is required not only to establish these internal levels but also to maintain them. The energetic cost of regulating ary parameter is a function of i 1, the difference betvjoen tha maintained level and the envirormental level 2, the degree to which the animal is open to the parameter 3, the efficiencies of the controlling mechanisms. The osmoregulatory system is one by which body fluid concentration can be maintairied at a relatively constant level irrespective of variatioiis in the concentration of the external roedixim. General aspects of osmoregulation have been revievred by Krogh (1939) t Sober jst.^. (19'^'5) » Baldidn (19^) , and Potts and Parry (19^^) • Osmoregulation in teleost fishes has been revievred by Smith (1932), Black (1951 t 1957). Gordon (196^) , and Pany (I966) . To st\34y 'the problem of teleost osmoregulation, I have chosen two congenoric Cyprinodont species vrhich inhabit areas of different salinity conditions. The euiyhalins Fvindulu.3 similis (Baird and Girard) lives in salt marshes where salinity conditions are highly variable.

PAGE 12

The comparativoly stenohaline Fimdulus chrysotxis (Kolbrook) has been foiind occasionally in waters of up to 2^,7 ppt (Kxlhy, 1955), ^t large popxilations are restricted to fresh water. The energetic cost of osmoregulation in fislios is the sum of the transport v/ork performed by the kidneys, gills, and intestinal tract. The gills account for the majority of the energetic expendit\iro (Ilargaria, 1931; Potts, 195^) • In order to estimate the energetic cost of osmoregulation, I have maasm^od the oxygen constmption of intact animals and excised gills as a function of the concentration difference bettieen the animal and the environment. The degree to which the osmoregulatory system of an animal is open to the environment is termed penraability. Because the energetic efficiencies of the controlling mechanisms are not considered in this study, they have been combined with permeability to yield a compo\ind factor which I call apparent permeability. If the efficiencies are high or the permeability is low, the apparent permeabiJlity is low. If tha efficiencies are low or the permeability is high, then the apparent permeabiltiy is high. Apparent pei'meability vairies directly with permeability and inversely with the efficiencies of the controlling mechanisms, Pany (1958) has suggested that body size is an important factor affecting the ability to osmoregulate. This factor has been almost completely ignored in osmoregulatory studies. In the present study, body size is considered by making plots of log oxygen constimptlon-log body vreight at different salinities. This typo of analysis has been carried out by Hickman (1959) for fishes and by Gilchrist (1956) , Rao (1958) , and Lumbye (1958) for vairLous invertebrates. For tissue res-

PAGE 13

piration studies i the dry gill tissue weight is correlated with oxygen consumption and with body weight in order to consider the influence of body sizet Piirposes The purposes of this study arei 1, to determine the precision of osmorogiilation of two closely related species of fish which are from habitats of different salinity conditions 2, to consider the energetic cost of osmoregulation for these two species by measuring the oxygen constmiption of intact animals and excised gills as a function of salinity 3, to calculate apparent permeability as a fxmction of body size and salinity k, to conqjare the magnitude and direction of changes in the oxygen consumption of intact animals and excised gills as a function of body size and salinity.

PAGE 14

MATERIjMS and ^STKODS Collection, liaintenance , and Acclimation of Fish Seines t-roro used to collect F. similis from tidal creeks near Cedar Key, Florida. The salinity at times of collection varied from 8 to y^ ppt. Specimens of F, chrysotus trere collected in the Gainesville, Florida, area from Lake KcCloud and River Styx with dipnets, seines, and small dredges. Fish were maintained in 10-gallon aquaria, Water was aerated and filtered. Temperature was regulated betxroen 20 and 25° C, During the studies vri.th intact animals, a 12-hoxir light and 12-hour dark cycle was maintained. For the blood plasma studies and tissue metabolism experiments, light was not controlled. Fish were fed commercial shrimp pellets, Z» simj.lis was acclimated to the different salinities in a stepwise fashion. Usually the salinity at collection was near that of ftCLl sea water. Therefore, the fish wore first kept in fvill sea water from Marineland^ of Florida (35.5 Ppt) , Two days later, 3/4 of the fish were transferred to a dilution of 2/3 sea water to 1/3 fresh spring water. In another two days, 2/3 of these fish irere transferred from 2/3 sea water into a I/3 sea water mixture. Because F. similis could not survive 100^ spring water, l/2 of the fish in I/3 sea water were transferred into a mixture equaling ca, 1 ppt saliaity. After all transfers had been completed, a period of two weeks was allowed for

PAGE 15

fvill accliin.ation, Pany (.19^6) has suggested that only a few hours are required for fish to becopie acclimated to salinity, F. ch rysotus was also accliinated in a stepi;d.sQ fashion. The process began in fresh Xi^ater and proceeded into l/3t 2/3i and full sea water, A period of two days was allo^Ted between transfers and a period of two weeks was allowed for full acclimation. Plasma Samples Plasma sarrples were obtained using heparinized capillary tubes. Fish wore removed from the aquaria and excess moisture in the gill region was absorbed with tissue paper, A heparinized capillary tube (1,4-1,6 ram in diameter) was placed adjacent to the isthmus and gently twisted and pushed into the heart, Tf/lien the capillary ttibo met reduced resistance and the heart had been pierced, the tube was slightly retracted, VJhole blood entered the tube. Samples were centrifuged at 10,000 rev/min for 15 uninutes to separate the plasma from the blood cells. For large samples (all F, sitnilis and soEO F, chrysotus ) , the plasma v/as separated from the cells by brealdng the tube at the plasma-cell interface. Then the plasma was traiisf erred to a new capillary tube. Such a separation xfas too difficvilt for small samples and the plasma was frozen in a vertical position with the column of cells under it, DeteiTiin a tion of Blood Plasma Concentration The determination of blood plasma concentration was modeled after Gross (195^) , Hoar (I96O) , ard Pavlovskii (1964) , This method is per-

PAGE 16

fonned by comparing tha tirao of melting of frozen solutions of linknown osmolar concentration with solutions of knovm osnolar concentration when alloired to warm very slowly and linearly in a cold brine (saturated NaCl) bath. Because only 0.001-0,01 ml of sanple is needed to fill a small portion of a capillajry tube, this method is desirable for small qxiantities, A l-gallon battery jar was suspended in a 2,5-gallon bucket which had a 12,5 cm diameter hole in the bottom, StyrofoaiS^ insulation was placed between the bucket and battery jar. The bucket was elevated trythree legs. Reflected light was passed through the battery jar containing cooled brine. One polaroid plate, 0,030 inches thick and 30^ light transmission, was placed immediately below the batteiy jar and another was placed above the battery jar in the "crossed" position. This allovTed 1$ light transmission and caused crystals to appear to glow. Gentle circulation of the brirw was provided by an air-driven stirrer, For suspending samples in the battery jar, a capillary tube holder vras made fi*on a plastic soap dish. Standard sol\itions vrere mixed to known concentrations as follows according to Hamed and Otran (1953, page 492) i Molarity Osmotic Coefficient Osmolarity 0,1 KaCl 1,86 ,186 0.2 NaCl 1,84 .368 0.3 NaCl 1.83 .549 0,5 NaCl 1,81 ,905 An experimental run consisted of these foxu* standards plus two to four unknovms. All volvimes were approximately equal. The standards and unlcnowns were placed in the holder and frozen with diy ice. The

PAGE 17

brins was cooled to betxTsen -8 and -10° C •with dry ice. The capillary tube holder was suspended in the cooled bath and the polaroid plates were positioned. The bath was allotred to warm at the rate of ca, 1^ C every half hour during the critical temperature range from -2 to 0° C, The thawing of ice crystals was observed and time was recorded as the last crystal molted, Tlien the concentrations of the unloioTms could be determined from a graph of osmolar concentration and time of thaiving of standards. The accuracy of this metliod is + 5~10 aosm/liter. Activity The system for measuring the effects of experimental handling on the activity of fish consisted of a 6,3 volt light source with an infrared filter and a Hxmter^ photorelay connected to an Esterline AngrilS' ©vent recorder. The experirantal fish was placed in a flask (either 250 or 500 nil, depending upon the size of the fish) between the infrared light source and the photorelay. Whenever the fish broke the beam, the event was recorded, Durir^ an experiment, water was kept aerated with an aLrstone and temperatur-e was maintained constant at 20° C, JOJL experiments were continued for 2^ hours in the dark. Five individuals of each species v/ero used. The number of events on the recorder paper was grouped into 1-hour intervals. Data from each of five fish of the same species were added together for each hour interval. The total number of times that the boara was broken per hour was graphed against time. The period of minimal activity was thtis determined.

PAGE 18

8 Intact Animal O^iy.'^en Consnrrptlon Apparatxts Tho systoa for measuring tho oxygen consumption of intact aninals was modified from that of Fry and Hart (19^7) . The respiration chamber consisted of a one-arm suction filtration flask fitted with a one-hole rubber stopper tlu'ough which a glass tube was passed. The end of the glass tube inside the flask was positioned as far away from the arm as possible (see Figure 1), Two sizes of flask Trere used (250 and 500 ml) t depending upon tho size of the fish, A 19-liter aquarium served as both the temperature bath and the resevoir for the circulating medium. One end of the aquarium was removed and replaced by plexiglass through which a hole was drilled, A glass tube, 5 cm long, was passed through tlds hole around vrliich aquarium cement was applied to prevent leakage. Both ends of the glass tube ifore connected to tygon tubing. The longer tube on the outside of the aquarium passed through a circulation pump and back into the aquarluiii. The shorter tube on the inside was connected to the glass tube of the respiration flask, V/hen the circulation punp was txirned on, water left the flask via the glass tubing and was replaced by crater entering through the arm of the flask. See Figriro 1 for flow directions • V/ater circulated through the cooling coil from a constant teraperatwe bath held at 20° C, An air pump vented tlirovtgh airstones maintained a high oxygen concentration in the aquarium water. To provide insulation and deo-kness, the aquarium was placed in a large Styrofoan^ cooler which v;as painted flat black on the inside.

PAGE 19

cS O t-, o «H bO a :;1

PAGE 20

10

PAGE 21

u Determnation of Dissolved Oxyj^en The Pracision'j' galvanic cell o:
PAGE 22

12 At the beginning of an experiment, a 100 ml sample of water was removed through the outside tygon tubing and oxygen concentration was determined. The system was then stopped from flowing for 30 minutes and a second sample was then taken from inside the flask. The dead space (tygon and glass tubing) was cleared and a 100 ml water sample was collected through the outside tube. The oxygen concentration was determned and subtracted from the first determination. The system was then allowed to flow for 30 minutes and the entile procedure was repeated from the beginning. Five such deterroinations were made during a 4,5-hour period. Fish were superficially dried with paper towels and weighed after e3qperim3nts on a TorsionS^ balance to a precision of 0,01 g, E^erlments ivith blue dye demonstrated that no mixing of water entering and leaving the flask occurred wliile drawing samples. The average of five readings was considered to bo an estimate of rountine oxygen consumption as defined by Beamish and Kookherjii (1964) • The log of oxygen consumption (ml/hr) was correlated i/ith the log of body xraight (g) for each species at four different salinities. Log oxygen consumption = A log body weight + log B where I A = increase in log oxygen consumption relative to increase in log body t'reight B = amount of oxygen consvmiod by a 1,0 g fish. Special Considerations Fry (1957) has suggested that the folloid.ng variables rr.aj affect the oxygen consumption of fisht 1, experimental temperature and thonnal history 2, oxygon and carbon dioxide tensions

PAGE 23

13 3, seasonal influences kt diurnal variations 5, activity 6, nutritional state 7, sex 8, boc3y -weight 9, environmental salinity. In this study, an attempt was made to consider each of these factors a)id to allow only body size and environmental salinity to vary. Diurnal variations, sex, and seasonal influences are not discussed elsewhere in this paper. E^eriments were performed between 0800 and 1200 (Eastern Standard Time) and between 2000 and 2^0 (Eastern Standard Time) . Ho difference was apparent in the resxits betv/een these two groups. Sex of individuals was determined, but was found not to significanUy affect o^qsrgen consumption. The duration of these studies did not permit the observation of possible seasonal influences on oxygen consumption. ' Oxygen Consuicption of Exci sed Gills Experimental Setup The oxygen consumption of excised giUs was determined using a Gilson differential respirometer. This is a closed system in which the reaction vessels are separated from a compensation chamber by a manomster (Umbreit et ^., 1956). Fifteen ml capacity flasks v;ere used. The center well of the flask ccntaiivad a piece of folded filter paper which had been satui-ated with 10^ KOH. The main compartment of the flask contained the gills and 2.0 ml of water of the desired salinity.

PAGE 24

1^ Experimontal Procedure Tho gill apparatus was excised as a vnlt, A single cut v;as made at the caudal end of the isthrius. The isthmus uas pulled cephalad exposing the gill apparatus. After the gill apparatvis had been teased loose using a scapel and dissecting needles, it was separated from the fish ^d.th a forceps and divided into right and left halves. This division allowed the tissue to lie flat in the nanomoter vessels and be completely covered by the mediujri. Oxygen consumption was corrected to standard conditions. Measurements vrere made at 20° C and barometric pressvire was recorded at the beginning and end of each experiment, A period of thirty minutes was allov?ed for equilibration and then readings were taken at 15-minute intervals for a period of 2,0 hours. At the end of each experiment, the gills wore removed from the manometer vessels and the surplus liquid was absorbed from the tissue ifith paper toirels. The gills were placed in small unstoppered vials and dried for one vreek at 110° C in a drying oven. At which time, the dry tissue weight was determined with a KettlerS) analytical balance to a precision of 0,1 mg. Analysis For each species in each medium, a plot of log dry gill iraight (mg) against log oxygen consumption (,/il/hr) was made. Log oxygen consumption = A log dry gill weight + log B, The wet body weight of each fish was also recorded and log dry gill weight (mg) was plotted as a function of wet body weight (g). Log dry gill weight = A log iret body weight + log B,

PAGE 25

15 Special Considerations Krebs (1950) has discussed two important aspects of tissm metabolismt 1, the choicQ of modiiim 2, treatment of tissvies. In the present study, the medium was an artificially prepared sea water (Svedrup et jl., V)^2) and dxl.utions i^ere made with distilled water to produce salinities which would be consistent ^vith those of the blood plasma studies. The giLls were excised as an intact unit and tissue damage was minimal. Gill Surface Area The method of determining the surface area of the gills was according to Gray (195^) and H^oghes (1966). Intact fish .Tere weighed and their gills removed. The number of filaments on each gill arch was counted for the gill apparatus on one side of the fish. The average length of thase filaments was determined by measuring every fifth filament on each gill ai'ch. The average number of lamellae per mm was determined by making five counts on the filaments of each giU arch. The average area of the laiaellae was determined by sampling ten lamellaa per fish. These were dra^m by means of a ZeichentubuP' and a planimeter was used to determine surface area. The lamellae are triangular in cross section. Because these drawiiigs eo^ two dimensional, their areas were multiplied by a factor of three to get total surface area. It is believed that this is an underestimate of the true surface area because the ti^angular cross sections have curved sides. The formula for giU surface area is:

PAGE 26

16 SA = 8X#FXLXlXsa x^here i SA = total gill surface area in mm^ 8 = total nuribor of gill arches vfF = avaorage number of filaments per gill arch L = average length of filatnents in mm 1 = aver?^e number of lamsllae per mm sa " average surface area of larcellao in mm^. For each species, log surface area {mrr) was correlated vdth log wet body '^jeight ( g) , Log surface area = A log vret freight + log B. Fish and gills were stored in 10^ formalin during those irieasuremenbs and correction factors for shrinkage and vnight change vjere applied according to Parker (19^3) . Permeability Gill permeabilities xrare inferi^d from other data by using the equation of Potts (195^) i W = KA (B-ll) R 7 In B/li where I W = work in cal/hr K = permeability in moles/ram^-nolar differenco-hr A = siArface area in mm'^ B = blood concentration in moles/liter M = medium concentration in moles /liter R = 1.937 cal/°C-mole T = absolute temperature in '^

PAGE 27

17 This equation may be rewritten! K = A (B-H) R T In B/H, Work was derived from gill osygen consumption readings by multiplying data by 4,3 cal/inl 02» It was decided better not to stibtract a small amount of energy used for maintenance of the gill cells, because Harvey et al. (19^7) have shoxm that this portion of the metabolism is also instrumental in ion transport. Surface area of the gills was known from the relationships in Table l8. Molar concentrations of the medium and blood were calculated from osmolar concentration data. Assumptions upon which these calculations are based are considered in the discussion. Statistical Methods Regression lines were calculated according to the least squares method for cun/ilinear regression (Killer and Freund, 19^5) • Regression coefficients sxq given xvith one standard error of the estimate and are compared by the method given in Bailey (1959) • Intercepts are given with one standard deviation according to Snedecor (1965) • Both the variation around the sample mean and the variation of the regression coefficient are taken into accoxint. Comparison of intercepts involved the use of "pooled" variances from the regression and the test statistic for intercepts according to Killer and Freund (I965) • Sample standard deviations from regressions were calculated for each regression line according to Snedecor (1965)* Means of the blood plasma samples were compared using a standai^ "t" test statistic for hypotheses concerning two means after Miller and Freund (I965) ,

PAGE 28

RESULTS Activity Both species, are most active immediately after being placed in the respiration flasks • Activity then decreases to a minimal level and eventually increases until the end of the experiment (see Figures 2 and 3) t Between 11 and 15 hours after the beginnir^g of the experiments, F. siiiiilis exhibits minimal activity which is approximately kO^ of the original level of activity. The period of minimal activity for Ft chrysotus occurs between 7 and 18 hours after being placed in respiration flasks, and is loss than 1^ of the original activity, Moasurements of rountine oxygen consumption were made diiring these periods of minimal activity. Concentration of Blood Plasma As the concentration of the medium is raised from 25 mosra/litor to 1,000 mosm/liter, the concentration of the blood plasma of £• sirailis increases from 339 + 15.9 mosra/litor to 397+ 7.1 mosm/liter (Figure h) , At QQ = 370 mosm/liter, the mean fish plasma concentration equals that of the mediiun. All means ai*e significantly different from each other except those of fish in l/3 and 2/3 sea water. The concentration of the blood plasma of F, chrysotus increases from 2'l-6 + 22,8 mosm/liter to 459 + 26,3 mosm/liter as the medium 18

PAGE 29

Figure 2. Activity i^ecord of F, sirailis after placement in respiration chambers. Data are the totals from records of five fish. The period of minimal activity is indicated.

PAGE 30

20 4So!-T Tima hours

PAGE 31

Figure 3. Activity record of F. chrysotus after placement in respiration chambors. Data are the totals from records of five fish. The period of minimal activity is indicated.

PAGE 32

22 Timehours

PAGE 33

3 o f-^

PAGE 34

2k Oi CO N

PAGE 35

25 is increased from 15 riosm/liter to 660 Kosra/liter. At [m] = 980 mosm/liter, tho blood loLasma concentration drops to 297+ 25.3 mosm/ liter (Figm-e 4) . The point of osmoneutrality is at [h] = ^5 mosm/ liter = [b^ . All means are significantly different from one another (P<.001) . Intact Animal Ox^F-.en Consw-ption There are significant variations in the ojcygon consumption of intact F. sitrolis in response to variations in salinity. The results are presented graphically in Figures 5 8 and are smnarizcd in Table 1. The slopes of the log oxygen ccnsumption-log body weight regressions are lowest in l/3 sea water, 0,k9l6, and increase as |b Ml increases. In fresh water, the slope is O.6896, an increase of kO^. In 2/3 sea water, the slope increases 10;^ to 0.5^32 aixi in sea water it is 0.5801, an increase of 3«^. There are no significant difference among these slopes (Table 2), The intercepts of these regression lines increase from ~1.0655 in fresh water (0.086 mL/hr) to -0,8239 in f^^ll sea water (O.I5O ml/hr). m intercepts are statisticaDy different from each other, except those in l/3 and 2/3 sea water (Table 2). The slopes of the log ojcygen consuraption-log body weight regressions for F. chrysotus follow the same pattern as for F. .similis. See Figures 9 12 and Table 3. The slopes are Ddnimal in l/3 sea water, 0.3'+30, 8JkI increase as |b m| increases. In fresh water, the slope is 0.3578, a IQf^ increase over the slope in l/3 sea water. In 2/3 sea water, it increases 12>^ to 0.i*'119 and it is 0.5077, a 15^ increase, in full sea water. Statistically, the slope in sea water

PAGE 36

Figure 5. Oxygen consumption of intact F. similis in fresh water as a function of body weight. Slope = O.6896 + 0,0755, Intercept = -1,0655 + O.O'j-36, Correlation coefficient = 0,91.

PAGE 37

27 Body Weight -g

PAGE 38

Figure 6, Oxygen constmiption of intact F, siwilis in I/3 sea water as a function of body weight. Slope = 0,'i-9l6 + 0.0772, Intercept = -0,9508+ 0,027^. Correlation coefficient = 0,81.

PAGE 39

29 Body V^oight-g

PAGE 40

Figure 7. Oxygon consvtmption of intact F, similis in 2/3 sea water as a function of body weight. Slope = 0, 5^4-32 + 0,092?, Intercent = -0.9281 + 0. 029^4-, Correlation coefficient = 0.77.

PAGE 41

31 0.02 0.01 0.1 2/3 sea vi/ater a2 Body Wfilght-g

PAGE 42

Figiire 8, Oxygen consumption of intact F, sinili s in full sea vrater as a fvmction of body weight. Slope = O.58OI + O.O625, Intercept = -0.8239 + 0.0^69. Correlation coefficient 0.89.

PAGE 43

33 %^>'* ' * saa water ^2 0.5 5 » f.iw^w 10 20 Body Weight— g

PAGE 44

3^ §3 (X, (0

PAGE 45

35 TABLE 2 STATISTICAL COIiPARISONS OF SLOPES AIID IKiTERCEPTS OF THE D0U12LE LCGARITffilC RELATIOIJSIUP BSTVEEN OriGEI-I COIJSUIiPTION AND ^vEIGHT OF lOTACT F. sJxillis AT FOUR SALIiaTIES Compai'ison Slopos Intercepts f-1/3 1^.05 *P<.05 f-2/3 P>.05 *P<.01 f-3/3 P>.05 *P<.001 l/>2/3 P>.05 P>.05 1/3-3/3 P>.05 *P<.01 2/>3/3 P>.05 *P<.05 •* Considered to be statistically significant.

PAGE 46

Figure 9» Oxygen consuraption of intact F, chrysotus in fresh water as a function of body woight. Slope = 0,3578 + 0,03^+8, Intercept = -0,9208+ 0,0118, Correlation coefficient = 0.91.

PAGE 47

37 0.40 030 4^0.20 o §0.10 a E 3 M u e o 0.02 0.01 0.1 frash water J— 0.2 as lllf ! IIIWIIWI^ !* II 2 3 4 Body Weight -g

PAGE 48

Figure 10, Oxygen consumption of intact F, chrysotus in l/3 sea water as a fxmction of body freight. Slope = 0,3^30 + 0,0518, Intercept = -0.9066+ 0,01^, Correlation coefficient = 0,81,

PAGE 49

39 0.40 0.30 O 0.20 e 9 •^ 0.10 a E 3 CO S o O 0.05 e > M o 0.02 0.01 0.1 1/3 sea water J 1 0.2 0.5 3 4 Bcdiy Weight -g

PAGE 50

Figure 11, Oxj'-gon cons^Imption of intact F, chrysotns in 2/3 sea water as a fvmction of body vreight. Slope = 0.4119 + 0.0511, Intercept = -O.9066 + 0.0215, Correlation coefficient = 0.86.

PAGE 51

41 0.40 aso jB 3'0.20 sa e 9 •^ 0.10 a E 3 M K O W 0.05 e o O 0.02 0.01 o 2/3 soa water 2 ae i Body Wo(ght-g

PAGE 52

Figure 12, Oxygen consumption of intact F, chrygotus in fxxLl sea water as a function of body vreight. Slope = 0.50??+ 0,0^-2^, Intercept = -1.017?+ 0.0126. Correlation coefficient = 0.93.

PAGE 54

44 I

PAGE 55

^5 differs from both the sloi^e in fresh water (P<.01) and the slope in 1/3 sea water (P<.05) . The hypothesis that any other differences in slopes exist is rejected (P>.05). See Table k. The intercepts of the regression lines for F. chrysotus do not show as much variation as those for F. simlis. They are -0.9208 (0.120 na/hiO in fresh water and -O.9O66 (0.12^^ ml/hr) in l/3 and 2/3 sea water. Ko-^rever, the intercept decreases in full sea water to -1.0177 (0.096 ra/lir), a result which is significantly different from all other intercepts (P<.001), See Table k^ An interspecific coopsrison of slopes at each salinity shows that they differ in fresh water (P<.00l). In all other salinities, a difference in slopes does not e^dLst (P>.05) . The intercepts differ in fresh and full sea water (P<.00l) . but do not differ in l/3 and 2/3 sea water (P>,05). See Table 5. Tables 6 and 7 show oxygen consumption of F. similis and F. chrysotus of five sizes at fo-or different salinities. Thesevalues are calculated from the relationships given inTables 1 and 3. Charges are calculated relative to values in l/3 sea water which is nearest to being osmotically equivalent to the blood plasma. 0:?ygen consumption is lowest in fresh water for F. similis weighing from 0.5 to h,Q g. A ^+.0 g fish has approxiinately the same oxygen consumption in fresh and l/3 sea water. A 10.0 g fish shows a 21.1^ increase in oxygen consumption in fresh water. In 2/3 sea water, fish of all sizes show increases in oxygen consumption (1.6^ to 18.6^). In full sea water, increases range from 25.0^ for a 0.5 g fish to 63.8^ for a 10.0 g fish. All fish in a hyperosmoUc medium show increases in oxj-gen consumption relative to l/3 sea water. Small

PAGE 56

46 TABLE ^ ST/iTISTICAL C0IPARIS0N3 OF SLOPES AND INTERCSFTS OF TIE DOUELE LOGARITICIC RELATIONSHIP BETIfESN OXYGEM COi;SU]!PTION AND VffilGKT OF niTACT F. chrysotus AT FOUR SAL.IiJlTIES Comparison Slopes Intercepts f-1/3 f-2/3 f-3/3 l/>2/3 1/3-3/3 2/3-3/3 P>.05 P>.05 *P<.01 P>.05 *p<,05 F>.05 P>.05 P>.05 *P<.001 P>.05 *P<,001 *P<,001 *Corisiderod to be statistically significant.

PAGE 57

hi TABLE 5 niTSRSPECir.05 P>.05 2/3 P>.05 ' P>.05 3/3 P>.05 *P<.ooi ''Considered to be statistically significant.

PAGE 58

^ W >H CO a H CO O Q O CO H ^ I o ^^ II ^-N (D + o II .-N
PAGE 59

^9 CO ^li

PAGE 60

50 fish (loss than ^,0 g) in a hypoosraotic ncdiira shox7 deci'oasos in oxygen consunption, while lerge fish (more then ^.0 g) in the sarao salinity show increases in oxygen consumption. The oxygen constnnption of F, chrysottts is relatively stable from fresh to 2/3 sea v/ater and then drops in full sea water. In fresh water, all fish show slight decreases in oxygen consumption ranging from li-,Z% for a 0.5 g fish to 2,Zjl> for a 2,0 g fish. In 2/3 sea x/ater, fish below 1.0 g show a slight decrease in oxygen consumption , and fish above 1.0 g show a slight increase in oxygen consiunption. In full sea water, all fish show decreased oxygen consumptions with the greatest depressions occvirring in the smallest fish. The slopes obtained for the double logaidtmic relationsliip betvjeen oxygen consumption and body weight are lo-vTer for both species in all salinities than those reported for other fishes. Job (1955) found slopes of 0.802 O.856 for Salvelinus f ont i nalis . Hickman (1959) found a slope of 0.86 to represent the effect of weight on the oxygen consraption of the starry flo^Jinder, Platichth ys stellatus. He also found slope values of 0.8-'+ for lemon sole, P,a£oph;vs vetulus , and 0,90 for sanddab, Cithari chthys sti^aeus , O'Hai'a (I968) found slopes of 0,71? and 0.710 for Lep^-'^is mscliroehiriis and Lepomis p;i.bbosu5 respectively. Beamish cxvi I'ookher jii (19^) fotmd an average slope of 0,85 for CarassiTi s aiu'atus . Only the value inferred from data of Wells (1935) for Fundulus parvipirnis approaches the values in this study. His data imply a slope of 0,50, All other studies concerning the dependence of oxygen consumption on weight in fishes have been conducted using larger fish than those in this study and only data of V/ells are for fish of a comparable size range.

PAGE 61

51 0>:ygen Consvimption of Excised Gills The slopes of tlie regrsssion lines of log oxygen consvrraption-log dry gill weight for F, sinalis are maximal (0,5817) in l/3 sea water and decrease as JB MJ inci^ases (FigtU'es 13 l6 and Table 8), In fresh water, the slope decreases 46fo to 0,3151« iJi 2/3 soa water, it is 0,^1^, a 29^ decrease. In full sea water, the slope is minimal at 0,2^53» a 5?^ decrease. Statistically, the slope in full sea water differs from both the slope in l/3 sea water (P<,001) and the slope in 2/3 sea water (P<,05) , See Table 9, The intercepts of these regression lines for F, sixrdlis are minimal in l/3 sea water (0,^751) and inci'ease as JB K^ increases. In fresh water, the intercept increases 50^ to 0,7121. Intercepts are 0.7526 and 0.9531 in 2/3 and 3/3 sea water respectively. These corx^espond to increases of S7f> and 101;^ over the intercept in l/3 sea water. The intercept in l/3 sea water differs from the intercept in 2/3 sea water (P<,02) and from the intercept in full sea water (P<.001) , See Table 9. The glUs of F. chrysot xrs vrare more difficult to handle because they were smal l er (1 20 mg dry weight) than gills of F. similis (1 157 mg dry waight) , Only 5$^ (66 of 120) of the excised gills sur-\rived the operation and consxtmed oxygen when placed in the respirometers. Therefore, the follovjing results are of questionable value . The slopes of the regression of log oxygen consumption-log dry gill weight for F. clrt-ysot-gs vai'y neither directly nor indirectly T-dth |b m| (Figures 17 20 aijd Table 10). The slope is minimal in

PAGE 62

Figure 13. Oxygen consumption of excised gills of F, similis in fresh water as a function of dry weight of gills. Slope = 0.3151+0.1311. Intercept = 0.7121 + 0.1325. Correlation coefficient ~ 0.^7,

PAGE 63

53 2Cfe .2 10 * Q. E a e O 5 s 9 & > H O fresh ^Aratar 10 20 wJLMUtMimJ!^ 40 Dry GiSI Woiyhi— mg

PAGE 64

Figure 1^, Oxygen consvunption of excised gi.Ils of F, sii^ii lis in l/3 sea vrater as a f miction of dry weight of gills. Slope = 0.5817+ 0.0673. Intercept = 0,^751 + O.O685. Correlation coefficient = 0.8?, "

PAGE 65

55 Dry Gill Woight -mg

PAGE 66

Figtire 15 1 Oxygen consuraption of excised gills of F, si milis in 2/3 sea water as a function of dry vraight of gills • Slope = 0.4li{4 + 0.0?64. Intercept = 0,7526 + 0.0888. Correlation coefficient =0,77. "

PAGE 67

51 Dry GiJI Weight — mg

PAGE 68

Figure l6. Oxygon consvtmption of excised gills of F, similis in f\ill sea water as a function of dry iraight of gills. Slope = 0.2453+0.0592. Intercept = 0.9531 + 0.0685. Correlation coefficient = 0.66,

PAGE 69

200 59 J ICO 5^ ^ s i IE 3 M e e o e s sea viator OryGSH Walght-mg

PAGE 70

60 CO CO Q CO + 1 -P o o I H + 1 o H CO CO o §° © w o o C Q o — o o

PAGE 71

61 TABLE 9 STATISTICAL COl'IPAjaiSONS OF SLOPES AJID lOTERCEPTS OF THE DOUHLE LOGARITm'lG RSLATIONSlilP BETVEEW OXTOEN CONSUI'T^riON Ii}JD DRY VffilGHT OF EXCISED GILLS OF F. simnis AT FOUR SALINITIES Comparison Slopes Intercepts f-1/3 P>.05 P>.05 f-2/3 K>.05 F>,05 f-3/3 R>.05 P>.05 1/3-2/3 P>.05 *P<.02 l/>3/3 *P<.ooi *p<.ooi 2/>3/3 *P<.05 P>.05 *Consid©red to "bo statistically significant.

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Fig\ire 17. Oxygen consxanption of excised gills of F. cbj.ys otus in fresh water as a f'oaiction of dry vieight of gills. Slope = 0.3if36+ 0,1216. Intercept = 0.6022+ 0.0801, Correlation coefficient = 0.60, "

PAGE 73

63 e o B. g 3 «) G O y c o Dry GiBl Weight -mg

PAGE 74

Figm-e 18, Oxygen consumption of excised gills of F, chry sottis in l/3 sea water as a function of dry weight of gills. Slope = 0.1383+ 0.3^102. Intercept = 0.7599 ± 0,1991. Correlation coefficient = 0,11,

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20 65 e o i. 3 5 «» c o u e > O 2 )/3 sea watGr It 10 Dry Gill ^Veight-mg

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Figure 19. O'^'gon consumption of excised gills of F, c hrysotus in 2/3 sea water as a function of dry weight of gills. Slope = 0.5173 + 0,18^5. Intercept = 0,5533 + 0.0979. Correlation coefficient = 0.59.

PAGE 77

5" 20 (il c o Q. g 3 C 9 u • ^« 2/3 sea wator 2 10 20 Dry Gill Weight -mg

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Figure 20, Oxygen constiPTption of excised gills of F, chr^^sotus in fidl sea water as a function of dry weight of gills. Slope = 0.^58fJ +. 0.1315. Intercept = O.6302 + 0.0937. Correlation coefficient = 0,67.

PAGE 79

69 L arm e o 3 « e o u > O 2 sea water MWiMMiirm X -L 10 20 30 Dry Gill Weight -mg

PAGE 80

70 o H m Q 00 CO + 1 •a o o + 1 O iH CO ^ v< O 1^ ^ -P O §§ o o C E o v^ o O Vt »r\ CM o o ^ H OS eg o VO H VO H Cvvo f-l

PAGE 81

71 1/3 sea water, 0.1383. In fresh water, it increases 1^^ to 0.3^1-36, In 2/3 soa water, the slope is 0.5173f a 27^ increase. However, in full sea water the slope drops to 0A5^, a 231'/ increase over the slope in l/3 sea water. There are no statistical differences a^o^S these slopes (Table 11). The intercept of this relationship for F. c^Zso^iSS ^s minimal in 2/3 sea water, 0.5533. It increases 9^ to 0.6022 in fresh water. In 1/3 sea water the slope is iiis.-dnal at 0.7599» on increase of 38^ over the slope in 2/3 soa water. The intercept for the relationsliip in ftai soa vrator is 0,6302, 5^ greater than the intercept for the relatioi^sMp in 2/3 soa water. Statistically, none of these intercepts differs from each other (Table 11), Copiparing slopes ar^d intercepts at each sal3jiity for the two species shows that the intercepts differ in soa water (P<,01) , See Table 12, Tables 13 and 1^^ show the aiuotint of oxygon consumed by excised gills of various sizes of fish in fottr different salinities. Gill vieight is calctaatod for each size fish based on the relationships in Table 18, Oisygen co-osumption is derived from ths relationships i-n Tables 8 and 10, For a small F. slMlis (0.5 g) . oxygen consumption of the gills increases as JB Ii| i)?creases, A 1.0 g fish also shows increases relative to l/3 sea water when it is in a hyperosmotic medium, but there is no rise in gill o^gon cons-v.a-.:ption when it is in a hypoosmotic medium. Fish above 1,0 g show decreases in gill osygen consumption in fi-esh water. Fish \rp to ca, '^-.O g show increases in gill o:xygen consumption during hypoosmotic regulation. Above this weight, decreases in gill oxygen consxuTrption occor and

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72 TABLE 11 STATISTICAl COITARISONS OF SLOPES AMD INTERCEPTS OF TIffi DOUBLE LOGARITffilC RELi\TIONSHIP BETVEEN OXTGEN CONSUl-PTION AND DRY V/EIGHT OF EXCISED GILLS OF F. chrysotiis AT FOUR SALINITIES Comparison Slopes Intercepts f-1/3 P>.05 P>.05 f-2/3 I>.05 1^.05 f-3/3 P>.05 P>.05 1/3-2/3 P>.05 P>.05 1/3-3/3 P>.05 P>.05 2/3-3/3 P>.05 P>.05 * Considered to bo statistically significant.

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73 TABLE 12 INTERSPECIFIC S^^^ICTICAL COLTARISOKS OF SLOPES Aim lOTERCEPTS OF TKE DOUBLE EOGARITHKLC RKLATI0N3KIP BET/EEN OXYGEN CONSUllFTION MD DRY i'iLTGIiT OF EXCISED GILLS AT FOUR SALINITIES Kediura Slopas Intercepts f P>.05 P>.05 1/3 P>.05 P>.05 2/3 P>.05 P>'05 3/3 P>.05 *P<.01 Considered to be statistically significant.

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7^ r^ w Q O CO C=, o I o II ^^ I O "^ (X) 3 *

PAGE 85

15 10 o I f^ CO °« H O CO ^^ H « P. o r-i4 o °^ s o O H ' O s o o 00 g II m CM 5 » M o <3 CM ,c! I o ^^ to §^^ VO A + o JL o ^ — P5 id © * CM ^ + O II ^_ ^ C\J ^ I O "^ s o O + CN SO i c^ CO VO CO 'A -:3la^ + H <^ ^ + + + VO CJv CO Pi + • CO C3V -P hJO o o id o o o o o CM

PAGE 86

become greater as JB k| increases. The results for F, .chrjrsotus show increases in gill oxygen constsmption relative to I/3 soa water for all fish in full sea water aiid for fish laa-ger than 0,5 g in 2/3 sea water. A 0,5 g fish shov;s a decreased gill oxygen consumption in 2/3 sea water. Fish iTeighing 1.0 g or less have depressed oi'gen consuriiptions , but animals above 1.0 g show decreased gill oxygen consuimptions , In 2/3 sea water, increases in the oxygen cons\amption of intact animals are

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11 TABLE 15 COI'TARISON OF PERCEOTATrE CIL^IIGE IN OXYGEN CONSDT-iPTION OF INTACT AK!II-:;'iLS AND EXCISED GILLS AS A FUNCTION OF SALINITY

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?8 present in all sizes of fish and tho percentage increase becoines larger as body size increases. Oxygen consiimption of excised gills also shows increases, but the percentage doci^ases vdth increasing body si?.e, until at 10,0 g there is an 8,^% reduction in gill oxj'-gen consumption. In 3/3 sea water, all F, similis show increased oxygen consmiption by intact aniiaals and the percentage change relative to 1/3 sea water increases td.th increasing body size. Fish weighing 2,0 g or les.':' shovj increased gill oxygen consurnption, but ^.0 and 10,0 g fish show doci'easod gill oxygen consumption. All sizos of F, clnysotus show decreased oxygen consumption of intact animals in fresh water. Animals i-.'eighing 1,0 g or less also show decreased gill oxygon concu'nption, Hovrever, animals ^reighing either 1,5 or 2,0 g show increases in gill oxygen consumption. In 2/3 sea water, oxygen consumption of intact animals decreases for animals of loss than 1,0 g and increases for animals vreighing more than 1,0 g. Oxygen consumption of gills of animals of all sizes except 0,50 g shows increases. In 3/3 soa water, oxygen consumption of intact anunals of all sizes is reduced, v;l-iile gill oxygen consumption of all animals is elevated. There is no apparent correlation between changes in oxygen consumption of intact animals and changes in oxygen consumption of excised gills for either of the species. Apparent Permoabilit y Apparent permeabilities which have been calciilated with the equation of Potts (195^) are given in Tables I6 and 17. In all

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79 2,00 TABLE 16 APPAREI'IT PEPJIE^VBILITY OF F. sinjJ.is AS A FUNCTION OF BODY SIZE MD SALimi V7t Surface area Perrnoability change Perineability B-M = +31^ B-H = +25^ Perrnoability change Perineabili-t^ (g) (mm?) O'/ima^Al-hc) % (H/mra^^M-hr) 0,50 150 4.11 X 10-^ -99. (^ 3.84 X 10"^ 1,00 251 2,82 X 10"^ -99.1$S 3.17 X 10"^ 490 1.93 X 10"^ -99.3^ 2,6l x 10"^ 4,00 387 1.32 X 10"*^ -99.45^ 2,51 X lO"'^ 10.00 1,941 0.80 X 10"^ -99. 55^ 1.66 X lO"'*' tf^Difference baUreen the medium concentraticn and blood plasna concentration d.n railliosnoles/liter, . Hi lioles per square railliineter per molar concentration difference per hoxir.

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80 TABLE 16 (Extended)

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81 TABLE 17 APPARiiNT PERI^EABILITY OF F. chz-jsotvis /\S A FUIICTION OF BODY SIZE~A1«) S/iLIHITY 3-H = +231^ B-II = +66^^ Wt Surface area Perriie ability* change Permeability* (g) (rm^) {Vi/mm^'-ll-hr) ^ (ii/mra^-II-hr) 0.50 102 if. 36 X 10"^ 9^!-.5^ 7.36 X 10"-5 9^.0^ 5.1^ X 10~^ 93.6^ 3.82 X lO-^ 93.1^ 2.51 X 10-5 92.?^ 1.85 X 10~-5 0,75

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82 TABLE 1? (Extended)

PAGE 93

83 salinities for both spocies, the smallest fish have the greatest permeabilitiost Percentage decreases relative to permeabilities in 1/3 sea water are also given in Tables l6 and 17, The percentage decrease from l/3 sea water increases with body sise in all salinities for F, similis . For F. chrysotus , this factor decreases Td.th body size in all salinities. Anato r oical Rolationshj-ps The log gill surface area-log body vreight relationship for . F, jlJnilis indicates that gill surface area increases less than proportionately as body weight increases (Figure 21 and Table 18) . • The slope eqvials 0.8^^57 and the intercept is 2,^330, A similar analysis for F, chrysotus shovrs that gUl surface area increases more than proportionately as body Tveight increases. The slope equals 1,1753 and the intercept is 2,3623 (Figure 22 and Table 18). The slopes differ statistically from each other, as do the intercepts with P<,01 in both cases (Table 2^) , The most notable studies concerning gill stirface area of fishes are by Gray (195^) , Steen and Kruysse (1964) , and Hughes (1966) , Gray (195^) found surface ai'eas ranging from 188 mm^/g for Lophopsetta maculatus to 1773 mm^/g for Erevoortia t yranm is . Steen and Kruysse (1964) found that a 550 g eel had 530 cm2 of gill svirface area or 1,075 ram^/g, Hughes (1966) foiorjd surface areas ranging from l68 mmVg for a 24 g C alliony mlus lyra to 845 mm^/g for an 85 g Clupea harengus . The surface area of F, similis ranges from 15^,3 mm^/g for a 9*25 g fish to 313.^ mm^/g for a l,^f6 g fish. The surface area of F, chry sotus ranges from 148,6 mnr/g for a 0,36 g fish to 2?9,7 iraa^/g for a 3.88 g fish.

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Figtire 21, Svirface area of gills of F, simllis as a function of body weight. Slops = 0.&J-57+ 0.0307. Intoi-cept = 2.^330 + 0.0204, Correlation coefficient = 0,99,

PAGE 95

85 I s < 3 <9 Body •J©lf25tt-g

PAGE 96

FigTore 22, ourfaco area of gills of F. p hxyso tus as a function of body woi.'iiht. Slope = 1.1753 + 0,0295, Intercopt = 2,3623 + 0,0133, CorrelatiorTcoefficient = 0,99,

PAGE 97

87 L g 2C0 a o < 9 t 3 (A 1CH5 5 Cody Wolght -g

PAGE 98

88 CO H C-i CO o o 9

PAGE 99

89 TABLE 19 INTERSPECIFIC COtiPARISON OF SLOPES AND INTERCEPTS OF MATOI^ICiURELATIONSHIPS Relationship Slopes Intercepts Svri&oe aroa-Body wt# *P<,01 *P<.01 Dry gill >;t-Boc3y wt^ ^ P>,05 *P<,001 * Doiible logai'itlimlc relationship, * Considered to be statistically significant.

PAGE 100

90 Only Prico (1931) has previously studied the gill surface ai'ea of a fish as a function of body weight and has found a slope of 0,78 for Kicroptemis dolomiou . This is lov;er than tho slopes of 0,8^6 for F. sitnilis and 1,175 for F. chi\ysotus . Dry gill freight increases proportionately with body weight in ' both F. siinilis and F, chrysotus (Figures 23 and 2k), The slopes are 0,987^ for F, sinilis and O.967I for F, clirysotus. These are not significantly different from each other (F>.05) . The intercejjts of this relationship are 0,89^ for F, siinilis and 0.7021 for F. chrysotus. The intercepts are significantly different from each other (P<,001) . These ar^toinical relationships and statistical cociparisons are reviewed in Tables 18 ajad 19.

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Figiu-e 23. Div vreight of gills of F, siriilis as a function of body Xs-Glght. Slope = 0.9874+ 0.05^45. Inter cept = 0.89'U) + 0,0201, Correlation coefficient = O.89, "

PAGE 102

92 200100 Bi

PAGE 103

Figtire 24-, Dry weight of gills of F, clirysotus as a function of body vraight. Slope = OlSoTl + 0,1022, Intercept = 0,7021+ 0,0Z&\-, Correlation coefficient = 0,65,

PAGE 104

9^ Body Weight-g

PAGE 105

DISCUSSION The capacity for precise osmoregulation is required of fish living in v:at8rs of rajbidly fluctuating salinity if conforrrdng to the external concentration means trespass across the internal limits of tolerance. In waters of stable salinity, there is little advantage in being able to osraoregvilate over a wide range of salinity, especially if such osmoregulatory ability requires additional energy to maintain the involved mechanisms. F. sirdlis , which naturally lives in waters of Tvidoly varying salinity, caji regulate blood plasma concentration precisely. Large populations of F. chrysotus inhabit fresh water only. This species possesses a less precise pattern of regxilation. In the fresh-water environment, individual.s may bo effective stenohaline hyperregulators, but they are not necessarily the bast regulators over a wide range of salinities. The concentration of tha blood plasma of F, similis , living in sea water, is ca, 390 mosm/liter. This is comparable to ottier values for salt-water fishes (Carrey, 1905j Dakin, I9O8; Duval, 1925} Black, 1951 J Vinogradov, 1953} Black, 1957; Hickman, 1959). The concentration of the blood plasma of F, chrysotus , living in fresh water, is 2^6 Kcsm/liter, This is comparable to, but slightly lower than, values for other fresh-water teleosts (Duval, 1925} Krogh, 1939? Black, 1951 I Robertson, 195^; Black, 1957). This slight depression may result from a reduced energetic expend! tui'e in fresh water. 95

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96 Tho pattoi'n of regulation found for F, sitrJllis has boon reportod foi' othor tcleosts (Duval, 1925; Keys, 1933; Hickman, 1959; Gordon, 196^}-; Valentine and Killer, 19^9) • The pattern of a gradual increase in blood plasma concentration up to some critical salinity, followed by a sudden decrease, iras shovm for F. chrysotus« Lotan (19^6) has previously described a sitiiilar pattern for Tilapia aurea and has concluded that the sudden decrease in blood plasma concentration may be the result of decreased porcBability, The depressed oxygen consumption of intact F» chrysotus in fvill sea water may be the result of decreased permeability, Stanley and Fleming (19^6) have found this pattern of regvGLation in Ftmdu lus kansao when fish are rapidly trajisf erred from fresh water to water of h3.gher saliriity, HoviTavor, TJhon fish are complotely acclimated to solinity, F, kansae regulates in a fashion similar to F, ^ similiB , Z» c hiy sotus is ecologically a stenohalina fresh-water specios, but it possesses a pattern of regulation simlnr to T» aux^^a axxi F, kansoi^_ , both of v.'hich are euryhaliiie species, F, chrysoti-is has tho physiological capacity to survivo indefinitely in waters of high salinity and its energy expenditure is similar to that of F, simllis . However, only occasional fish are foij/id under such conditions normally. Some other factor of an ecological or physiological nature, such as competition for food or salinity tolerance of the eggs, must be ijnportant in liroiting the range wMch F, chry sotus nornally inhabits, A possible error in the blood plasma concentration studies arises from the fact that most sarTp3.es from F, siJdJ li s xcei'e larger than those from F, chrysotu s. This was vmavoidable because of tho difference between the size ranges of tho two species.

PAGE 107

97 In Tables 6 and 7f salinity-dopondont changos in tho oxygen consumption of intact animals do not appear to bo correlated with changes in osmotic work for the follo^d-iig reasons i 1. AsGXuning Potts (195^) and Potts and Parry (196^^') have correctly estiBiated the energy necessary for osmotic work, the changes in oxygen consumption of intact animals are too lai'ge to be explained by differences in ion transport. Potts (195^) has stated that between 0,3^ and 1,:^ of the total energy requirement is used for osmotic work. Potts and Parry (196^) have estimated 7$ for tliis value and further have claimed thati ,,,the increase in metabolism consequent on tho increased ion transport would not be detectable against the backgromid of the metabolism of the ^Aole animal, (p, 337) Z» sim ilis show changes in intact animal oxygen consumption ranging from a 3^o docreass for a 0,5 g animal in fi*8sh water to a 64^ increase for a 10,0 g animal in sea water, F, chrysotus values range from a 31^ decrease for a 0,5 g animal in sea vrater to a 5/S increase for a 2,0 g animal in 2/3 sea water, 2. Thei*e is no change in the oxygen consumption shown by a 1,0 g F, chrysotus in 2/3 sea water even though JB MJ has increased to three times what it v/as in 1/3 sea water, 3. There are large decreases in oxygen consimption shown by F, siTTa.lis in fresh water and F, chrysotus in sea water. If changes in intact animal oxygen consim^tion . are associated with changes in osmotic i?ork, then as

PAGE 108

98 JB K] increasoSf oxygen consxunption should also increase vjiless permeability is varying. Significant changes in the o3
PAGE 109

99 aro of thQ proper sizo to account for changes in osmotic work, thoir direction is not ali-rcys parallel to the changes in \B ~ m\ • Somo decreases in gill oxygen consxiaiption occur as |B Vi\ inci>3as3s. At loast tivo explanations aro possible, 1, Potts and Parry (196^1-) have stated j ,t,it is possible that transport systems can adjust to different demands either by 'changing gear* at each trai-irpoz't site, or by having a vai'iety of sites of different capacity, (p, 39) Thus an identical amount of oxygen consumption can result in various amounts of salts being transported depending on the demands placed on the system, 2. Smith (1967) has suggested that permeability of meinbranes laay vary as a function of salinity. If gill permeability is decreased sufficiently, then it is possible for a decreased energy expondituro to be associated x^th an increase in JB M|» Both factors may bo responsible for the fact that changes in gill oygen consuiaption do not consistent3.y parallel the changes in \B k| . Since this stuc3y was not carried out on an ion flux level, the first factor cannot be considered. But giJJ. perineability can ba calculated according to Potts (195^+) t as outlined in the materials and methods section. Because the possibility of a transport puinp with a changing efficiency is not considered in this paper, these permeabilities are better termed apparent penncabilities, A necessary assumption in calcvilating permeabilities in this manner is that salt gained by the gills equals salt lost by the gills. For a fish in a hypoosmotic mediun, this means w© must assume that salt lost in the urine equals salt gained through the intestinal

PAGE 110

100 tract, Sinco theso two values are sKall both quantitatively and energetically relative to flvix through tho gills (Grafflin, 1933 1 Potts, 195^; Black, 1957)1 this is a reasonable assumption. In a hypo rosTiio tic nedium, tho urine of fishes is nearly isosmotic to the blood (Baldwin, 19^8) , The Icidnoy neither dilutes nor concentrates the blood, SwalloTd.ng as an important osmoregulatory mechanism in sa3.t water vras first suggested by Smith (1930) • Recently, Potts and Evans (I966, 19^7) and Ilotais and Maetz (19^^^) have questioned the relative importance of drinking sea water. Potts et _al, (19^7) have estimated that 13.3,» of sodium influx in Tilapia mossambica is throxigh the gut iviaen the fish is living in 100^ sea water. Although drinking is essential in sea water, it is of much less iiriportance quantitatively than gill activity. Because the intestinal tract does play some rolo in hypoosmotic regulation, work estimated for fish in salt v/ater -aay be a little low and therefore apparent permeability may also be low. For both species, the apparent permeability is greatOvSt in water that is nearest to being isosmotic with the blood plasma (Tables I6 and 17) • Apparent permeability decreases as |B m| increases. If we assvuae that gas, ion, ard water permeabilities are related to each other, this sviggests that gill pei^neability is a compromise between gas exchange ani osmoreg-ulation, A higlily permeable gill is desirable for gas exchange and a relatively impermeable gill is a requirement for precise osmoregvilation, VJhen the concentration gradient between the blood plasma and the external medium is low, the fish gill is highly permeable, allowing enhanced gas exchange, Ho;rever, if the concentration gradient is groat, the fish gill becoiiKJs more impermeable, decreasing tho energy necessary for osmoregulation.

PAGE 111

101 In both species, apparent psnaQability decreases •with increasing body size at all salinities. Tliis result seems reasonable for F. chrvsotus because its sm-face area per unit v.nsight increases as M * _. I. L Ml I I body weight increases. F. similis shows a decrease in both apparent permeability and weight specific surface area as body freight increases. This enables large meiiibers of tliis species to regulate at reduced energetic costs. Potts (195^0 has given values for KA (svirface area X permeability), osmotic work (cal/g-hr) and its percentage of the total energy expenditua-e. These values are given in Table 20, The KA factor varies from 3,312X10'^ to l.il-2:a0'"^ moles/molar concentration difference-lir. In this study, KA varies from 3.85X10"^ to 3.23X10-1 moles/molar concentration difference-lir for F. similis (Table 21) and from 1.20X10 to 9.62X10-3 moles/molar concentration difference-hr for F. chi-yso tus (Table 22) , Potts has given values for osmotic work varying from Z.klXlO-^ to 1.26X10-3 caJ./g-hr, F. similis uses 9.69X10-3 to 1.21X10-^ cal/g-hr and F. chrr/sotus uses from 1.90a0"'2 to 6,32X10"2 cal/g-hr. These values are larger for two reasons! 1, Potts discusses values for 60 g animals. Values given for this study are for F, s imjais -vreighing from 0.5 g to 10.0 g and for F. chrysotus ireighing from 0.5 g to: 2,0 g. That these values decrease vdth increasing body size may be seen in Tables 21 and 22, 2. Potts states that his values are thermodynamically minimal for performing the necessaa^y osmotic work. Osmotic vroi'k varies as a percentage of the total Trork expenditure from

PAGE 112

102 TABLE 20 ENERGY EXPEUDITURE FOR OSMOREGULATION (POTTS, 195^) Species

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103 TABLE 21 El^ERGY EXPENDITURE OF F. si mil 5 FOR OSf'OREGULATION

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10^ TABLE 22 ENERGY EXPEIJDITURE OF F. clirysotus FOR OSMOREGULATION

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105 0.?/. to 1.2^ according to Potts. In the present study, oxygen consumption of e::cised gills varies from ^,^1^ to 1^.^^ of the total oxygen consumption for F. sn.niilis and from 5.0^ to 9.7^ of the total oxygen consumption for F. chrysotus. These values are also higher than those of Potts due to differences in sizes of the ani-^als and the efficiencies of the systems. Data in Table 21 aiid 22 suggest that F. simnis pays a higher price for its precise pattern of osmoregulation than F. chrysotus_ pays for its sloppy pattern of regulation. Comparing fish of equal sizes, the differences in energetic costs can bo estimated. The gills of 0.5 g and 2.0 g F. sird-lis utilize more energy than gills of 0.5 g and 2.0 g F. cln-ysotus at all salinities. The diffei^nce ranges from l,06n0-2 to 5.89X10"^ cal/g-hr for a 0.5 g fish and from 0.83X10-2 to 1.62X10-2 cal/s-hr for a 2.0 g fish. As body size increases, the discrepancy botv?BGn the price for precise regulation and sloppy regu3.ation decreases. Changes in the oxygen consumption of intact animals do not consistently parallel changes in the oxygen consuir^ption of excised gills because they are directly dependent upon different variables. Oxygen consumption of intact apdmals depends priJJ'.arily upon the degree of musculcr activity (Spoor, 19^6). That activity is a function of salinity has been suggested in a previous paragraph by the fact that animals under the greatest osmotic stress ere inactive and consuna small amounts of oxygen. It is assumed that a major factor determining the oxygen consumption of excised gills is the amount of osmotic work being performed vrtuch in turn is a function of salinity. Although oxygen consur5>tion of excised gills is a function of osmotic work, the

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106 relationshi.p is not a simple ono as tlio efficioncy of transport piunps and permeability may also vary as a function of salinity. Intact animals and excised gills consume ojygen at rates uliich indirectly depend on salinity, Hovrever, the factors ttpon v^liich those rates directly depend are distinct. The effect of temporatiire on the standard oxygon consumption of fishes has been shoim to be independent of body size (Job, 1955? Beamish and llookhorjii, 196^) • Hickman (1959) has made the only study of the effect of body size on the salinity-oxygen consumption relationship in fishes. His data indicate that body size is an important factor, but he only cor^-idors two salinities. The present study suggests that the effect of saD-inity on the oxygen consumption of both intact animals and excised gills is a fimction of body size. Since intact anical oxj-gen consumption depends on rmiscular acti\'ity and o:>qygon ccnsvimption of excised gills on osmotic v7ork, the effect of salirdty on muscul.ar activity s.nd osmotic vrork i!iust also be a function of body size, VJlionevor possible, body size should bo considered in osmoregulatory studies. Conclusions In any homeostatic system, the energy expenditure may be varied in order to control the level of the system in the face of external fluctuations, Hotraver, the constant of proportionality may also be varied. This may lead to energy changes which are mor« than propoj>tional or less than proportional to changes in external levels. It is even possible to find decreases in the energy oxpenditui'e as the difference between the level of a parameter in an animal and the

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10? environment increases if the constant of proportionality is decreased stifficiontly. In this study, osraoregtilatory ability is shovm to be a resxiLt of varying the energy expenditure and also varying the apparent permeability. Energy expenditure varies both as a function of |B MJ and as a function of body size. Apparent permeability, likei-dse, is a function of both JB M| and body size. Good osmoregulation results when proper adjustments in these tvro factors are made as the concentration of the medivim varies. Summary 1, F, siiidlis wliich inhabits salt marsh tidal creeks is a precise osnoregulator over the range of salinity from fresh to full sea water. The fresh~i-rator F, chrygotus has a less precise pattern of osmoregulation over tlie s^me rar^ge. This pattern is similar to those of the euiyhaline fishes, Tilapia atpr ea (Lotan, I966) and Fui'idulus kansao (Stanley and Fleming, I966) , F, clnysotus has the physiological capacity to osmoregulate in a manner similar to some ei'xyhaline species, but it is stonohaline in an ecological sense, 2, Oxygen consumption of intact animals varies significantly as a function of salinity, Hoirever, these changes cannot be correlated iijith the energetic costs for osmotic work because they are up to 30 times the mininatm amount necessary to perform osmotic I'rork as estimated by Potts (195^) , This indicates an efficiency of ca, Jp which is much lower than the efficiencies (20/J to 80^) of other transport systems (Potts and Parry, 19<^) .

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108 It is concliided that salinity nay caiise an activity response which results in changes in o:
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LITERAl'URE CITED Bailoy, N. 1959. Statistical mathods in bioloQ'. The English Universities Press I London, 200 p, Baldwin, E, 19'4-3, An introduction to comparative bi ochondstry , 3rd ed,, Cambridge Univ. Press, London and New York, IWp^ Beamish, F. VJ. H, and P. S. Kookherjii, 196^. Respiration of fishes with special emphasis on standard oxygon consumption. I. Influence of weight and temperature on respiration of goldfish, Carassius auratus L. Canad. J, Zool. ^2sl6l-175» Bertalanffy, L, von and W, J. Perozynski. 1951. Tissue rnotabolism and body size. Science 113t 599-600, Bertalanffy, L. von and \-I, J. Perozynski, 1953. Basal mtabolism, tissue respiration and gro^rt-h. Biol. Bull. 10512^^0-256, Black, V, S. 1951. Osnotic regulation in teleost fishes. Pubis. Ont. Fish. R es. Lab 71:53-89, Black, V. S, 1957. Excretion and osmoregulation, l6>205. Ini The p hyslolo °:y of fis hes, I, ed, Bro^^. Academic Press, New York, Dakin, W, J, I9O8. The osmotic concentration of the blood of fishes taken from sea water of naturally varying composition, Biochem. J. 3t 258-278. Dehnel, P. A. and D. A. KcCanghran, 196^. Gill tissue respiration in two species of estuarine crabs, Comp, Biochem. Physiol. 13:23>259. Duval, M. 1925. Recherches physico-chimques et physiologiques ^ur le milieu interieur des animaux aquatiques. Ann. Inst. Oceanogr. 2j232-M)7. Fry, F, E. J. 1957. The aquatic respiration of fish, 1-6^, Im T he physioloCT" of fishes , I, ed. Brown, Academic Press, Hew York, Fry, F, E. J, and J, S, Hart. 19^7. The relationship of temperature to oxygen consumption in goldfish, Biol. Bvill, 9^t66-77, Gai'rey, W, E, I905, The osmotic pressure of sea water and of the blood of marine animals. Biol. Bull. 8; 257-270, 109

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110 Gilchrist, B. K. 1956. Tha oxygon consvuription of Arteni a salina (L.) in different salinities, Hydrobiolorrica 8t5'^S3, Gordon, l], S, 196^1-, Anirials in sqiiatic em-irorErantsj fishes and aiiiphibians, 69>713, In: Handbook of p .iysiolo,
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ni Krebs, At H, 1950. Body cizo and tissue respiration, Biochim, et Biopliys. Acta 'A: 2^9269 , Krogh, A. 1939. Osmotic re.pjulati on in aquatic animal s, Cambridge Univ. Press, C&nbridgo, 2^2 p. Lotan, R, I966. Oxygen consuraption in the gills of Tilg.pja aurea (Staindachner) in various saline conditions, Israel J. Zool. 15!3>37. Lumbye, J, 1958. The oxygen consumption of Theodoxus fluviatilis L, and PotamopyrpT^ s J Q.rf 'rf-^si in brackish water and fresh v;ater, Hydi-obiolog:ica "lO s 2^5-2*22 . Margaria, R. 1931* The osmotic changes in some marine animsls. Proc. R. Soc. B. 107i6o6-624-, Miller, I, and J. Freiuid, I965. Probability and statistics for enf?:ineers . PrenticeHall, Englewood Cliffs, ^32 p, Kotais, R, and J, Kaetz, 196k, Action des honnones neurohypophy^ sa3.ros svr les echanges sodium (measui-es a I'aide du radiosodium ZH' Via) choz. tel^st^on etuyhalin Platichthys flesus . Gen. Comp. Endocrin. k i 210-22^ , 0*Hara, J, 1968, The influence of x^ight and tejiiperature on the metabolic rate of sunfish. Sc olo;^ ^9 » 159-161. Parker, R. R. IS^J, Effects of formalin on length and weight of fishes. J. Fish. Res. Rd. Can^a 20:l^'4l-1^55. Parry, G. 1958 • Size and osmoi-egulation in fishes. Na ture ,,_ London 188:1218-1219. Parry, G, I966, Osmotic adaptation in fishes, Biol . Revs, of Ca m. Phil. Soc. ^lj392-Jf'-!4. Pavlovslcii, E. N. 196^. Technique s for the i nvestigatio n of fish physiology, S, Monson, Jerusalem, 313 p. Potts, VJ, T, W, 195^. The energetic of osmotic regulation in bracldsh and fresh-water animals. J. Exptl. Biol. 31s6l8-630, Potts, W. T. V;. and D, H. Evans. I966. The effects of hypophysectoray and bovine prolactin on salt flixxes in fresh-water adapted Fundulios heteroclitus , Biol. Bull. 131{36>368, Potts, V7, T. W, and D. H. Evans, 19^7, Sodivsm and chloride balance in the killifish, Fundulus heteroclitus , Biol, Bull. 133 t ^11-^25. Potts, W, T, v., Foster, K. A., Rudy, P. P., and G. P. Howolls. 1967. Sodium and water ba3.ance in the ciclilid teleost, Tilapia mossambica. J. Exptl. Biol. ^7:^6l-^l70,

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112 ? -^ o ' Potts, W, T. W, and G, Parry, V^G\, Osmotic and ionic rop'tilation in animals . Pormagon Press, New York, Wl'S p. Price, J, W, 1931 • Grovrth and gull development in the sraallmouthod black bass, Kicroptervis dolomieu , Lacepode, Ohio State Univ. Studies hx'L-^r€7 Rao, K. P, 1958. Oj;ygan consumption of goldfish. Biol, Bii ll. 91 « 312-325 • Stanley, J, G. ar^d V/. R. Fleming. I966. Effects of hypophyssctomy on the function of the kidn^oy of the eur^irhaline telcoot, Fundulus kansae . Biol. Bull. 1 30 j h,j)^.li^\\\ . Steen, J. B, and A. Krxiysse. 1964. The respiratory function of teloostean gills. Comp. Biochen, Physiol. 12 j 127-142, Svedrup, H. U., Johnson, M. W. , and R. H. Fleming. 19^2. The oceans, their physics, chemistry, and p;eneral biolofry , Prentice-Hall, Englewood Cliffs. 108? p. Umbreit, W. V/., Burris, R. H., and J, F, Stauffer. 1956. I'lanometric techniques and tissue metabolisn . 3rd ed., Burgess Publishing Co., Minneapolis. 338 p. Valentine, D. W. and R. Miller. 1969t Osmoregulation in the California killifish, Fundtilus parvipinnj.s . Calif. Fish and Ga£3 58:20-25. Vornberg, F. J, 1954. The respiratory metabolism of tissues of marine teleosts in relation to activity and body size. Biol, Bull. 106 J 360-370.

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113 Vornbergi F, J. 1956. Study of tho oxygon constuuption of excised tissue of certain raarina decapod Crustacea in relation to habitat, ^MsiS^JiS^L*. 29:227-23^, ^^c/^^s Vinogradov, A. P. 1953 » The elenentar y cot^po sition of marine o rganisras , Bis-nco Luco's Printing, Coi:ienhagGn, 6^1-7 Pt Wolls, N. A. 1935. Variation in tho recpiratory metabolism of the Pacific kjj-lifish, Fundu lus parvipinnis , due to size, season and continited constant temperature, PPxysiol, Zool, 8: 318-335* Weymouth, F, I*/,, Crimson, J, M, , Hall, V, E,, Belding, H, S., and J. Field II, 19^4, Total and tissue raatabolism in relation to body weight, Physiol, Zo ol, 17:50-71,

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BIOGRAPHICAL SKETCH Dale Fi'ederick Burnside was born Kay 15 1 19^3 1 at Cincinnati, Ohio. Ho graduated from Walnut Hills High School in Jvme, I96I, I'lr, Eurnside entered tho University of Cincinnati in September, I96I and received a Bachelor of Arts degree vdth high honors in June, I965, In September, 19^5 » he entered the Zoology Depai'tment at the Univerity of Florida and served one year as a graduate teaching assistant. During the surarjer of I966, I-lr, Burnside attended the National Science Foujidation organism-sedinent seminar in Berinuda* Upon his retiu^n to the University of Florida, llr. Burnside contjjnued studies toward the degi^ee of Doctor of Philosopliy xrith the siipport of a National Defense Education Act Title IV Fellowship, He pai'ticipated in the National Defense Education Act traveling scholar program during the suirmer of 1967» when ho attended a coxu*se in the pliysiology of fishes at tho University of South Florida. In the spring of 1969* he presented a paper to the Florida Acaden^y of Sciences, Mr, Burnside is married to tho former Lavira Virginia Sjodahl, He is a member of Phi Beta Kappa and Phi Sigma, 11^

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This dissertation was prepared under the direction of the chairman and the co-chairman of the candidate's supervisory committee and has been approved by all members of that coiranittee. It was submitted to the Dean of the College of Ai'ts and Sciences and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy, August, 1969 F - Dean, College M" ^J^ts /and Sciences V, Dean, Graduate School Supervisory Committee 1 Chairman -?^ Co-chairman

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