Responses made by the salt marsh teleost Cyprinodon variegatus (Atherinomorpha: Cyprinodontidae) to live in a variable s...

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Responses made by the salt marsh teleost Cyprinodon variegatus (Atherinomorpha: Cyprinodontidae) to live in a variable salinity environment
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Haney, Dennis Charles, 1962-
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Sheepshead minnow   ( lcsh )
Zoology thesis, Ph. D
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Thesis (Ph. D.)--University of Florida, 1995.
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Includes bibliographical references (leaves 108-122).
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by Dennis Charles Haney.
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Typescript.
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Vita.

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RESPONSES MADE BY THE SALT MARSH TELEOST CYPRINODON
VARIEGATUS (ATHERINOMORPHA: CYPRINODONTIDAE) TO LIFE IN A
VARIABLE SALINITY ENVIRONMENT













By


DENNIS CHARLES HANEY


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


UNIVERSITY OF FLORIDA


1995


/-l /" /













ACKNOWLEDGMENTS


The work described in this dissertation was the focus of my activities for many

years. Throughout this time I have been fortunate to meet and interact with an extraordinary

variety of people, many of whom played integral roles in the successful completion of this

dissertation.

I would first like to acknowledge the assistance of the members of my graduate

committee: Frank Nordlie, Steve Walsh, Brian McNab, Harvey Lillywhite, Ken Sulak, and

Tom Crisman. I would also like to thank a founding member of my committee, Michelle

Wheatly, who, unfortunately for me, left the University of Florida before I was able to

complete my dissertation. All of these people were instrumental in the completion of this

work. In particular, I owe an incredible debt of gratitude to the chair of my committee, Frank

Nordlie. His unfailing friendship, help, and guidance over the years was truly an inspiration

to me, and I can safely say I would not have made it through to the end without him. Steve

Walsh also deserves special recognition for his much-needed assistance. Steve has been a

close friend and colleague for many years, and I am proud to be the first student to complete

a dissertation with Steve as a member of his committee!

So many members of the Zoology Department have helped out that its difficult to

know where to begin. Brent and Sylvia Palmer, John Matter, Frank Hensley (my fusiform

twin!), and Lou Somma were all invaluable in helping me to maintain good mental health

over the years. We spent many hours trying to figure out life, the universe, and everything.

We also had lots of fun looking for unsuspecting fish and herps. Lisa Gregory has been a

friend I could always count on no matter what! John Binello was my trusted field

companion. We spent many days tromping through the salt marsh (and other bodies of








water) in search of numerous fishes including the wily pupfish, sailfin molly, Florida

flagfish, and fat sleeper. Thanks also go to Leo Nico, Adele Hensley, Pam Fuller, Paula

Cushing, Patricia Harrison, Lianna Jarecki, Becky Thompson, Vince DeMarco, Kevin

Baldwin, Ellen Burroughs, Chris Kardish, Mark Hostetler, Doug Weaver, Frank Jordan,

John Anderson, Carol Binello, and everyone else from Zoology!

For the past 4+ years I have worked with many people at the Department of the

Interior's Gainesville laboratory (presently the National Biological Service, formerly U.S.

Fish and Wildlife Service). While working full-time for the past few years certainly slowed

down my progress towards finishing my Ph.D., the extra time was worth it (mostly!). I

formed many new friendships and learned lots of things I wouldn't have otherwise. Jim

Williams showed me that freshwater clams were actually kind of neat. Noel Burkhead

helped remind me that fish were still much cooler! Les Parker, Jayne Brim-Box, and I had

great fun diving in zero visibility water. Howard Jelks, Gary Hill, Ann Foster, Tina Yanchis,

Rob Whiteford, and Bill Stranghoener were all inspiring at one time or another. Special

thanks go to my office mates, Leslie Straub and Cindy Timmerman, for putting up with me

for so long. Cindy especially has been a great friend and confidante.

Last, but certainly not least, I want to thank my family for supporting me all these

years. Mom and Dad never doubted me, though it seemed like I would never finish. Their

encouragement really did help! My brother Scott helped to show me you really can finish a

dissertation and be successful afterwards. For this, and everything else, I can never thank

him enough.


iii












TABLE OF CONTENTS


LIST OF TABLES ........................................................................................................... vi

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

ABSTRACT ......................................................................................................................... x

CHAPTERS

1 INTRODUCTION ................................................................................................. 1

Experimental Animal..............................................................................................7...
Questions.......................................................................................................... 10
Study Site ............................................................................................................... 1 1

2 INFLUENCE OF ENVIRONMENTAL SALINITY ON ROUTINE
METABOLIC RATE AND CRITICAL OXYGEN TENSION OF
CYPRINODON VARIEGATUS ..................................................................... 12

Introduction ............................................................................................................ 12
M ethods............................................................................................................. 14
Results.................................................................................................................... 18
Discussion ........................................................................................................... 31

3 INFLUENCE OF SIMULATED TIDAL CHANGES IN AMBIENT
SALINITY ON ROUTINE METABOLIC RATE IN CYPRINODON
VARIEGATUS ............................................................................................... 39

Introduction ......................................................................................................... 39
M ethods................................................................................................................ 40
Results....................................................................................................................46
Discussion........................................................................................................... 46

4 INFLUENCE OF A FLUCTUATING SALINITY REGIME ON
OSMOREGULATION IN CYPRINODON VARIEGATUS.........................55

Introduction ......................................................................................................... 55
M ethods............................................................................................................. 56
Results....................................................................................................................58
Discussion ........................................................................................................... 66

5 INFLUENCE OF ENVIRONMENTAL SALINITY ON BLOOD
OXYGEN LEVELS OF CYPRINODON VARIEGATUS..............................70

Introduction ......................................................................................................... 70
M ethods............................................................................................................. 71







Results ....................................................................................................................73
D discussion ........................................................................................................... 79

6 SU M M A RY A N D CO N CLU SIO N S ................................................................ 87

A PPEN D ICES ...................................................................................................................91

1 CRITICAL OXYGEN TENSION FIGURES....................................................91

2 FIELD M EA SUREM ENTS................................................................................102

LITERA TURE CITED ................................................................................................... 108

BIO G RA PHICA L SK ETCH ........................................................................................ 123













LIST OF TABLES


Table page

1-1. Phylogenetic classification of the cyprinodontiform fishes (modified from
Parent, 1981)...........................................................................................................8.......

2-1. Relationships of routine metabolism (RMR), critical oxygen tension (Pc), and
slope in the conformation region at a series of ambient salinities. Values are given
as m means se ........................................................................................................ 19

2-2. Measurements of oxygen concentration (mg L- 1), salinity (ppt), and temperature
(oC) taken at four sites in the Cedar Key area from June 1990 through June 1991
(see text for details on location of sites). Values are given as means, se (in
parentheses), sam ple size........................................................................................... 32

3-1. Acclimation and final salinities used in simulated tidal change study........................42

3-2. Mean routine metabolism (mg 02 h-1) before (acclimation salinity) and
following (final salinity) a simulated tidal change. Values are given as means se.
Groups exhibiting a significant change in metabolism are indicated with an
asterisk..........................................................................................................................47

4-1. Salinity trials used in cyclical salinity study. The group maintained at 30 ppt was
split into three groups following cycle 10 (day 20); groups CD, C, and CQ (see text
for details).....................................................................................................................58

4-2. Results of salinity fluctuations experiment. Values in the top row of each cell
represent hematocrit measurements (% erythrocytes), values in bottom row of each
cell represent plasma osmolality measurements (mOsm kg-1). Sample sizes are
n=5 for each cell. All values are expressed as means se. See text for explanation
of group abbreviations............................................................................................... 60

5-1. Hematocrit (Hct), hemoglobin concentration ([Hb]), erythrocyte count (RBC),
mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and
mean corpuscular hemoglobin concentration (MCHC) as a function of salinity for
Cyprinodon variegatus. All values are expressed as means se. See text for
further details on blood indices .................................................................................. 74












LIST OF FIGURES


Figure page

2-1. Mean adjusted routine metabolic rates (RMR) over a range of salinities in
Cyprinodon variegatus (metabolic rates were mass-adjusted using an analysis of
covariance; bars indicate se; numerical values above the points in the figure
indicate sample sizes at each salinity)......................................................................... 21

2-2. Relationship between mean adjusted routine metabolic rates (RMR) and mean
plasma osmolality over a range of salinities in Cyprinodon variegatus (metabolic
rates were mass-adjusted using an analysis of covariance; bars indicate se;
plasma osmolality data from Nordlie, 1985) .............................................................. 23

2-3. Mean critical oxygen tension (Pc) measurements over a range of salinities in
Cyprinodon variegatus (bars indicate se; numerical values above the points in
the figure indicate sample sizes at each salinity)......................................................... 26

2-4. Relationship between mean adjusted routine metabolic rates (RMR) and critical
oxygen tensions (Pc) over a range of salinities in Cyprinodon variegatus
(metabolic rates were mass-adjusted using an analysis of covariance; bars
indicate se). ......................................................................................................... 28

2-5. Relationship between mean critical oxygen tensions (Pc) and mean plasma
osmolality over a range of salinities in Cyprinodon variegatus (bars indicate se;
plasma osmolality data from Nordlie, 1985). ............................................................. 30

2-6. Relationship between mean adjusted routine metabolic rates (RMR) and mean
plasma osmolality over a range of salinities in Adinia xenica (metabolic rates were
mass-adjusted using an analysis of covariance; bars indicate se; numerical
values above the points in the figure indicate sample sizes at each salinity)................35

3-1. Schematic diagram of respirometry apparatus used for routine metabolism
experiments. See text for detailed description of system............................................44

3-2. Results of metabolic trials where salinity was increased over the course of the
trial. Bars represent groups listed in Table 3-2 for which final salinity was greater
than initial salinity. The height of each bar signifies the magnitude of the salinity
change for each metabolic trial and the asterisk indicates at which of the salinities
(for each metabolic trial) the routine metabolic rate (RMR) was highest. The x axis
has no scale and serves only to visually separate groups........................................... 49


vii






3-3. Results of metabolic trials where salinity was decreased over the course of the
trial. Bars represent groups listed in Table 3-2 for which final salinity was less
than initial salinity. The height of each bar signifies the magnitude of the salinity
change for each metabolic trial and the asterisk indicates at which of the salinities
(for each metabolic trial) the routine metabolic rate (RMR) was highest The x axis
has no scale and serves only to visually separate groups.. ......................................... 51

4-1. Mean plasma osmolality values measured for groups experiencing decreases in
salinity during the course of the experiment. Group designations are as follows:
DI, salinity fluctuated between 30 ppt and 2 ppt; D2; salinity fluctuated between
30 ppt and 10 ppt; D3, salinity fluctuated between 30 ppt and 20 ppt; CD; salinity
constant at 30 ppt for days 0-20, decreased to 2 ppt following subsample on day
20; C, salinity constant at 30 ppt Sample sizes are n=5 for each group. See
text for details of experimental procedure.................................................................. 62

4-2. Mean plasma osmolality values measured for groups experiencing increases in
salinity during the course of the experiment Group designations are as follows:
I1, salinity fluctuated between 30 ppt and 40 ppt; I2; salinity fluctuated between 30
ppt and 50 ppt; 13, salinity fluctuated between 30 ppt and 60 ppt; CI; salinity
constant at 30 ppt for days 0-20, increased to 60 ppt following subsample on day
20; C, salinity constant at 30 ppt. Sample sizes are n=5 for each group. See text
for details of experimental procedure......................................................................... 64

5-1. Mean erythrocyte (RBC) count over a range of salinities in Cyprinodon
variegatus (bars indicate se; numerical values above the points in the figure
indicate sample sizes at each salinity)......................................................................... 76

5-2. Mean hemoglobin concentration ([Hb]) over a range of salinities in Cyprinodon
variegatus (bars indicate se; numerical values above the points in the figure
indicate sample sizes at each salinity)......................................................................... 78

5-3. Mean hematocrit (Hct) over a range of salinities in Cyprinodon variegatus (bars
indicate se; numerical values above the points in the figure indicate sample sizes
at each salinity). .......................................................................................................81

Al-1. Plot indicating the calculation of the critical oxygen tension (Pc) for an
individual Cyprinodon variegatus in water at 0 ppt ..................................................93

A 1-2. Plot indicating the calculation of the critical oxygen tension (Pc) for an
individual Cyprinodon variegatus in water at 50 ppt ................................................95

A 1-3. Plot indicating the calculation of the critical oxygen tension (Pc) for an
individual Cyprinodon variegatus in water at 100 ppt ..............................................97

A 1-4. Generalized critical oxygen tension (Pc) plots at each salinity used in the Pc
experiments. Plots were produced by using the mean Pc, mean routine metabolic
rate (RMR), and mean slope in the conformation region for each salinity group .........99


viii







A2-1. Oxygen concentration (mg L'l), salinity (ppt), and temperature (oC) at four
sites in the Cedar Key area taken between June 1990 and June 1991. Values are
given as means, bars indicate + se. a) Measurements taken on the bottom at site 1;
b) measurements taken on the surface at site 1; c) Measurements taken on the
bottom at site 2; d) measurements taken on the surface at site 2; e) Measurements
taken on the bottom at site 3; f) measurements taken on the surface at site 3; g)
M easurements taken on the surface at site 4 ............................................................ 104












Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


RESPONSES MADE BY THE SALT MARSH TELEOST CYPRINODON
VARIEGATUS (ATHERINOMORPHA : CYPRINODONTIDAE) TO LIFE IN A
VARIABLE SALINITY ENVIRONMENT

By

DENNIS CHARLES HANEY

December, 1995



Chairman: Frank Nordlie
Major Department: Zoology


Cyprinodon variegatus, a common coastal resident of the western Atlantic Ocean

and Gulf of Mexico, lives in ambient salinities ranging from freshwater to 142 ppt. Fish

used in this study were obtained from a Gulf of Mexico salt marsh near Cedar Key, Florida.

In a steady-state experiment, routine metabolic rate (RMR) and critical oxygen tension (Pc)

were determined at salinities ranging from 0 tol00 ppt. Salinities between 0 and 40 ppt had

little influence on RMR or Pc. However, at salinities above 40 ppt, RMR declined, and Pc

increased. The reduction in RMR and rise in Pc correlates with a reduced ability of C.

variegatus to osmoregulate effectively at high salinities. The variations in RMR and Pc at

high salinities suggests that C. variegatus responds by reducing energy expenditures,

effectively increasing the time that individuals can tolerate hypersaline conditions. The

metabolic patterns of C. variegatus as influenced by simulated tidal changes in salinity were

then measured, with RMR unaffected by changes in salinity between 2 and 40 ppt.

However, at extremely high or low salinities metabolism was affected by changes in ambient






salinity. Individuals of C. variegatus responded to fluxes at salinity extremes by reducing

general activity and energy expenditures-essentially waiting for conditions to return to

normal, where they responded by increasing metabolic activity. Cyprinodon variegatus

efficiently regulated plasma osmolality, even when fishes were exposed to large fluctuations

in salinity. However, prior exposure to salinity fluctuations did impart an osmoregulatory

advantage. Fish previously exposed to large salinity fluctuations regulated plasma

osmolality better than fish that previously experienced no or small changes in salinity.

Increasing salinity had a greater impact on osmoregulation than did decreasing salinity.

Salinity also had a significant effect on blood oxygen carrying capacity in C. variegatus,

although differences were only noted at the very highest (60 80 ppt) and lowest (0 ppt)

salinities tested. Oxygen carrying capacity was highest in the group acclimated to 0 ppt.

Erythrocyte count was the most consistent indicator of the influence of salinity on blood

oxygen, with hematocrit the least consistent measure.














CHAPTER 1
INTRODUCTION

Salinity is a crucial physicochemical factor that exerts an important influence on

aquatic life, particularly on estuarine and salt marsh organisms that are exposed to

unpredictable salinity fluctuations diurnally and seasonally. Salt marshes are intertidal beds

of rooted vegetation that are alternately flooded and exposed by rising and falling tides.

Vegetation on higher ground develops a complex network of branching channels through

which water, nutrients, and aquatic organisms move during the tidal cycle. Intertidal salt

marshes are inhospitable yet interesting environments due to their unstable water levels and

to the extreme variability of their physical, chemical, and biological processes (Cooper,

1974; Adam, 1990; Allen and Pye, 1992).

Factors that may influence salinity in salt marshes include precipitation, wind,

frequency and extent of tides, shoreline height, and coastal topography (Remane and

Schlieper, 1971; Wheatly, 1988). Due to the harshness of the physical environment, salt

marshes tend to have low species diversity, but often high abundance of selected species

(McClusky, 1989; Dunson and Travis, 1994).

Whereas variable salinity habitats are also characterized by fluctuations in other

environmental factors, such as temperature, dissolved oxygen, and pH (Vernberg, 1983;

Wheatly, 1988), the distribution and abundance of fishes in these habitats is largely

determined by salinity (McClusky, 1989; Davenport and Sayer, 1993; Gill and Potter,

1993). Not surprisingly, animals respond to fluctuations in salinity in complex ways.

Salinity affects osmoregulation, ventilation, metabolism, acid-base balance, growth,

reproduction, development, and other biological processes (Wheatly, 1988). Some of the

primary responses of teleosts to changes in salinity are reviewed here.








Avoidance is the first line of defense to variable salinity conditions. If behavioral

responses do not sufficiently minimize exposure to variable salinities, aquatic organisms

must rely on physiological and biochemical responses to tolerate environmental changes

(Beitinger and McCauley, 1990). This may be by passive tolerance (osmoconforming) or

active osmoregulation (Truchot, 1987; McClusky, 1989). Only fishes that are capable of

osmoregulation can tolerate wide changes in salinity, and these euryhaline teleosts will be

the focus of the rest of this review.

The basic patterns of osmoregulation in fishes have been extensively reviewed in

recent years (Parvatheswararao, 1970; Eddy, 1982; Evans, 1984; Zadunaisky, 1984;

Karnaky, 1986; Foskett, 1987; Pisam and Rambourg, 1991; Ventrella et al., 1992; Evans,

1993; McCormick, 1994; Wood and Marshall, 1994). Euryhaline teleosts regulate their

blood osmolality at about one-third the concentration of seawater (35 ppt), and thus face

severe osmotic problems whether in freshwater (0 ppt) or seawater. Body fluids of a teleost

in freshwater are hyper-osmotic to the external environment, whereas in seawater they are

hypo-osmotic. Thus, euryhaline fish possess mechanisms for osmoregulating in both

hyper-osmotic and hypo-osmotic conditions.

Teleosts in seawater are susceptible to a loss of body water to the external

environment, and balance water loss by actively drinking large amounts of seawater.

However, both water and salts are absorbed together across the gut. Ingested excess salts

are actively excreted, divalent ions mostly in urine and feces, and monovalent ions by the

gills.

Active excretion of salts by the gills takes place via chloride cells (Zadunaisky, 1984;

Kamaky, 1986; Foskett, 1987; Pisam and Rambourg, 1991). Chloride cells in seawater-

acclimated fish are located at the base of the secondary gill lamellae, and are large,

columnar-shaped cells usually extending from the basal epithelium to the external

environment. They are characterized by numerous mitochondria (for this reason they are

often referred to as "mitochondria rich cells") and an extensive tubular reticulum continuous








with the basolateral membrane (Pisam and Rambourg, 1991). The transport enzyme

Na+,K+-ATPase is restricted to this tubular system and to the basolateral membrane. This

ion pump creates a large Na+ gradient (low in the chloride cell cytoplasm) which drives a

NaCI co-transporter by which Cl- enters the cell. The Cl- accumulates sufficiently in the cell

such that it is able to exit to the external environment across the apical membrane, with Na+

following passively down its electrochemical gradient between adjacent chloride cells

(Evans, 1993; McCormick, 1994; Wood and Marshall, 1994).

In freshwater, teleosts continuously face an efflux of salts and an influx of water.

Hence, their osmoregulatory response is to actively transport salts from the external

environment via the gills, to avoid drinking water, and to excrete copious amounts of dilute

urine. Our present understanding of the mechanisms involved in the uptake of Na+ and Cl-

from the environment in freshwater teleosts is somewhat unclear and incomplete. It appears

that Cl- is actively taken up, with Na+ uptake being a passive process. Whether this takes

place via a freshwater-type chloride cell, or through interactions with acid-base balance is

unclear, as evidence exists for both (Wood and Marshall, 1994).

What is unique about osmoregulation in euryhaline fish is not that they possess a

structurally distinct osmoregulatory mechanism, but that they have the ability to function

efficiently in variable salinities. They must not only be able to hypo- and hyper-

osmoregulate, but they must be able to alter their pattern of osmoregulation quickly if they

live where salinity rapidly fluctuates.

Osmoregulation is controlled largely by the endocrine system. In teleosts, hormones

known to exert osmoregulatory effects include prolactin, growth hormone, cortisol,

angiotensin II, arginine vasotocin, atrial natriuretic peptide, thyroid hormones, urotensins,

vasoactive intestinal peptide, insulin, calcitonin, catecholamines and sex steroids (Jenkins,

1981; Bern and Madsen, 1992; Leloup and Lebel, 1993; Sakamoto et al., 1993; Takei, 1993;

McCormick, 1994). The hormone most involved with osmoregulation under freshwater

conditions is prolactin, with arginine vasotocin, urotensin II, catecholamines, and atrial








natriuretic peptide also playing roles (Hirano et al., 1987; Bern and Madsen, 1992; Takei,

1993). Control of osmoregulation in seawater is largely via the effects of cortisol and

growth hormone, together with thyroid hormones, angiotensin II, vasoactive intestinal

peptide, atrial natriuretic peptide, and urotensin I (Balment et al., 1987; Bern and Madsen,

1992; Takei, 1993). Although the exact mechanisms and interactive effects of many of these

hormones are unclear, it is well established that both rapid and long term control of

osmoregulation under hyper-osmotic and hypo-osmotic conditions is mediated via the

endocrine system.

Osmoregulation is not the only physiological process affected by salinity. Salinity

adaptation is a complex event that involves a number of physiological and behavioral

responses, including energetic. Many studies have shown that salinity influences

metabolism of fishes, but little agreement exists with respect to the magnitude or direction of

these effects (Nordlie, 1978; Febry and Lutz, 1987; Nordlie et al., 1991; Swanson, 1991;

Morgan and Iwama, 1991; Kirschner, 1993). One reason for a lack of agreement among

studies is that many have focused on the energetic costs associated with osmoregulation,

while ignoring other related physiological and behavioral processes. For example, many

studies have used measured differences in metabolism at different salinities to represent

differences in osmoregulatory costs. The rationale is that osmoregulation is an energetically

costly event, with the energetic costs being related to the osmotic gradient between the fish

and its environment (e.g., Madan Mohan Rao, 1968; Muir and Niimi, 1972; Furspan et al.,

1984; Zadunaisky, 1984). However, this is probably an oversimplification, since other

factors, such as activity, food intake, and permeability changes may also be influenced by

salinity, and in turn, affect metabolism (Swanson, 1991). For this reason metabolic

measurements represent the overall costs associated with living in a particular salinity

environment, with comparisons among salinities reflecting these total costs, not simply the

cost of osmoregulation.








Several attempts have been made to categorize the patterns of metabolic responses

of teleosts to altered salinities (Kinne, 1967; Remane and Schlieper, 1971; Nordlie, 1978;

Morgan and Iwama, 1991). One commonly observed pattern is that the lower rates of

metabolism in response to salinity are associated with environments for which the species

(and life stage) are presumably best adapted for, and in which they are normally found.

Teleosts that inhabit widely variable salinity environments are uniquely characterized by

having metabolic rates that are constant over a wide range of salinities. This allows

euryhaline teleosts to avoid the large metabolic costs normally associated with physiological

adjustment to changing salinity.

Another physicochemical parameter that may act synergistically with salinity is

dissolved oxygen. The dissolved oxygen content of many bodies of water is subject to large

natural fluctuations. This is especially true in shallow water, such as salt marshes, where

chronic and/or periodic hypoxia may be common (Renaud, 1985; Dejours, 1987;

Toulmond, 1987; Graham, 1990). Variations in oxygen can dictate the distribution of some

species in aquatic ecosystems (Boutilier, 1990). Changing environmental salinity may

directly influence respiratory function by affecting both the oxygen solubility in water

pumped over the gills and the solubility of gases dissolved in plasma. Changes in the ionic

composition of the body fluids could also interact with oxygen to influence tolerance to

variable salinity conditions (Truchot, 1987).

Exposure of fish to reduced oxygen tensions initiates physiological responses

mostly directed at increasing the amount of oxygen available to the tissues (Boutilier et al.,

1988). The transfer of oxygen from the environment to the tissues can be characterized as a

series of processes. Gill ventilation is the first step, followed in turn by branchial diffusion,

blood oxygen transport, and diffusion into the tissues (the last of which, due to a lack of

available information, will not be discussed further) (Perry and McDonald, 1993).

Gill ventilation is a function of the frequency and depth of breathing, and is

normally increased when low oxygen tensions are encountered. While ventilation does not








appear to be directly limiting to the uptake of oxygen, substantial cost is involved with

increased ventilatory pumping, which ultimately means that any additional oxygen acquired

is used to fuel the ventilatory apparatus itself (Boutilier et al., 1988; McMahon, 1988;

Cameron, 1989; Perry and McDonald, 1993).

Two processes are largely available to increase branchial oxygen diffusion:

increases in functional gill surface area and increases in the mean water to blood oxygen

partial pressure gradient. This tradeoff is particularly important in regard to salinity, as fish

in waters of low oxygen tension must balance the advantage of maximizing branchial

oxygen diffusion with a disadvantage in osmoregulation due to the accompanying increases

in ion and water exchange (Perry and McDonald, 1993). Increasing blood gas transport is

likely the primary route used by most fish to increase the amount of oxygen delivered to the

tissues. Oxygen transport by the blood in teleosts depends on the respiratory pigment

hemoglobin. Blood oxygen transport is normally increased by increasing the concentration

of hemoglobin, increasing the number of erythrocytes in circulation, and/or by adjusting the

affinity of hemoglobin for oxygen (Davis, 1975; Wells et al., 1989; Jensen et al., 1993;

Perry and McDonald, 1993).

All of the processes described above can be modified to optimize oxygen transport

under a variety of environmental conditions. One additional strategy that can be utilized in

conjunction with the above is the lowering of metabolism in concert with reductions in

oxygen. This potentially minimizes the impact of the lowered oxygen tension, but also

reduces aerobic metabolism and therefore the amount of energy available for physiological

processes.

Most fish would be described as metabolic oxygen regulators, as they maintain a

constant metabolic rate over a range of oxygen tensions extending downward from

atmospheric levels to some low level that has been defined as the critical oxygen tension

(Pc). Below the Pc, metabolism is dependent upon oxygen tension, and decreases linearly

with decreases in oxygen. The Pc was probably first documented by Hall (1929), but was








not considered an important index for fishes until formalized by Fry (1947). However, this

is an extremely important variable relating to habitat selection and overall metabolic patterns

of fishes, and calculations of the Pc have since been made for a number of species under a

variety of environmental conditions.

Experimental Animal


The subject of this study was the sheepshead minnow, Cyprinodon variegatus.

Cyprinodon variegatus is a member of the family Cyprinodontidae, a large and diverse

family containing over 650 species in 80 genera (Parenti, 1981; Parker and Kornfield, 1995;

see Table 1-1). Members of this family are found in fresh, brackish, and salt water, and

distributed pantropically as well as throughout North America. Cyprinodon variegatus is

the type species for both the genus Cyprinodon and the family Cyprinodontidae.

The genus Cyprinodon comprises a group of approximately 30 species of small,

oviparous fishes commonly known as pupfishes. They exhibit remarkable tolerance to

harsh environmental conditions (Miller, 1981) occurring throughout North and Central

America, the Caribbean Sea, and Venezuela (Darling, 1976; Turner and Liu, 1977; Parenti,

1981; Duggins et al., 1983; Barus and Wohlgemuth, 1993). The genus is characterized by

limited genetic divergence and few unique alleles, even in morphologically distinct species

(Kodric-Brown, 1989). Much of the research on the genus has focused on species

inhabiting North American deserts, where the largest concentration of species occurs. Most

of these desert species have small, allopatric distributions (Miller, 1981; Duggins et al.,

1983). Members of this genus also occur in the coastal brackish and marine waters of

eastern North America, where C. variegatus is the dominant pupfish species.

Cyprinodon variegatus is the only pupfish species having an extensive geographic

range. It is found along the Atlantic coast from Massachusetts to the Florida Keys, and

throughout the Gulf of Mexico. A disjunct population occurs along the Yucatan peninsula

(Johnson, 1974; Darling, 1976; Duggins et al., 1983). Populations are also located in the








Table 1-1. Phylogenetic classification of the cyprinodontiform fishes (modified from
Parenti, 1981).


Order Cyprinodontiformes

Suborder Aplocheiloidei

Suborder Cyprinodontoidei

Section 1

Family Profundulidae

Section 2

Division 1

Family Fundulidae

Division 2

Superfamily Poecilioidea

Family Anablepidae

Family Poeciliidae

Superfamily Cyprinodontoidea

Family Goodeidae

Family Cyprinodontidae

Subfamily Cyprinodontinae

Tribe Orestiini
Genus Orestias
Genus Kosswigichthys
Genus Aphanius

Tribe Cyprinodontini
Genus Cyprinodon
Genus Megupsilon
Genus Jordanella
Genus Floridichthys
Genus Cualac








Bahamas, West Indies, and Cuba (Duggins et al., 1983). Cyprinodon variegatus has also

been introduced into several areas, where they have negatively impacted native pupfish

species (Echelle and Echelle, 1987; Echelle and Connor, 1989; Kodric-Brown, 1989; Wilde

and Echelle, 1992). Genetic variability within C. variegatus is as large as the amount of

genetic divergence within the entire genus Cyprinodon, with most of this genetic divergence

occurring in populations north of Cape Hatteras-very little genetic divergence is displayed

among southern populations (Darling, 1976; Schwartz et al., 1990).

Cyprinodon variegatus is a numerically dominant and ecologically important

species throughout most of its range, especially in salt marsh and estuarine waters (Kilby,

1955; Simpson and Gunter, 1956; Relyea, 1975; Naughton and Saloman, 1978;

Subrahmanyam and Coultas, 1980; Stout, 1985; Nelson, 1992; Ross and Doherty, 1994).

Due to its importance, C. variegatus has been an important research animal in diverse

disciplines. These include investigations on the species behavior (Itzkowitz, 1974; Itzkowitz,

1978; Mettee and Beckham, 1978; Itzkowitz, 1981, Dwyer and Beulig, 1991), ecology (Doll

and Bast, 1969; Martin, 1970; Martin, 1972; Able, 1976; Harrington and Harrington, 1982;

Fyfe, 1985; Shipley, 1991; Avila et al., 1992; Wright et al., 1993), evolution (Elder and

Turner, 1994), life history (Warlen, 1964; De Vlaming et al., 1978; Berry, 1987; Able, 1990;

Echelle and Echelle, 1994), physiology (Martin, 1968; Karnaky et al., 1976;

Subrahmanyam, 1980; Nordlie, 1985; Barton and Barton, 1987; Nordlie, 1987; Peterson

and Gilmore, 1988; Nordlie and Walsh, 1989; Peterson, 1990; Price et al., 1990; Nordlie et

al., 1991; Dunson et al., 1993), and reproduction (Raney et al., 1953; Warlen, 1964; Berry,

1987; Kodric-Brown, 1987; Conover and DeMond, 1991). The species has also been used

in voluminous toxicology experiments because of its extreme hardiness (e.g., Schimmel and

Hansen, 1975; Hawkins et al., 1984; Battalora et al., 1985; Linton, 1992).

Cyprinodon variegatus has been called "the toughest fish in North America"

(Gunter, 1967) due to its extreme tolerance of harsh environmental conditions. It is found in

waters ranging from freshwater (Ager, 1971; Johnson, 1974; Kushlan, 1980) to salinities of








142 ppt (Simpson and Gunter, 1956), although it typically inhabits brackish water and

coastal salt marshes. Cyprinodon variegatus is tolerant of temperatures ranging from about

1 oC (Berry, 1987), to 41 oC (Strawn and Dunn, 1967), and oxygen levels approaching

anoxia (Odum and Caldwell, 1955). Thus, it is an exceedingly useful experimental subject

for studying how teleost species respond to harsh environmental conditions.

Although certain organisms can withstand greater changes in environmental

conditions than others, the ability to respond to natural environmental changes is a basic

characteristic of all living systems. Unfortunately, the terminology used to describe these

responses is not uniform. Various researchers have attempted to define the terms adaptation,

acclimation, acclimatization, and accommodation (e.g., Prosser, 1955; Kinne, 1962; Prosser,

1975; Smit, 1980; Fontaine, 1993). I will use the term "adaptation" in its broadest sense,

defining it as a modification of the characteristics of an organism that facilitate an enhanced

ability to survive and reproduce in a particular environment. In this way I recognize that

adaptations involve both genetic and physiological (phenotypic) components, while not

attempting to separate these components from one another. The term acclimation will be

used as defined by Prosser (1975), where compensatory changes are measured following

changes in single environmental variables.

Questions


This study was designed to examine some of the costs to C. variegatus associated

with living in variable salinity environments. Specifically, I asked the following questions:

(1) What are the metabolic costs associated with different ambient salinities? (2) How does

salinity influence the energetic response at low oxygen tensions? (3) What is the

osmoregulatory response to variable salinity environments? (4) How does salinity influence

blood oxygen levels? These questions were tested by the following: measurement of

metabolism in C. variegatus fully acclimated to a wide range of experimental salinities;

measurement of the critical oxygen tension in C. variegatus fully acclimated to the same








range of salinities; measurement of metabolism prior to, and following, simulated tidal

changes in salinity; monitoring of plasma osmolality in C. variegatus exposed to a group of

different cycling salinity regimes; and measurement of hemoglobin concentration,

erythrocyte count, and hematocrit in C. variegatus acclimated to a wide range of ambient

salinities.


Study Site


Fish used in this study were collected from tidal marshes of the Gulf of Mexico

near Cedar Key, Florida. The shore in the Cedar Key area is classified as a zero energy

sector in which wave energy is dampened over the broad, shallow limestone plateau of the

Gulf of Mexico bottom (Stout, 1985). This results in a wide intertidal zone along the coast.

Furthermore, the coastal physiography is extremely diverse due in large part to irregularities

in the shore line of the mainland, to the presence of numerous islands and oyster bars in the

tidal area, and to the maze of intertidal and subtidal creeks and channels (Kilby, 1955). No

significant sediment sources are found in this area, and tides occur on a semi-diurnal basis.

The dominant emergent vegetation in the area is Spartina alterniflora, with the salt marshes

dominated by Juncus roemerianus. Fish communities of the Juncus marsh are dominated

by atheriniforms, with C. variegatus, Fundulus similis, and Poecilia latipinna making up

50-90% of the catch throughout most of the year (Kilby, 1955; Simpson and Gunter, 1956;

Stout, 1985; pers. obs.).













CHAPTER 2
INFLUENCE OF ENVIRONMENTAL SALINITY ON ROUTINE METABOLIC RATE
AND CRITICAL OXYGEN TENSION OF CYPRINODON VARIEGATUS


Introduction

Most fishes are capable of tolerating only a narrow range of salinities. However,

some fishes live in areas that experience frequent variations in salinity. These euryhaline

species possess important physiological and behavioral mechanisms that enable them to

survive in variable salinity environments. One such fish is the sheepshead minnow,

Cyprinodon variegatus. This species ranges along most of the Atlantic coast of the U.S.,

throughout the Gulf of Mexico, and disjunctly along the Yucatan peninsula (Johnson, 1974;

Darling, 1976; Duggins et al., 1983). It typically inhabits brackish water coastal salt

marshes that undergo frequent salinity fluctuations. Cyprinodon variegatus is capable of

tolerating salinities ranging from 0 ppt (Ager, 1971; Johnson, 1974; Kushlan, 1980) to 142

ppt (Simpson and Gunter, 1956).

This study was designed to examine the metabolic response of C. variegatus over a

variety of environmental salinities. Whereas energetic responses to a number of variables

including temperature, body mass, oxygen, and activity level have been well studied, the

influence of salinity on metabolism of fishes has received less attention. Most previous

studies have found that salinity does affect the energetic of fishes. Unfortunately, the

magnitude and/or direction of these effects are equivocal (Febry and Lutz, 1987; Morgan

and Iwama, 1991; Nordlic et al., 1991; Swanson, 1991). This has led several authors to

categorize the general patterns of metabolic responses to altered salinities (e.g., Nordlic,

1978; Morgan and Iwama, 1991).








One general pattern of fishes that inhabit variable salinity environments is a stable

metabolic rate over a range of salinities, with the range of salinities most commonly tested

between freshwater (0 ppt) and seawater (35 ppt) (Morgan and Iwama, 1991). Physiological

stability enables such fish to have a euryhaline existence that is unfettered by large

metabolic costs associated with adjustment to salinity change. However, there are relatively

few studies of the metabolic response of euryhaline fishes over an even wider range of

salinities that they encounter in their natural habitats.

Fish metabolism is also strongly influenced by the partial pressure of oxygen

(P02). Metabolism of fishes is independent of P02, as they maintain a constant metabolic
rate over a range of P02 extending downward from high atmospheric levels to some lower

level defined as the critical oxygen tension (Pc). Below the Pc (conformation region),

metabolism depends on oxygen tension and decreases linearly with decreases in oxygen

(Fry, 1947). Oxygen consumption declines at the Pc because the gas exchange system can

no longer supply both the extra demands of the respiratory system and the oxygen

demands of the tissues (Hughes, 1964). Since Pc is a useful metabolic parameter,

calculations of the Pc have been made for a number of fishes under a variety of

environmental conditions (e.g., Hall, 1929; Tang, 1933; Ultsch et al., 1978; Ott et al., 1980;

Subrahmanyam, 1980; Donnelly and Torres, 1988; Rantin et al., 1992; Nonnotte et al.,

1993). However, the influence of ambient salinity on the Pc has not been specifically

studied. This is surprising since salinity and oxygen are important abiotic factors that may

act synergistically in affecting metabolism.

This study examined the influence of a wide range of environmental salinities on
routine metabolic rate (RMR) and Pc in C. variegatus. I hypothesized that both RMR and

Pc would be unaffected by alterations in salinity over the range of salinities commonly

encountered in natural habitats of C. variegatus. Salinities outside this range, but within the

range known to be tolerated, were predicted to cause increases in both parameters.








Methods


Fish used in this study were obtained by seining canals and ditches in the salt marsh

near Cedar Key, Florida (Gulf of Mexico). Specimens were transported back to the

laboratory in 19 L carboys containing water from the collection site. Upon arrival at the

laboratory, individuals were held overnight in this water with constant aeration. The

following day, fish were transferred into holding tanks (75 to 114 L aquaria) maintained at

the salinity at which fish were captured, and treated prophylactically for 7-14 days in a 5 mg

L"- solution of Acriflavine. Following treatment, groups of approximately 10 fish were

placed into experimental (38 L aquaria) tanks containing water at a salinity within 10 ppt of

that in which they were collected. Both holding and experimental tanks were equipped with

undergravel filtration and constant aeration, and were maintained in rooms on a 12:12

light:dark cycle. Fish were fed Tetramin flake food once each day. All experimental

aquaria were located in a constant temperature environment room that maintained aquaria at

20 1 oC.

Experimental aquaria were used to acclimate fish to salinities ranging from 0 ppt to

100 ppt (0 to 2860 mOsm kg-1). The initial acclimation period was 14 days, after which

fish were either used in a metabolic trial or were transferred to the next higher or lower

salinity in the series. Salinity changes were in steps of 5 ppt, with smaller increments used

to acclimate fish to 0 ppt. This procedure was repeated until determinations had been made

at all experimental salinities. Water used in the freshwater acclimation was obtained from

wells in Alachua and Levy counties, Florida (mean conductivity = 360 ILS cm 1).

Experimental salinities greater than freshwater but less than full seawater were prepared by

diluting filtered Atlantic Ocean seawater (obtained from the C.V. Whitney Laboratory of the

University of Florida, Marineland, Florida) with appropriate quantities of deionized water.

Salinities greater than 35 ppt were produced by supplementing seawater with appropriate








amounts of synthetic sea salt (Instant Ocean). Salinities were monitored daily with an

AO temperature-compensated refractometer, and adjusted as necessary.

Metabolic determinations were carried out in sealed, opaque flasks ranging in

volume from 0.6 to 1.175 L, with the volume selected based on the size of the fish being

tested. Rate of oxygen consumption was used to measure metabolism, with flasks being

used as closed respirometers. Measures of metabolism were considered to be routine

metabolic rates, as animals were sequestered in such a way as to minimize, but not eliminate

activity (Winberg, 1956; Fry, 1957). Experiments were performed with post-absorptive fish

in a resting state, but fish were unconstrained and capable of spontaneous motor activity

(Winberg, 1956; Fry, 1957). For a relatively inactive species such as C. variegatus

(compared to actively swimming salmonids, for example), RMR is perhaps the most

appropriate measure of metabolism, as it more closely reflects normal activity patterns of the

fish than does either active or standard metabolic rate.

Each flask was sealed with a rubber stopper through which two hypodermic needles

(No. 18) were inserted. Each needle was fitted on the inside with catheter tubing, one with a

piece long enough to reach the bottom of the flask, the other half this length. A 25 ml plastic

syringe filled with water at the salinity of the experimental aquarium was inserted into the

needle fitted with the short piece of catheter tubing, while an empty 10 ml syringe was

inserted into the other needle. A 1 ml water sample was drawn into the empty syringe for

each determination of PO2. As each water sample was withdrawn, an equal volume of water

was injected from the filled syringe into the flask. This method of sampling, along with

minor movements by the fish, effectively stirred the water in the flask (Nordlie, pers.

comm.). Determinations of PO2 were made with a Radiometer oxygen electrode

connected to a Radiometer PHM 71 acid-base analyzer.

Measurements of the rate of reduction in P02 were made at 0.5 h intervals, and

continued until fish had depleted the oxygen level to approximately 20 mm Hg (generally 5

to 9 h). Following the final P02 determination, each fish was removed from its flask, damp-








dried and weighed to the nearest 0.01 g. All metabolic determinations were made between

0700 and 1900 hours, and fish were not re-used in other metabolic trials.

To ensure that fish were post-absorptive at the time of testing, food was withheld

from experimental aquaria for 24 h prior to beginning a metabolic reading. Respirometers

were filled with water at the salinity of the experimental aquaria, and placed in a water bath

maintained at 20 1 oC. The entire metabolic apparatus was located in a small, semi-

darkened room in which no other activity took place. In order to allow time for the fish to

adjust to the respirometers, individuals were placed into the flasks (with constant aeration)

12 to 16 h before beginning a trial. At the beginning of the metabolic trial, aerators were

removed and the flask was sealed.

Calculation of oxygen saturation values for the experimental conditions (taking into

account salinity, temperature, relative humidity, and barometric pressure) were made using

the equations of Truesdale et al., 1955 and both RMR (mg 02 h-1) and Pc (mm Hg) were

calculated for each fish. Data used for calculation of metabolic rates were limited to values

obtained while the P02 in the respirometer was greater than 100 mm Hg, in order to ensure

that these calculations were made at oxygen tensions well above the Pc. All data were used

for calculation of the Pc. Determination of the Pc was made using a BASIC program to

calculate the critical point (Yeager and Ultsch, 1989). Following recommendations by

Yeager and Ultsch (1989), data for each fish were first plotted to ensure that the relationship

was a two-step function, following which the midpoint approximation was used to calculate

the Pc.

Oxygen consumption is strongly influenced by body mass, so RMR values were

mass-adjusted using an analysis of covariance (ANCOVA). Log mass-independent RMR

was used as the dependent variable and log mass as the covariate. Least square means

derived from the ANCOVA were used as adjusted RMR values. It was not possible to

perform an ANCOVA for the Pc values, so calculations of Pc were mass-corrected to the

value of the average mass (3.13 g) of all individuals used in this study. The exponent








describing the relationship between mass and metabolism for C. variegatus (Nordlie et al.,

1991), MR = kW-68, was used to correct oxygen consumption rates. Values were corrected

following the relationship MRc = (Wo0.32)(3.13-0.32)(MRo), where MRc is the mass-

corrected oxygen consumption, Wo is the observed mass, and MRo is the observed oxygen

consumption at mass Wo (Ultsch et al., 1978; Cech, 1990). Statistical analyses follow

procedures outlined in Winer et al., (1991) and Sokal and Rohlf (1995). All statistical

analyses were one way tests using the Tukey-Kramer post hoc comparison (p = 0.05), and

values are given throughout as means standard error of the mean (se).


Field Measurements


Salt marsh habitats are widely considered to experience unpredictable and

fluctuating abiotic conditions. However, actual physicochemical measurements are

infrequently reported. To address this issue, field measurements were made at four sites in

the Cedar Key area over a one year period. Whenever possible, measurements at each site

were taken both at the surface and on the bottom (generally 1-1.5 m deep). Dissolved

oxygen, salinity, and temperature were measured one to three times each month between

0700 h and 1700 h, for a total of 19 dates between June 1990 and June 1991. Sites 1, 2, and

3 were located deep in the salt marsh where C. variegatus was routinely collected. These

sites were located in close proximity to one another (< 10 m apart), and were interconnected.

Unlike many locations in the salt marsh, these sites were never completely isolated from one

another or from connections to the Gulf of Mexico, even during the lowest tides. Site 4 was

located directly on the Gulf of Mexico, in the town of Cedar Key, approximately five km

from sites 1, 2, and 3. Although C. variegatus is present at site 4, collections were not made

at this location. These field measurements were not intended to indicate the complete ranges

of oxygen, salinity, or temperature experienced by organisms living within the salt marsh.

Thus, the actual ranges of physicochemical conditions experienced by C. variegatus are

most likely greater than reported here. However, these values are a subset of the conditions








experienced by salt marsh inhabitants, and give an indication of some of the variability in

the measured physicochemical parameters.

Results



Routine Metabolism


Mean RMR was calculated for each fish and organized by salinity groups. Mean

values for unadjusted and adjusted RMR (from ANCOVA) are given in Table 2-1, and

adjusted RMR are plotted against ambient salinity in Figure 2-1.

In the range of ambient salinities between freshwater and 40 ppt, adjusted RMR

values were highest at 2 ppt and 40 ppt, being slightly lower and roughly equivalent at the

other measured salinities in this range. At salinities greater than 40 ppt, there was a

progressive decline in adjusted RMR. Overall, adjusted RMR ranged from a maximum of

0.97 mg 02 h-1 at 2 ppt, to a low of 0.64 mg 02 h-1 at 100 ppt, representing a 66%

decline. This decline corresponds with a decreased ability to regulate plasma osmolality at

elevated salinities (Figure 2-2; plasma osmolality data are from Nordlie, 1985).

A multiple linear regression analysis was used to generate a predictive model for

describing the effects of both salinity and mass on metabolism. In this model, Log body

mass (Log W; in g) and salinity (S; in ppt) were used as independent variables, and Log

mass-independent RMR (Log MR; in mg 02 h-1) as the dependent variable. The equation

that best described this relationship is

Log MR = -0.296 + 0.548 (Log W) 0.001 (S) (F2,110 = 85.445; P < 0.0001)

This model described 62% of all variability about the mean, and the random distribution of

the residuals suggests an absence of significant relationships that might have biased the

analysis.
















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Measurements of oxygen consumption (mg 02 h 1) for each fish at all time

intervals were mass-adjusted to the mean mass of all individuals used in this study. These

mass-adjusted values were then used to calculate the Pc for each fish. Mean values of Pc

were organized by salinity group and are given in Table 2-1. Plots showing the relationship

between Pc and ambient salinity and between Pc, RMR, and ambient salinity are shown in

Figures 2-3 and 2-4, respectively.

Mean Pc values in the range of ambient salinities from 0 ppt to 40 ppt were not

significantly different from one another (p = 0.95), similar to the pattern exhibited by the

RMR data. Mean Pc values increased at salinities greater than 40 ppt, with the highest levels

recorded at salinities 80 ppt and higher. Pc values ranged from a low of 51.49 mm Hg at a

salinity of 2 ppt, to a high of 79.50 mm Hg at 80 ppt, representing a 45% increase. The rise

in mean Pc values corresponds well with a decreased ability to regulate plasma osmolality,

again similar to the RMR pattern (Figure 2-5; plasma osmolality data are from Nordlie,

1985).

Below the Pc, metabolism depends on the oxygen tension and decreases as the P02

decreases. Mathematically, the slope of the resulting line in this conformation region

depends on three factors: the Pc, the RMR at the Pc, and the lethal P02. A comparison of

the slopes in the conformation region reveals that despite the changes noted above in RMR

and Pc values, the P02 at which the fish can no longer survive (under experimental

conditions) is essentially equivalent for all salinities tested. This is reflected in the

increasingly shallow slopes seen at salinities greater than 50 ppt (Table 2-1).


Field Measurements


The field measurements of oxygen concentration, salinity, and temperature revealed

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period (Table 2-2). For all sites combined, ranges of dissolved oxygen, salinity, and

temperature over the course of the year were 0.6 10.8 mg L-1, 1.0 29.0 ppt, and 9.0 -

38.0 oC, respectively. Even higher salinities (> 40 ppt) were encountered occasionally in the

salt marsh outside the sampling period. Other areas of the salt marsh probably encounter

even greater extremes of these variables. Cyprinodon variegatus was seen on all dates when

physicochemical measurements were made.

In addition to high overall variability, there were large differences among some sites

in close proximity to one another. For example, mean oxygen concentration measurements

taken during the Fall at sites one, two, and three were 2.94 mg L-1, 3.85 mg L-1, and 5.1 mg

L-1, respectively. All three physicochemical parameters also strongly varied temporally

among sampling dates. These data provide good evidence that the Cedar Key salt marsh is

an extremely variable habitat with respect to these physicochemical parameters.


Discussion


The family Cyprinodontidae is a diverse group of fishes with many species that

tolerate extreme environmental conditions (Lowe et al., 1967; Lotan and Skadhauge, 1972;

Naiman et al., 1976; Stuenkel and Hillyard, 1981; Chung, 1982). Cyprinodon variegatus is

perhaps the most physiologically tolerant member of the family. It has been called "the

toughest fish in North America" (Gunter, 1967) due to its extreme tolerance of severe

abiotic conditions. The species is found in waters ranging in salinity from freshwater (Ager,

1971) to 142 ppt (Simpson and Gunter, 1956), and can reproduce in waters as high as 100

ppt (Martin, 1972). They are known to tolerate temperatures ranging from about 1 C

(Berry, 1987), to temperatures greater than 41 OC (Strawn and Dunn, 1967), and to tolerate

near anoxic conditions (Odum and Caldwell, 1955). Thus, this species is an exceedingly

useful experimental subject for examining how teleosts respond to harsh environmental

conditions.












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Field measurements indicated that the Cedar Key salt marsh varies tremendously,

both spatially and temporally, in dissolved oxygen, temperature, and salinity, thus exposing

organisms living within the salt marsh to extreme environmental conditions. Cyprinodon

variegatus tolerates these conditions very well. Salinities between 0 ppt and 40 ppt have
little effect on the energetic of C. variegatus as measured by RMR and Pc. This species

fits into the Type I metabolic response pattern (species exhibiting no significant change in

metabolic rate over a wide range of environmental salinities) as categorized by both Nordlie

(1978) and Morgan and Iwama (1991). A similar pattern characterizes the related salt marsh

resident, Adinia xenica (D.C. Haney, unpublished data), in which RMR was more variable,

but relatively constant over salinities from 0 ppt to 50 ppt (Figure 2-6).

Cyprinodon variegatus exhibits a decline in RMR only at salinities exceeding 40

ppt. This result was somewhat unexpected, but is nearly identical to the pattern found by

Nordlie et al., (1991) in a study that also examined metabolism of C. variegatus over a wide

range of ambient salinities. Nordlie et al., (1991) concluded that the depression in

metabolism at high salinities is probably related to permeability changes of the gill

membrane and/or integument. The point at which metabolism is reduced corresponds well

with a diminished ability of C. variegatus to osmoregulate efficiently. If osmotic

permeability of the gills is reduced at high salinities to help offset ionic influx and osmotic

efflux, the potential for oxygen uptake may be reduced as well (Kristensen and Skadhauge,

1974; Skadhauge, 1974; Davenport and Sayer, 1993). Evidence for this hypothesis comes

from several recent studies. In the first of these, Kultz and Onken (1993) found that overall

in vitro permeability of the opercular membrane of the cichlid Oreochromis mossambicus

was reduced in hypersaline media, with a simultaneous reduction in passive ion fluxes.

More direct evidence comes from studies by Bindon et al., (1994a) and Bindon et al.,

(1994b), on the rainbow trout, Oncorhynchus mykiss. In these studies the authors

demonstrated that impairment of respiratory gas transfer coincided with chloride cell

proliferation induced by an osmoregulatory challenge.











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Changes in activity may also account for a depression in metabolism. In a study on

the milkfish, Chanos chanos, Swanson (1991) showed that a depression in metabolism at

an elevated salinity (55 ppt) was highly correlated with a reduction in activity. This may

have been a factor in this study although the method used here for metabolic determinations

minimized activity of fish at all salinities.
The main focus of this study was determination of Pc over a wide salinity range, as

no other study has investigated to any extent the influence of salinity on this parameter.

Critical oxygen tension can be affected by many factors. These include both physical and

biotic parameters such as temperature, salinity, size and activity, reproductive and nutritional

state, and experimental parameters, such as rate of oxygen depletion or manipulation and

disturbance of fish during measurements (McMahon, 1988).

Like other vertebrates, C. variegatus is a good oxygen regulator over a wide range

of oxygen tensions. Critical oxygen tension in C. variegatus was unaffected by changes in

salinity between 0 ppt and 40 ppt, similar to the result for RMR. However, above 40 ppt, Pc

increased with further increases in salinity. This seems reasonable, as fish might be

expected to encounter difficulties obtaining sufficient oxygen at elevated salinities.

Unfortunately, it is difficult to compare my results with those from other groups of

fishes. While the Pc has been measured for a number of freshwater, marine, and intertidal

fishes, only a few authors have considered the effects of salinity in their analyses. Job

(1969) examined oxygen consumption of Tilapia mossambica (= 0. nossambicus) at 0.4

ppt, 12.5 ppt, and 30.5 ppt, and found no effect of salinity on the Pc of small (5 g) fish at

either 15 oC, 30 oC, or 40 oC (Pc approximately 50 mm Hg). Salinity did affect Pc on

larger (80 g) individuals at 15 oC, where the Pc doubled from approximately 50 mm Hg to

100 mm Hg in fish acclimated to 0.4 ppt versus 12.5 ppt or 30.5 ppt However, Pc

calculations made by Job (1969) were extremely rough approximations, and are difficult to

compare with values in this study.








Subrahmanyam (1980) examined the influence of oxygen tension on the metabolic

rate of several salt marsh fishes including C. variegatus, with all measurements made at 25

oC and salinities of 17-21 ppt. All species tested in Subrahmanyam (1980) (C. variegatus,

Poecilia latipinna, Lagodon rhomboides, Leiostomus xanthurus, Fundulus grandis, and

F. similis) were oxygen conformers at oxygen tensions of 85 mm Hg and lower, a

somewhat different response than seen in this study. This difference may be due to the

small sample sizes, and relatively few measurements of oxygen consumption made by

Subrahmanyam (1980). In a study on the golden mullet, Liza aurata, Shusmin (1989),

measured the "oxygen threshold" at salinities ranging from freshwater to 50 ppt. He

showed that the oxygen threshold was relatively constant at all salinities between freshwater

and 40 ppt, with a large increase at 50 ppL Cyprinodon variegatus did not show this type of

response, as the lethal endpoint does not appear to increase at higher salinities in this

species.

Values of the Pc in this study compare fairly well with Pc measurements for other

groups of fishes. Intertidal marine species generally have lower Pc values than C.

variegatus, ranging from 20 to 26 mm Hg in Paraclinus intergripinnis (Congleton, 1980)

to 30 to 40 mm Hg in Gobius cobitus (Bridges, 1988) and Helcogramma medium (Innes

and Wells, 1985; Pelster et al., 1988; Quinn and Schneider, 1991). A number of African

cichlids ( e.g., Oreochromis niloticus, Cichlasoma urophthalamus, Eretmodus

cyanosticus, Dimidiochromis compressiceps) have either slightly lower, or roughly

equivalent Pc values to the approximate value (55 mm Hg) displayed by C. variegatus in

this study at most salinities (Ross and Ross, 1983; Becker and Fishelson, 1986; Palacios

and Ross, 1986; Verheyen et al., 1994). Donnelly and Torres (1988) found Pc values

ranging from 25 to 50 mm Hg for a number of midwater fishes from the eastern Gulf of

Mexico, again values near, or slightly lower than those of C. variegatus. Thus, C.

variegatus has Pc values that are fairly close to those of ecologically and evolutionarily

diverse teleosts.








The variations in RMR and Pc as a function of environmental salinity observed in

this study suggest that C. variegatus responds to high salinities by reducing energy

expenditures. Observations by myself and others indicate that hypersaline conditions are

encountered infrequently, and likely last for short (days) periods of time. Available evidence

suggests that high salinities are metabolically expensive, whether due to an influence on

activity, osmoregulation, or other physiological and/or behavioral processes. Low oxygen

conditions also commonly occur with high salinities. Decreased metabolism in conjunction

with increased Pc reduces energetic expenditures dramatically at elevated salinities. These

responses effectively increase the time C. variegatus can tolerate adverse conditions, albeit

at a cost of a reduction in energetic processes. However, this is an appropriate response to

harsh conditions that appear at variable and unpredictable time intervals, but that are present

for only short periods of time.

The type of metabolic response to salinity in C. variegatus fits the concept of

"scope for survival" as described by Hochachka (1990). This is a pattern of metabolic

response to environmental stressors characterized by a depression in metabolism,

sometimes below maintenance levels. The advantage of such a reduction in energy

expenditure is essentially the slowing of biological time, enabling survival despite the

temporarily imposed physiological stressor.

Cyprinodon variegatus is an extremely successful euryhaline fish that can survive a

large range of salinities, with typically encountered salinities having little effect on normal

metabolism or on the ability of individuals to osmoregulate efficiently. Fish accommodate

extremely high salinities by reducing energetic expenditures. Such a response increases the

amount of time sheepshead minnows can survive hypersaline conditions, enabling them to
"wait out" the difficult conditions.













CHAPTER 3
INFLUENCE OF SIMULATED TIDAL CHANGES IN AMBIENT SALINITY ON
ROUTINE METABOLIC RATE IN CYPRINODON VARIEGATUS


Introduction

Salt marshes often undergo large and rapid salinity fluctuations, a condition that

may significantly affect the distribution and abundance of organisms within these habitats.

Changes in salinity may dramatically influence the energetic of individual fish. Information

on the metabolic costs associated with salinity fluctuations may be useful in explaining or

predicting distribution patterns of some coastal fish species.

Energetic patterns of fishes have been examined relative to a number of abiotic

factors, most notably temperature and oxygen (e.g., Wells, 1935; Fry, 1957; Beamish, 1964;

Brett and Groves, 1979; Stuenkel and Hillyard, 1981; Johnston and Battram, 1993).

Comparatively fewer studies have been considered the influence of salinity on metabolic

patterns of fishes (e.g., Kinne, 1966; Madan Mohon Rao, 1974; Nordlie et al., 1991;

Swanson, 1991). In these studies, various metabolic responses to salinity have been

reported. Euryhaline fish exhibit one of the few consistent patterns, namely that metabolism

remains relatively unaffected over the range of salinities normally encountered (Nordlie,

1978; Morgan and Iwama, 1991).

Most studies that have examined the influence of salinity on fish energetic have

employed measurements from fish maintained at constant salinities. Data from these

experiments are very useful for understanding the overall metabolic patterns exhibited by

the species tested, as fish were usually fully acclimated to each experimental salinity prior to

testing. However, these experiments provide less information about more ecologically








relevant responses, as fish in their native habitats are often subject to rapid and dramatic

fluctuations in salinity.

Studies of the influence of salinity fluctuations on metabolism of fishes have

usually focused on species that generally experience maximal fluctuations in salinity only

between freshwater (0 ppt) and seawater (35 ppt) (e.g., Davenport and Vahl, 1979; Von

Oertzen, 1984; Moser and Gerry, 1989; Shusmin, 1989; Moser and Miller, 1994).

However, some salt marsh teleosts regularly encounter salinities outside this range. This

study examined the influence of salinity fluxes on routine metabolic rate (RMR) of the salt

marsh teleost, Cyprinodon variegatus, a species that regularly encounters salinities greater

than 35 ppt.

Fish used in this study were fully acclimated to a series of salinities ranging from 0

ppt to 60 ppt, followed by exposure to a simulated tidal change in salinity. The magnitude,

rate, and direction of salinity changes may be important determinants of how salinity

fluctuations affect metabolism. In this study, the direction of the salinity change in

conjunction with the ambient (acclimation) salinity was the primary focus. The magnitude

of the salinity changes were selected to simulate extremes known to occur in the habitat

from which fish were collected, and the rate was chosen to simulate a normal diurnal tidal

cycle. I predicted that C. variegatus would show minimal changes in RMR when the

salinity was changed within the range commonly encountered by the species. Salinity

changes outside this range were hypothesized to lead to depressions in metabolism.

Methods


Collections of fish, transportation back to the laboratory, and general laboratory

procedures were performed as described previously. Fish were first sequentially acclimated

to a series of salinities ranging from 0 ppt to 60 ppt (0 to 1715 mOsm kg-1). However,

unlike the previous experiments, these procedures were designed to measure RMR before








and following simulated tidal changes in salinity. Initial salinities (acclimation) and salinities

following the simulated tidal change (final) are shown in Table 3-1.

Metabolic determinations and changes in salinity were carried out in a flow-through

respirometer with an effective volume of 0.875 L (Figure 3-1). The respirometer was

immersed in a thermoregulated reservoir during metabolic trials that served to maintain a

constant temperature within the respirometer. A submersible pump maintained within a

second thermoregulated reservoir (38 L) was connected to the inlet of the respirometer. This

reservoir was vigorously aerated and was used to supply water to the respirometer. The

respirometer outlet emptied back into this main reservoir. A third reservoir (19 L) serving as

a salinity source was connected to the main reservoir via a peristaltic pump. Due to

limitations of the available equipment, the system was closed during oxygen partial pressure

(P02) determinations. Two ports within the respirometer were used to sample water for
determination of PO2. A 10 ml plastic syringe filled with water at the experimental salinity

was inserted into the first port (inlet side), while an empty 10 ml syringe was fitted into the

second port (outlet side). As a 1 ml water sample was drawn into the empty syringe, an

equal volume of water was injected from the filled syringe into the respirometer.

Determinations of PO2 were made with a Radiometer oxygen electrode connected to a

Radiometer PHM 171 acid-base analyzer.

To begin a metabolic trial, the main reservoir and respirometer were filled with water

at the acclimation salinity. Fish were placed into the respirometer 12 h prior to the

beginning of a metabolic determination. This allowed fish sufficient time to adjust to the

experimental setup. The respirometer was then sealed and immersed in the thermoregulation

reservoir, and the submersible pump was turned on at a flow rate of 1 L min-1. The
following morning, the pump was turned off, the system was closed, and P02

measurements begun. Measurements of the rate of reduction in P02 were made at 0.5 to

1.0 h intervals, and continued until fish had depleted the oxygen level to approximately 100

mm Hg (generally 4 to 6 h). Following the final P02 determination, the system was








Table 3-1. Acclimation and final salinities used in simulated tidal change study.


Acclimation Salinity Final Salinity
0 ppt 0 ppt (control)
20 ppt
2 ppt 2 ppt (control)
20 ppt
30 ppt
10 ppt 10 ppt (control)
30 ppt
20 ppt 0 ppt
2 ppt
20 ppt (control)
40 ppt
50 ppt
30 ppt 2 ppt
10 ppt
30 ppt (control)
50 ppt
40 ppt 20 ppt
40 ppt (control)
60 ppt
50 ppt 20 ppt
30 ppt
50 ppt (control)
60 ppt 40 ppt
60 ppt (control)


reopened, and the submersible pump turned on. At this time, either well water (0 ppt) or a

saline solution of variable concentration was placed into the salinity reservoir. The salt

concentration in this reservoir was determined so that its addition to the main reservoir

would change the salinity of the main reservoir and respirometer to the desired final salinity.












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The volumes of both reservoirs and the rate of pumping by the peristaltic pump were

adjusted so that the salinity would change at a uniform rate over a 6 h period. The peristaltic

pump was plugged into an automatic timer so that salinity changes took place between 1900

h to 0200 h.
On the second morning the system was re-closed and PO2 measurements were

made at the new (final) salinity. PO2 measurements at the final salinity were thus made 6-

12 h following completion of the salinity fluctuation, a period sufficiently long to be past

the stressful transition period (Von Oertzen, 1984). Following the final P02 determination,

the fish was removed from the respirometer, damp-dried and weighed to the nearest 0.01 g.

All metabolic determinations were made between 0700 h and 1500 h, and fish were not re-

used in other metabolic trials. Fish were thus held within the respirometer for a total of 40

to 44 h (including adjustment period).

Food was withheld from experimental aquaria for 24 h prior to beginning a

metabolic reading to ensure that fish were post-absorptive at the time of testing. Fish were

not fed while in the respirometer. The entire metabolic apparatus was located in a small,

semi-darkened room in which there was no other activity, with determination of metabolic

rates performed as previously described.

Because a metabolic determination for the two salinity treatments was performed on

the same individual, it was not necessary to mass-correct values in order to perform the

statistical analyses. However, to compare data obtained in this study more easily with

published literature, all RMR measurements were mass-adjusted to the value of the average

mass (2.94 g) of all individuals used in this study. Adjusted RMR values were calculated as

previously described. A repeated measures analysis of variance was used to compare

adjusted RMR's between acclimation and final salinities. Statistical analyses follow

procedures outlined in Winer et al. (1991) and Sokal and Rohlf (1995). All analyses of

variance were one way tests using the Tukey-Kramer post hoc comparison (p = 0.05).








Results


Comparison of RMR's at acclimation and final salinities revealed some interesting

patterns (Table 3-2). There were no significant differences (p > 0.05) in RMR between

acclimation and final salinities in any control trials (n = 4 each). Similarly, when both

acclimation and final salinities (2 ppt, 10 ppt, 20 ppt, 30 ppt, and 40 ppt) were in a range that

is typically encountered by C. variegatus, there was a significant change in RMR in only

one trial. Typical salinities in the wild range from 2-5 ppt through 30-35 ppt, with

hypersaline conditions (35-40 ppt) occurring much more frequently than salinities near 0

ppt (pers. obs.). When the acclimation salinity is high (50 ppt and 60 ppt), all groups

exhibited significant elevation of RMR in the lower, more typical, final salinity. The same

general pattern was seen when the final salinity was high, where fish in two of three groups

had depressed RMR at the highest salinities. RMR was depressed when either the

acclimation or final salinity was 0 ppt. The direction of salinity change strongly influenced

the metabolic response. When salinity was increased over the course of the trial (Figure 3-

2), fish were only affected metabolically at the very highest and lowest salinities, where

metabolism was depressed. When salinity was decreased over the course of the trial (Figure

3-3), fish again showed metabolic depression at extreme salinities, but, overall, more groups

exhibited changes in metabolism with changes in salinity than groups in which salinity was

increased.


Discussion


My results demonstrate that C. variegatus maintains a very stable metabolism when

exposed to typical salinity fluctuations seen in the salt marsh habitat at Cedar Key, Florida.

The experimental procedure resulted in no mortalities during or immediately following any

salinity trial. Metabolism was generally unaffected in salinity trials where both acclimation

and final salinities were in the range typically encountered by C. variegatus at Cedar Key. It








Table 3-2. Mean routine metabolism (mg 02 h-1) before (acclimation salinity) and
following (final salinity) a simulated tidal change. Values are given as means se. Groups
exhibiting a significant change in metabolism are indicated with an asterisk.



Acclimation Acclimation Final Final P value Response
Salinity RMR Salinity RMR
(ppt) (mg 02 h-1) (ppt) (mg 02 h-1)

0 ppt 0.466 0.07 20 ppt (n = 5) 0.584 0.09 0.04 Increase
2 ppt 1.242 0.18 20 ppt (n = 5) 1.244 0.23 0.99 No Change

1.024 0.13 30 ppt (n = 5) 0.978 0.06 0.69 No Change

10 ppt 1.084 0.20 30 ppt (n = 5) 1.082 0.19 0.97 No Change

20 ppt 1.348 0.14 0 ppt (n = 5) 0.906 0.09 0.01 Decrease

0.886 0.03 2 ppt (n = 5) 0.478 0.07 0.01 Decrease

0.948 0.08 40 ppt (n = 6) 0.785 0.17 0.33 No Change

1.025 0.11 50 ppt (n = 6) 0.745 0.07 0.01 Decrease

30 ppt 1.028 0.23 2 ppt (n = 5) 0.854 0.10 0.33 No Change

0.628 0.07 10 ppt (n = 5) 0.688 0.11 0.34 No Change

0.728 0.07 50 ppt (n = 6) 0.728 0.12 1.00 No Change

40 ppt 0.704 0.12 20 ppt (n = 5) 0.662 0.09 0.75 No Change

0.626 0.06 60 ppt (n = 5) 0.476 0.03 0.03 Decrease

50 ppt 0.620 0.04 20 ppt (n = 5) 0.904 0.11 0.05 Increase

0.850 0.12 30 ppt (n = 5) 1.076 0.14 0.05 Increase

60 ppt 0.452 0.07 40 ppt (n = 5) 0.578 0.05 0.01 Increase


was only when the acclimation or final salinities were outside the range normally

encountered (i.e., 0 ppt, 50 ppt, and 60 ppt) that changes in metabolism were found. In these

cases metabolism was depressed at each extreme salinity relative to the measurements made

at the more typical salinity.































Figure 3-2. Results of metabolic trials where salinity was increased over the course of the
trial. Bars represent groups listed in Table 3-2 for which final salinity was greater than
initial salinity. The height of each bar signifies the magnitude of the salinity change for each
metabolic trial and the asterisk indicates at which of the salinities (for each metabolic trial)
the routine metabolic rate (RMR) was highest. The x axis has no scale and serves only to
visually separate groups.










RMR's Not Significantly Different (p = 0.05)

RMR's Significantly Different (p = 0.05)


Salinity Increasing


D
60




50































Figure 3-3. Results of metabolic trials where salinity was decreased over the course of the
trial. Bars represent groups listed in Table 3-2 for which final salinity was less than initial
salinity. The height of each bar signifies the magnitude of the salinity change for each
metabolic trial and the asterisk indicates at which of the salinities (for each metabolic trial)
the routine metabolic rate (RMR) was highest. The x axis has no scale and serves only to
visually separate groups.










D RMR's Not Significantly Different (p = 0.05)

SRMR's Significantly Different (p = 0.05)


* *


*


Salinity Decreasing








These results allow for limited comparison because similar studies have examined

the influence of salinity over a much narrower range than examined in this study. The best

comparison is with a study by Wakeman and Wohlschlag (1983) on the red drum,

Sciaenops ocellatus. In their study, test animals were acclimated to a series of salinities

between 1 ppt and 50 ppt, and then abruptly transferred to salinities either 10 ppt higher or

lower than the acclimation salinity. Although Wakeman and Wohlschlag (1983) did not

specifically compare metabolic rates following the transfer with those taken at the

acclimation salinity, there were trends similar to results of this study. Fluctuations in salinity

over a moderate range caused little or no change in metabolism of S. ocellatus on

determinations made 12 to 24 h following the transfer. Only when either the acclimation or

final salinity was 50 ppt were changes in metabolism obvious, with metabolic rates elevated

in 50 ppt compared to 40 ppt. It thus appears that S. ocellatus responds to abrupt salinity

changes somewhat differently than does C. variegatus, as metabolism increased upon

transfer to 50 ppt in S. ocellatus, but decreased in C. variegatus. This difference may be

due to the smaller magnitude of the salinity changes performed by Wakeman and

Wohischlag (1983) (10 ppt vs 20 to 30 ppt in the present study). However, these results

could also be attributable to different sizes and activity patterns of experimental fish

between the two studies.

In a similar study, Shushmin (1989) studied the dynamics of oxygen consumption

in juvenile golden mullet, Liza aurata, following abrupt transfers from 18 ppt to a series of

salinities ranging from 0.4 ppt to 50 ppt. All salinity changes resulted in increased

metabolic rates for one to three days following the transfer, with metabolism generally

returning to near normal levels within three to four days. It is not surprising that salinity

changes are more stressful in the golden mullet, since the species has lower salinity

tolerance and osmoregulatory ability than C. variegatus.

Barton and Barton (1987) examined metabolism in juvenile (< I g) C. variegatus

collected from inland saline lakes of San Salvador Island. They measured metabolic rates of








fish at 10 ppt and 35 ppt following abrupt transfer from an acclimation salinity of 35 ppt.

Metabolic rates in fish measured at 10 ppt were significantly higher than those made at the

acclimation salinity. These results are somewhat different from those obtained in the present

study. However, measurements by Barton and Barton (1987) were made only 3 h following

abrupt transfer to the reduced salinity, whereas measurements in the present study were

made 6-12 h following a gradual change in salinity. Fish in this study thus experienced a

less stressful transition to the new salinity. An ontogenetic effect may also account for the

observed differences, as many investigations have demonstrated that adults and juveniles of

the same species may differ greatly in physiological competence (e.g., Kinne, 1966; Martin,

1968; Oikawa et al., 1991).

Other relevant comparisons can be made with data on juvenile croaker,

Micropogonias undulatus, and juvenile spot, Leiostomus xanthurus (Moser and Gerry,

1989; Moser and Miller, 1994). These studies examined effects on metabolism from

fluctuations in salinity between 0 ppt and 35 ppt. Like C. variegatus, a salinity decrease

from higher salinities to 0 ppt generally led to a decreased metabolism in L. xanthurus.

Similarly, when fish were acclimated to 0 ppt and salinity was increased, metabolism was

elevated at the higher salinities. Contrary to findings for C. variegatus, salinity fluctuations

between 15 ppt and 35 ppt elicited changes in metabolism in both L. xanthurus and M.

undulatus. Thus, these species are influenced to a greater extent by salinity fluctuations than

C. variegatus. However, rates of salinity fluctuation in the above studies were faster than

rates used in the present study (16 or 32 ppt h-1 vs 3.3 or 5 ppt h- 1), and C. variegatus

likely experiences greater fluctuations in salinity than do spot or croaker.

Acclimation state is the most important measured factor influencing the metabolic

response of C. variegatus to simulated tidal changes in salinity. However, direction of the

salinity change also influenced metabolism in C. variegatus. Like the results obtained by

Moser and Miller (1994) on juvenile L. xanthurus, it appears that C. variegatus adjusts to

increasing salinity more effectively than to decreasing salinity, as evidenced by the








responses to salinity changes between 20 ppt and 2 ppt, and between 50 ppt and 30 ppt.

Thus, when salinity is increased over the course of the trial, fish are only affected

metabolically at the very highest and lowest salinities. However, when fish are acclimated to

a high salinity, and salinity is then decreased to a more typical range, individuals take

advantage by increasing metabolism to more normal levels.

Thus, C. variegatus is well adapted to a varying salinity environment Its

metabolism is unaffected by changes in salinity over the typical range encountered, even

when salinity is changed rapidly. Furthermore, this corroborates my hypothesis that C.

variegatus tolerates extremes in salinity by lowering metabolism and decreasing energy

expenditures. Fish appear to wait for conditions to improve, and respond to these more

favorable conditions by returning metabolism to normal levels.

A decrease in energetic expenditures as just described is a potentially adaptive

response for fishes living in variable salinity environments like those of Florida coastal salt

marshes. While few data exist on responses of other salt marsh residents to wide ranges in

salinity, I suspect this may be a general pattern. Cyprinodon variegatus may be unusual

because of its broad tolerances, but is a useful experimental animal because of this as well.

Information gleaned from studies with C. variegatus may indicate areas of examination for

other important salt marsh teleosts which may have more limited salinity tolerance, but

which may follow the same general metabolic patterns seen in C. variegatus.













CHAPTER 4
INFLUENCE OF A FLUCTUATING SALINITY REGIME ON OSMOREGULATION
IN CYPRINODON VARIEGATUS


Introduction

Few areas in the field of fish physiology have received as much attention as the

study of osmoregulation. The basic patterns of osmoregulation are well understood and are

reviewed extensively by Evans (1984), Karnaky (1986), Ventrella et al. (1992), Evans

(1993), McCormick (1994), and Wood and Marshall (1994). The mechanisms of

osmoregulation in euryhaline fish are not unusual. Euryhaline fishes are unique in having

the ability to osmoregulate efficiently in waters of highly variable salinity. They can

osmoregulate in water both more and less concentrated than their own body fluids, and they

are able to alter their pattern of osmoregulation rapidly if they live in environments where

sudden fluctuations in salinity may occur.

Despite the voluminous research done in this area, few studies have examined

patterns of osmoregulation in fishes that may encounter salinities outside the range from

freshwater (0 ppt) to seawater (35 ppt). Such species tend to be small and of little or no

economic importance, with a notable exception being the milkfish, Chanos chanos (Ferraris

et al., 1988; Swanson, 1991). However, to understand osmoregulation patterns in fishes,

studies need to encompass the range of salinities encountered by species in nature.

One topic that has received little attention concerns the effect of fluctuating salinities

on osmoregulation in telcosts. Salinity is a limiting factor in certain estuarine and salt marsh

habitats, and may widely vary daily and seasonally. Despite this, most investigations of

osmoregulation in fishes involve studies at constant salinities. While such studies provide








important information on overall osmoregulatory patterns, they provide less information

about more ecologically relevant responses.

The aim of the present investigation was twofold. First, I examined the ability of

individuals of the euryhaline teleost, Cyprinodon variegatus, to regulate plasma osmolality

under the influence of a cycling salinity regime. Second I examined a hypothesis proposed

by Goolish and Burton (1988) in a study involving the intertidal copepod Tigriopus

californicus. Goolish and Burton (1988) suggested that species exposed to fluctuating

salinities would be able to respond more rapidly and completely to salinity stress. In other

words, could past exposure to changing salinity result not in improved osmoregulation at

any single salinity, but rather to improved performance immediately following another

salinity fluctuation? These hypotheses were examined by determining plasma osmolality

and hematocrit of individual C. variegatus subjected to fluctuations in salinity over a wide

range of ambient salinities.

Methods


Collections of fish used in this study were obtained from tidal creeks in the salt

marsh near Cedar Key, Florida. Specimens were transported back to the laboratory in 128 L

coolers supplied with aeration and filled with water obtained from the collection site. Fish

were obtained in two collections made during September 1994. The salinity of the collection

site was approximately 25 ppt for both collections. This study was conducted at the

Southeastern Biological Science Center (SBSC), National Biological Service, Gainesville,

Florida. Fish were held overnight in the coolers used for transportation (with aeration)

before transfer to a 1.2 m diameter fiberglass holding tank containing water at 30 ppt Fish

were treated prophylactically for 7-14 days in a 5 mg L-1 solution of Acriflavine.

Following treatment, all fish were transferred to experimental aquaria (30 ppt salinity = 860

mOsm kg-1) located within a constant environment room maintained at 20 1 oC and on a








12:12 h lightdark cycle. Both holding and experimental aquaria were equipped with sponge

filters providing continuous aeration, and fish were fed flake food once each day.

Experimental aquaria were used to subject fish to a cycling salinity regime. Seven

salinity trials were performed (Table 4-1). All fish were maintained in experimental aquaria

located on bank 1 for eight days prior to initiating the first salinity change. Salinity changes

were effected by carefully netting fish from the experimental aquaria located on bank 1, and

immediately transferring individuals into the appropriate experimental aquaria located on

bank 2. Fish remained in experimental aquaria located on bank 2 for 24 h before transfer

back to tanks located on bank 1. Fish then remained in bank I experimental aquaria for 24

h before again being transferred to aquaria on bank 2. Thus, one complete cycle began with

24 h in bank 2 aquaria, and ended following 24 h in bank 1 aquaria. The procedure was

repeated so that a total of 10 cycles was performed (20 days). A subsample of fish was

removed for testing from each experimental aquarium just prior to the first change in

salinity (cycle 0), and at the end of cycles 1 (day 2), 5 (day 10), and 10 (day 20). All

subsamples were thus taken following a 24 h period in bank I aquaria (30 ppt).

Following completion of the 10th cycle, fish remaining in all decreasing salinity

groups (groups DI, D2, and D3) were transferred to aquaria at 2 ppt and fish remaining in

all increasing salinity groups (groups Ii, 12, and 13) were transferred to aquaria at 60 ppt

Fish in the aquaria maintained at 30 ppt were split into three groups at that time: one third of

the group was transferred to 2 ppt (group CD), one third transferred to 30 ppt (group C),

and the final third transferred to 60 ppt (group CI). Fish remained in these salinities for 24

h and were then removed for testing (day 21). Salinities were checked daily with a Leica

temperature compensated refractometer and adjusted as necessary.

Hematocrit (Hct) and plasma osmolality were determined at each sampling interval.

Fish were first carefully netted from their experimental aquaria and blotted dry. Blood was

taken by sternal cardiac puncture using heparinized microhematocrit tubes drawn to a fine

point, and fish were weighed and standard length determined. The tubes were then







Table 4-1. Salinity trials used in cyclical salinity study. The group maintained at 30 ppt was
split into three groups following cycle 10 (day 20); groups CD, C, and CI (see text for
details).


Group Direction of Salinity Change Salinity in Bank 1 Salinity in Bank 2
(ppt) (ppt)

DI Decreasing Salinity (n=25) 30 2

D2 Decreasing Salinity (n=25) 30 10

D3 Decreasing Salinity (n=25) 30 20

C, CD, and Ci No Change (n=35) 30 30

II Increasing Salinity (n=25) 30 40

12 Increasing Salinity (n=25) 30 50

13 Increasing Salinity (n=25) 30 60


centrifuged in a micro-hematocrit centrifuge for 10 minutes to separate plasma from cells.

Hematocrit was read using a micro-capillary reader before plasma was isolated from formed

elements by scoring the tube with a file and retaining only the portion containing plasma.
Plasma osmolality (mOsm kg-1) was determined on 5 pl samples using a Wescor 5500

vapor pressure osmometer. Fish were used without regard to sex, and all blood samples

were taken between 0600 h and 1000 h. Statistical procedures followed Winer et al. (1991)

and Sokal and Rohlf (1995). All statistical analyses were one way tests using the Tukey-

Kramer post hoc comparison (p = 0.05)



Results

All fish entered into the experimental procedure survived the entire duration of the

experiment Fish in all groups ate normally throughout the course of the experiment, and no

discernible changes in behavior were observed during the experimental procedure for any








group. Fish appeared to have no difficulty in tolerating the imposed salinity fluctuations,

even in groups Di and 13, which were subjected to daily changes in salinity of 28 ppt and

30 ppt, respectively. The influence of body mass on both Hct and plasma osmolality was

evaluated using an analysis of covariance, and for no analysis was body mass a significant

covariate. Sizes of fish used in this study ranged from 0.75 g to 5.887 g (2.8 mm to 5.8 mm

standard length, mean 4.1 0.038 mm), with a mean mass of 2.245 0.066 g. Subsamples

of groups were not significantly different in mass.

Hematocrit and plasma osmolality values were compared both between salinity trials

for each time period sampled (i.e., all groups were compared to one another on days 0, 2,

10, 20, and 21), and within a single trial over the time course of the experiment. Groups

experiencing increases in salinity (groups Ii, 12, 13, and CI) were compared separately from

groups experiencing decreases in salinity (groups DI, D2, D3, and CD), with all compared

to the group maintained at a constant salinity (group C). Data from days 0 through 20 are

discussed separately from day 21 data as follows.


Day 0 through 20

Hematocrit values showed no obvious trends either between salinity trials or within

any single trial over the time course of the experiment (Table 4-2). There were no

statistically significant differences (p > 0.05) within any group (between days 0 and 20), or

among groups on days 0, 2, 10, or 20. There was a slight trend towards increased variance

in Hct values over the course of the experiment in all groups, peaking at cycle 5, but

differences were non-significant in all cases.

Plasma osmolality data, unlike the Hct data, exhibited consistent trends. No

significant differences existed among groups on days 0, 2, 10, or 20. However, comparisons

within groups over time revealed a different pattern. All groups, regardless of salinity

regime, showed a trend towards slightly elevated plasma osmolality between days 0 and 20

(Table 4-2; Figures 4-1 and 4-2). For groups C and II, this was statistically significant, with










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the values on day 20 significantly higher than values from day 0 and day 1 (p<0.05); all

other groups exhibited the same trend, but differences were statistically non-significant.

This appears to be related to the experimental manipulation of fish, and not to the salinity

regime experienced by each group. All groups regulated plasma osmolality effectively. No

differences in regulatory ability could be seen among any of the groups tested, regardless of

the magnitude or direction of the salinity fluctuation.


Day 21


The above addresses the question of how effectively C. variegatus regulates plasma

osmolality in the face of salinity fluctuations. Transfer of individuals to either 2 or 60 ppt

following 20 days of exposure to a variety of salinity fluctuations addresses the second

question posed earlier Does past exposure to large salinity fluctuations result in improved

osmoregulatory performance, compared to animals experiencing little or no salinity

fluctuation, immediately following a fluctuation in salinity? All measurements here were

taken 24 h (on day 21) following the final salinity fluctuation.

Hematocrit results showed no consistent trends on day 21 samples. Only one

significant difference was noted, with the Hct value for group C1 being significantly elevated

compared to the value for group C (p<0.05). All other comparisons showed no significant

differences in Hct.

There were significant differences in plasma osmolality for day 21 measurements.

Here groups which had experienced greater fluctuations in salinity over the course of the

experiment showed a much better ability to osmoregulate compared to the control group or

groups which had undergone small salinity fluctuations. Increasing salinity seemed to have

a greater impact on plasma osmolality than did decreasing salinity (Figures 4-1 and 4-2).

Plasma osmolality in groups DI, D2, and D3 all exhibited declines on day 21

relative to day 20, but these differences were not statistically significant. No differences in

regulatory ability among these three groups, or with the control group (group C), could be








discerned. However, group CD, which had been maintained at 30 ppt for the first 20 days of

the experiment, did show a significant decrease in plasma osmolality on day 21 relative to

values measured on days 0 through 20. Furthermore, this group had lower osmolality

values on day 21 than all other groups, although this difference was only statistically

significant when compared to groups C and D3.

More striking differences existed for groups II, 12, 13, and C1. In all cases plasma

osmolality was elevated on day 21 relative to measurements taken from the same group on

day 20 or earlier. Group 13, which had experienced daily fluctuations in salinity of 30 ppt,

showed the smallest increase in osmolality values, being significantly higher than

measurements from day 0 only. Group 12 showed a similar result, with the plasma

osmolality value from day 21 significantly higher than values from day 0 and day 2 only.

Groups Ii and C1 showed the largest changes in plasma osmolality on day 21. Both groups

had osmolality values that were significantly higher than all previous measurements taken

from their respective groups. The value for group I1 was elevated compared to groups 12, 3,

and C, although this increase was statistically significantly only when compared to group C.

Group C1 was significantly elevated when compared to all other groups (including group

II).

Discussion


The ability to adjust rapidly to altered salinities would be an obvious advantage to

salt marsh organisms. Physiological responses of euryhaline fishes exposed to rapid

changes in salinity can be grouped into two phases (Holmes and Donaldson, 1969): an

adaptive period and a regulatory period. During the adaptive period, plasma osmolality

varies, gradually returning to values approaching original levels. In the regulatory period,

plasma osmolality is more finely controlled as the fish adjusts to the altered salinity and

reaches ionic homeostasis. Fishes which reach the regulatory period quickly (i.e., have short

adaptive periods) should be best able to tolerate alterations in ambient salinity. Although the








length of the adaptive period was not measured in the present study, results nevertheless

suggest that it is relatively short for C. variegatus.

Nordlie (1985) showed that C. variegatus was an excellent osmotic regulator over a

salinity range from 0.3 ppt to 70 ppt. Plasma osmolality values changed only slightly over

this range of salinities in his study, varying by only 40 mOsm kg-1. However, fish in his

study were fully acclimated to each experimental salinity prior to testing. The present study

indicates that C. variegatus is an excellent regulator of plasma osmolality even when fishes

are exposed to large daily fluctuations in salinity. Although fish in all groups in the present

study regulated at slightly higher levels than seen by Nordlie (1985), plasma osmolality

values varied similarly, with differences of less than 40 mOsm kg-1 for all groups on days

0 through 20. The transfer process itself elicited much of this variation, with slight increases

in plasma osmolality seen over the time course of the experiment. A similar trend was seen

in a study by Woo and Wu (1982) on the red grouper, Epinephelus akaara, and the black

sea bream, Mylio macrocephalus.

The influence of a single alteration in salinity on osmoregulatory ability has been

studied for a number of fishes, with changes in plasma osmolality observed in the present

study of similar magnitude to those seen in other euryhaline fishes (Wakeman and

Wohlschlag, 1983; Engel et al., 1987; Ferraris et al., 1988; Mancera et al., 1993; Yoshikawa

et al., 1993; Altimiras et al., 1994; Tort et al., 1994). Few studies have examined how

fluctuating salinities influence osmoregulation of fishes. The best comparison of my results

are with a study on Blennius pholis by Davenport and Vahl (1979). In their study B. pholis

were exposed to alternating periods of freshwater and seawater, with each period lasting 6 h.

Similar to the results from the present study, plasma osmolality did not vary significantly

over the course of the experiment.

No consistent differences in Hct were observed over the course of the experiment.

Nordlie et al., (1995) found that C. variegatus efficiently regulates water content over a

wide range of salinities, with a difference of only 4% observed between 0 ppt and 100 ppt.








These results suggest that there are no large movements of water between the blood and

tissues as a result of fluctuations in salinity over the range tested. The lack of response in

hematocrit in this study may also indicate that within the range of salinities studied C.

variegatus is subject to normal, or tolerance physiological processes, as an increase in

hematocrit values would be expected if C. variegatus were exposed to conditions leading to

resistance processes (Swift, 1982).

It has been hypothesized that fishes occurring in a habitat where salinity often

fluctuates may be able to respond more quickly and completely to alterations in salinity, and

that the limits of tolerance are farther apart if salinity fluctuates periodically (Gunter, 1967;

Spaargaren, 1974; Johnston and Cheverie, 1985). My results provide evidence that prior

exposure to fluctuations in salinity does impart an osmoregulatory advantage. Fishes

previously exposed to large fluctuations in salinity regulated plasma osmolality better than

fishes that had previously experienced no change or small changes in salinity. Increasing

salinity had the greatest impact on regulation of plasma osmolality. Group C1, which had

experienced no prior change in salinity, and group Ii, exposed to the smallest fluctuations in

salinity prior to being transferred to 60 ppt showed large increases in plasma osmolality

compared to the control group and groups which had previously experienced large

fluctuations in salinity. Decreasing salinity groups exhibited the same pattern, but

differences in osmolality were less pronounced. Only group CD, which had not been

previously exposed to fluctuations in salinity, showed a significant decline in plasma

osmolality after transfer to 2 ppt Salinities between 2 ppt and 35-40 ppt are typically

encountered by this population of C. variegatus in its native habitat, with salinities as high

as 60 ppt rarely encountered. Thus, it is not surprising that transfer to 60 ppt elicited greater

changes in regulation of plasma osmolality.

Variations in salinity imposed on fish in this study caused no dramatic effects. No

adverse behavior or mortalities were noted throughout the course of the experiment. Thus,

despite the differences seen in regulation of plasma osmolality between some of the





69


experimental groups, these differences were not large enough in magnitude to cause

observable distress in the experimental animals. These results indicate that C. variegatus is

well adjusted for life in variable salinity conditions. It would be interesting to compare

results from the present experiments with studies examining the influence of fluctuating

salinity on osmoregulation in fishes that can tolerate changing salinity, but that experience

infrequent salinity variations in their native habitat.













CHAPTER 5
INFLUENCE OF ENVIRONMENTAL SALINITY ON BLOOD OXYGEN LEVELS OF
CYPRINODON VARIEGATUS


Introduction


The sheepshead minnow, Cyprinodon variegatus, is a euryhaline teleost whose

typical habitats are brackish water, coastal salt marshes that experience frequent salinity

fluctuations. Variations in environmental salinity may directly affect the respiratory system

of fishes in at least two ways: by affecting the solubility of oxygen in the water pumped

over the gills, and by affecting the solubility of oxygen dissolved in plasma. Changes in the

ionic composition of bodily fluids may also interact with oxygen to influence tolerance to

variable salinity conditions (Truchot, 1987). Furthermore, fishes in saline water with low

oxygen tension must balance maximizing branchial oxygen diffusion with greater

osmoregulatory demands due to the accompanying increases in ion and water exchange

(Perry and McDonald, 1993). Additionally, the oxygen content of many aquatic habitats is

subject to large natural fluctuations, so oxygen is a potentially limiting factor by itself

(Dejours, 1987; Graham, 1990). This is especially true in shallow waters, where chronic or

periodic hypoxia may be a common phenomenon (Graham, 1990). Salt marsh habitats are

often exposed to hypoxic conditions (Renaud, 1985; Toulmond, 1987).

Cyprinodon variegatus is an extremely competent euryhaline teleost. Over a range

of salinities from freshwater (0 ppt) to 40 ppt, very small changes in metabolism occur

(Nordlic, et al., 1991; this study, Chapter 2). Both oxygen consumption and critical oxygen

tension (Pc) are essentially unaffected by changes in salinity over this range. Salinities

above this range cause metabolic adjustments, with increases in Pc and decreases in

metabolism observed (this study, Chapter 2). The mechanisms involved in maintaining








constant metabolism and Pc at low to moderate salinities, and the alterations of metabolism

and Pc at higher salinities likely involve adjustments in oxygen transport. Not only do high

salinity waters have lowered concentrations of dissolved oxygen, but salinity itself seems to

elicit physiological responses similar to those resulting from lowered levels of oxygen.

Increasing blood oxygen levels with increasing salinity, possibly leveling off at the highest

salinities, could lead to the observed responses in metabolism and Pc in C. variegatus.

Increased blood gas transport is likely the primary mechansim used by most fishes

to increase the amount of oxygen delivered to tissues. Blood oxygen transport in most

teleosts is dependent upon the respiratory pigment hemoglobin. Blood oxygen transport is

normally increased by increasing the concentration of hemoglobin, increasing the number

of erythrocytes in circulation, and/or adjusting the affinity of hemoglobin for oxygen

(Davis, 1975; Wells et al., 1989; Jensen et al., 1993; Perry and McDonald, 1993). I

examined hemoglobin concentration, erythrocyte count, and packed cell volume (hematocrit)

over a wide range of salinities for individuals of C. variegatus to evaluate the influence of

salinity on blood oxygen levels in this extremely euryhaline species.

Methods


Collections of fish, transportation back to the laboratory, and general lab procedures

were performed as described previously. Using the same protocol described earlier, fish

were sequentially acclimated to a series of salinities ranging from 0 ppt to 80 ppt (0 to 2285

mOsm kg- 1). At the end of the acclimation period, fish were sacrificed to determine

hemoglobin concentration ([Hb]), hematocrit (Hct), and erythrocyte (RBC) count.


Blood Sampling


Fish were first carefully netted from their experimental aquaria and blotted dry.

Blood was taken by sternal cardiac puncture using freshly heparinized microhematocrit

tubes drawn to a fine point. Blood from each fish was collected in two microhematocrit








tubes. Once the first tube was filled with a volume > 20 jpl, the blood was immediately

dispensed into a small ceramic crucible. Eppendorf micropipettes were then used to

dispense aliquots of blood from the crucible into test tubes used in the determination of

hemoglobin concentration and erythrocyte count. A second microhematocrit tube was then

filled and used for determination of the hematocrit, and mass and standard length of fish

were then determined. Fish were used without regard to sex, and all blood samples were

taken between 0700 h and 1100 h. Food was withheld from experimental aquaria for 24 h

prior to testing to ensure that all fish were post-absorptive. Statistical procedures followed

Winer et al. (1991) and Sokal and Rohlf (1995). All statistical analyses were one way tests

using the Tukey-Kramer post hoc comparison (p = 0.05).

Hemoglobin Analysis

Hemoglobin concentration was measured spectrophotometrically on 10 ptl samples

using the cyanomethemoglobin method (Brown, 1993). As recommended by Innes and

Wells (1985), the cyanomethemoglobin solutions were centrifuged for 10 min at 5000 g

prior to colorimetric determination to remove erythrocyte debris. This method gives reliable

results when used with fish blood (Blaxhall, 1972; Blaxhall and Daisley, 1973; Coburn,

1973; Innes and Wells, 1985).


Erythrocvte Count


Erythrocytes were counted immediately following dilution in test tubes containing

Natt and Herricks solution (Campbell and Murru, 1990). This solution acts as both stain

and dilutent and is routinely used for counting erythrocytes of fish (Campbell and Murru,

1990). A 1:200 dilution was used and cells were counted in an improved Neubaucr

hematocytometer following precautions outlined in Brown (1993).








Hematocrit

Hematocrit was measured to determine the packed cell volume of the erythrocytes

contained in the blood. Immediately after filling the second microhematocrit tube one end

was sealed and the tube placed into a micro-hematocrit centrifuge. Tubes were then

centrifuged for 10 min to separate plasma from formed elements. The hematocrit was read

using a micro-capillary reader and expressed as percent erythrocyte.

From the test values obtained, mean corpuscular volume (MCV), mean corpuscular

hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were

calculated for each fish as follows (Brown, 1993):
Hct x 10' [Hb] (g/ 1) [Hb] (g / dl)
MCV BC MCH B- MCHC- H
RBC/1 'RBC/1 Hct

These erythrocyte indices are used to further define the relationship between hemoglobin

content and size of the erythrocyte.

Results


The various measures of blood oxygen are arranged by salinity group in Table 5-1.

Significant differences over the range of test salinities were found for all parameters except

MCHC. Body mass had no significant influence on any of the measured or calculated

blood oxygen indices.

Salinity exerted the greatest influence on erythrocyte count (Figure 5-1). Values

obtained at 80 ppt and 0 ppt were significantly higher than all other salinities. Erythrocyte

count was next highest at 60 ppt and 70 ppt, being significantly elevated compared to values

in fish acclimated to 2, 10, 20, and 30 ppt Fish acclimated to salinities from 2 ppt through

50 ppt exhibited no significant differences in erythrocyte count.

Measurements of hemoglobin concentration exhibited a similar pattern, although

fewer significant differences were noted (Figure 5-2). Mean values of fishes acclimated to 0

ppt were highest and significantly different from fish acclimated to salinities from 2 ppt











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through 50 ppt. Hemoglobin concentration was also elevated in fish acclimated to 60 ppt, 70

ppt, and 80 ppt, although these values were only significantly higher than the value of fish

acclimated to 20 ppt.

Hematocrit measurements showed less dependence upon salinity (Figure 5-3).

Mean hematocrit was highest in 0 ppt, and was significantly different than mean values of

fish acclimated to all salinities except 60 ppt and 70 ppt No other significant differences in

hematocrit were noted.

Calculated erythrocyte indices indicated a slightly different pattern. The average

concentration of hemoglobin in the erythrocyte (MCHC) did not vary significantly among

salinity acclimation groups. However, both the average weight of hemoglobin in the

erythrocyte (MCH) and the average volume of the erythrocyte (MCV) were lowest in fishes

acclimated to 80 ppt. MCH was significantly depressed in fish acclimated to 80 ppt when

compared to groups acclimated to 2, 30, 60, and 70 ppt, with MCV values at 80 ppt

significantly lower than values obtained for groups at 10, 20, 30, 50, and 70 ppt.

Discussion


Changes in environmental salinity can exert profound effects on blood oxygen

transport. Increases in salinity confront fishes with the necessity of satisfying oxygen

requirements under conditions of reduced oxygen availability. Fishes may exploit multiple

strategies to optimize blood oxygen transport. The amount of oxygen delivered to the

tissues by the blood per unit time is a product of the cardiac output, the oxygen tension

difference between arterial and venous blood, and the blood oxygen capacitance coefficient

(Jensen, 1991; Jensen et al., 1993). The capacitance coefficient reflects the hemoglobin's

oxygen transporting properties, and can be adjusted in what have been termed 'qualitative'

and 'quantitative' ways (Jensen, 1991). Regulation of hemoglobin-oxygen (Hb-O2) affinity

represents the primary method for qualitatively altering oxygen carrying capacity, with

control of hemoglobin concentration the primary quantitative mechanism (Jensen, 1991).












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Blood oxygen carrying capacity can also be increased quantitatively by release of stored

erythrocytes, by accelerating maturation of immature erythrocytes, and/or by production of

new erythrocytes (Murad et al., 1990); release of erythrocytes from storage organs (e.g.,

spleen) appears to be the most likely scenario (Soivio et al., 1980; Wells et al., 1989). Fish

exposed to water of changing salinity would be expected to experience variability in their

blood oxygen capacitance coefficient and blood oxygen carrying capacity (Jensen et al.,

1993). Quantitative mechanisms for adjusting blood oxygen carrying capacity were

examined in this study.

Few studies have examined the influence of salinity on oxygen carrying capacity of

fishes. Guernsey and Poluhowich (1975) examined the blood oxygen capacity of American

eels (Anguilla rostrata) acclimated to 0 ppt, 24 ppt, and 34 ppt. As in C. variegatus,

hematocrit was highest in eels acclimated to 0 ppt. However, while oxygen capacity of

acclimated eels was higher in 0 ppt than 34 ppt, the highest oxygen capacity was seen in

eels acclimated to 24 ppt. In a similar study with the cichlid Oreochromis niloticus, Sun et

al., (1995), observed a similar effect of salinity on measures of blood oxygen, with

hemoglobin concentration significantly higher in 0 ppt than in higher salinities (5 to 20

ppt).
Other factors may also contribute to variations in oxygen carrying capacity of

fishes. Hall and Gray (1929) were among the first to note that there is a general correlation

between the habits of fishes and the hemoglobin concentration of their blood. More recent

studies have shown that this generalization also applies to erythrocyte count and hematocrit

(e.g., Haws and Goodnight, 1962; Coburn, 1973; Larsson et al., 1976; Putnam and Freel,

1978). In general, highly active fishes and those that regularly encounter hypoxic conditions

have elevated blood oxygen carrying capacity relative to other species. Values determined

for C. variegatus in the present study are comparable to other fishes with similar activity

levels, and indicate that C. variegatus does not possess exceptionally high oxygen carrying









capacity at any salinity tested (Hattingh, 1972; Coburn, 1973; Larsson et al., 1976; Putnam

and Freel, 1978; Smit and Hattingh, 1979; Pelster et al., 1988b).

Salinity had a significant effect on blood oxygen carrying capacity in C. variegatus.

However, differences were seen only at the very highest and lowest salinities tested. As

expected, oxygen carrying capacity increased at high salinities. Elevations were seen at 60

ppt through 80 ppt when compared to measures made at salinities between 2 ppt and 50 ppt.

One finding of interest was indicated by MCV measurements. These results showed that at

80 ppt overall size of erythrocytes had decreased, implying the production and/or release of

smaller, possibly immature erythrocytes. One possible explanation for this is that smaller

erythrocytes have been found to be more effective in oxygen exchange than larger

erythrocytes (Coburn, 1973) due to a larger surface area per unit volume, which may allow a

faster rate of gas exchange. Energetic constraints related to viscosity of the blood may also

play a role. A balance must exist between the advantages for oxygen transport from

increased hematocrit with a disadvantage due to increased viscosity (Wells and Weber,

1991). Hematocrit began to increase as salinity reached 60 ppt, but declined back towards

normal values at 80 ppt. It is possible that at extremely high salinities even small increases

in hematocrit lead to significant viscosity problems. Utilizing smaller erythrocytes at

extreme hypersalinities may help offset this problem.

Unexpectedly, oxygen carrying capacity was highest in the group acclimated to 0

ppt, as indicated by measurements of all blood indices. Although freshwater is tolerated by

C. variegatus, previous work has shown that acclimation to freshwater is difficult and may

result in significant mortality unless decreases in salinity are made in small steps (Nordlie

and Walsh, 1989; Nordlie et al., 1991). Survival in freshwater requires many of the same

responses that are necessary at extremely high salinities, with both freshwater and

hypersaline conditions imposing difficult osmoregulatory problems for C. variegatus. In

both situations, proliferation of mitochondria rich cells on gill epithelia is needed to

maintain ionic balance (Evans, 1984; Evans, 1993; Wood and Marshall, 1994). However,








this extends the surface involved in ionic exchange at the expense of gill epithelia involved

in gas exchange. Recent studies have shown that such proliferation of mitochondria rich

cells does impair respiratory gas transfer (Bindon et al., 1994a; Bindon et al., 1994b).

Mechanisms to increase oxygen carrying capacity of the blood would be expected under

such conditions.

However, freshwater conditions differ significantly from hypersaline conditions in

several ways. Most importantly, metabolism is reduced at extreme hypersalinities (this

study, Chapter 2). In conjunction with elevated Pc, depressed metabolism at these

hypersalinities greatly reduces energetic expenditures, partially alleviating the need for

increased oxygen carrying capacity. Thus, whereas measures of blood oxygen are elevated

at salinities of 60 ppt and higher, increases were moderated by a reduction in overall

energetic expenditures. Fish acclimated to 0 ppt exhibit insignificant reductions in

metabolism, so possible increases in oxygen needs in freshwater can not be compensated

for in this manner. Furthermore, a number of studies have indicated that Hb-02 affinity is

decreased in freshwater conditions (e.g., Benditt et al., 1941; Weber et al., 1976; Woo and

Wu, 1982). Thus, oxygen carrying capacity may have to be increased in quantitative ways if

oxygen affinity of the hemoglobin molecule is decreased in dilute media.

Another finding from the present study was that C. variegatus exhibits little change

in quantitative measures of oxygen carrying capacity over the range of salinities between 2

ppt and 50 ppt. Previous studies have shown that salinities within this range have little

effect on the metabolic rate, Pc, and osmoregulatory ability of C. variegatus (this study,

Chapters 2,3, and 4). However, as oxygen needs would be expected to rise as salinity was

increased over this range, oxygen carrying capacity was expected to increase as well.

Several possibilities may explain why blood oxygen carrying capacity did not

increase over this range of salinities. First, increases in salinity may require little

compensation in oxygen carrying capacity until extreme hypersalinities are reached.

However, it is more likely that the lack of a response may have been due to the fact that








large variations in salinity alone seldom occur under natural conditions. A larger response

would be expected if salinity were varied together with oxygen and/or temperature. Another

possibility is that C. variegatus, like many fish species, may utilize multiple hemoglobins.

Weber (1990) noted that the use of multiple hemoglobins can extend the range of

conditions under which oxygen is transported efficiently, thus enlarging the range of

available habitats. If C. variegatus utilizes such a mechanism, large quantitative changes in

oxygen carrying capacity might not be seen.

Finally, C. variegatus may exhibit mostly qualitative changes in response to salinity,

i.e., the primary response to changing salinity may be better reflected in Hb-02 affinity.

Changes in Hb-02 affinity as a result of environmental changes are extremely common in

fishes (e.g., Johansen and Weber, 1976; Wells et al., 1980; Weber, 1981), although this has

rarely been examined with respect to salinity. However, studies on Anguilla anguilla

(Weber et al., 1976) and Salmo salar (Maxime et al., 1990) did find that increases in

salinity between freshwater and seawater led to increases in Hb-02 affinity. A high affinity

hemoglobin molecule might also be advantageous under hypersaline conditions, although it
may be ineffective during activity (McMahon, 1988). As Hb-02 affinity was not measured

in this study, direct correlation with C. variegatus is purely speculative at this time.

Nevertheless, changes in Hb-O2 affinity would correlate well with the large decreases in

energetic expenditures exhibited by C. variegatus at high salinities. However, other factors

could also be involved. First, C. variegatus may exhibit low salt sensitivity over the normal

range of salinities encountered to reduce dependence of blood oxygen affinity on

environmental salinity (Weber et al., 1976). Second, the primary mechanism used to alter

Hb-02 affinity results from swelling of erythrocytes (Jensen, 1991; Wells and Weber,

1991; Jensen et al., 1993). Swelling of erythrocytes is normally accompanied by a rise in

cell pH and decreased red cell hemoglobin, NTP and ATP concentrations, all of which serve

to increase the oxygen affinity (Wells and Weber, 1991). However, as seen by the MCHC

and MCV values determined in this study, erythrocytes did not swell in response to





86


increased salinity in C. variegatus. Thus if Hb-O2 affinity is altered with changes in

environmental salinity, it must be changed in some other manner.

This study clearly indicates that salinity does influence the oxygen carrying capacity

of the blood of C. variegatus. Quantitative differences in hemoglobin concentration,

hematocrit, and erythrocyte count were noted in response to changing salinity. As discussed

above, it also seems likely that salinity may influence qualitative changes in blood oxygen

transport in C. variegatus. Further research is needed to better understand the influence of

salinity on blood oxygen levels in euryhaline teleosts.













CHAPTER 6
SUMMARY AND CONCLUSIONS

This study examined costs associated with life of a teleost in a variable salinity

environment, represented here by a salt marsh. Cyprinodon variegatus was used to examine

the influence of salinity on routine metabolic rate (RMR), critical oxygen tension (Pc),

osmoregulation, and blood oxygen carrying capacity. Results are summarized below.

1) Field measurements in the Cedar Key salt marsh indicated that this habitat undergoes

extensive variation in salinity, temperature, and oxygen.

2) RMR was relatively constant over a range of salinities from 0 ppt to 40 ppt. At higher

salinities RMR began to decline, and was significantly depressed under hypersaline

conditions.

3) Following sequential acclimation to experimental salinities, PC was unaffected by

changes in salinity between 0 ppt and 40 ppt, with Pc increasing at higher salinities.

4) Reduction in metabolism and rise in Pc corresponded well with a reduced ability of C.

variegatus to regulate plasma osmolality efficiently. Osmotic permeability of the gills may

be reduced at high salinities to offset osmotic losses or ionic gains to/from the environment,

indirectly reducing the potential for oxygen uptake as well.

5) Variations in RMR and Pc as a function of environmental salinity observed in this study

suggest that C. variegatus responds to high salinities by reducing energy expenditures.

These responses effectively increases the time C. variegatus can tolerate such conditions,

albeit at a cost of a reduction in energetic processes. This strategy fits the concept of scope

for survival, as described by Hochachka (1990).

6) When C. variegatus was exposed to simulated tidal changes in salinity, RMR was

unaffected in salinity trials where both acclimation and final salinities were in the range








typically encountered by this population in its native habitat. Where the acclimation or final

salinities were extremely high (50 and 60 ppt) or extremely low (0 ppt), RMR was

depressed.

7) Acclimation state was the most important factor determining the metabolic response to

simulated tidal changes in salinity. However, direction of the salinity change also influenced

metabolism in C. variegatus, with increasing salinity dealt with more efficiently than

decreasing salinity.

8) Simulated tidal experiments corroborate the hypothesis that C. variegatus tolerates

extremes in salinity by lowering metabolism, and hence decreasing energy expenditures.

Following adverse conditions metabolism returns to normal levels.

9) Cyprinodon variegatus is an excellent regulator of plasma osmolality even when

exposed to large fluctuations in salinity within the range of salinities typically encountered.

Daily fluctuations in salinity of up to 30 ppt elicited no significant differences in

osmoregulatory ability when compared to control fish.

10) Prior exposure to fluctuations in salinity does impart an osmoregulatory advantage.

Fishes previously exposed to large fluctuations in salinity regulated plasma osmolality

better than fishes that had previously experienced no or small changes in salinity.

Increasing salinity had a greater impact on regulation of plasma osmolality than did

decreases in salinity.

11) Salinity had a significant effect on blood oxygen carrying capacity in C. variegatus,

although differences were only noted at the very highest (60 to 80 ppt) and lowest (0 ppt)

salinities tested. Oxygen carrying capacity and all blood indices were highest in the group

acclimated to 0 ppt.

12) C. variegatus exhibited little change in oxygen carrying capacity over the range of

salinities between 2 ppt and 50 ppt. Possible reasons for this include: (a) increases in

salinity may require little compensation in oxygen carrying capacity; (b) detectable changes

may only occur when salinity is varied in conjunction with variations in oxygen and/or








temperature; (c) C. variegatus may utilize multiple hemoglobins, and/or; (d) the primary

mechanism to increase oxygen carrying capacity may instead be through adjustment of Hb-

02 affinity.

13) Erythrocyte count was the most consistent and hematocrit the least consistent measure

of the influence of salinity on blood oxygen level.

Competition or predation pressure may be less intense in harsh, fluctuating

environments, and that certain species may avoid these pressures by evolution of wide

physicochemical tolerances and the use of such environments (Matthews and Styron,

1981). Cyprinodon variegatus seems to fit this mold well, as this species appears to be a

generalist that very successfully inhabits harsh and variable habitats where it does not have

to be very efficient to compete with other species of fishes (Martin, 1972; Berry, 1987).

This argument may explain why C. variegatus does not invade freshwater in more locales,

and why they are not very abundant in most freshwater systems; except in south Florida

freshwaters where temperature and dissolved oxygen are variable and extreme.

Furthermore, it is believed that C. variegatus has conquered its wide geographic

range by physiological flexibility rather than by local accommodation in physiology or life

history (Berry, 1987). This hypothesis remain to be rigorously tested, although this study

lends further evidence for the physiological flexibility of C. variegatus. Examination of the

physiological ability of other populations of C. variegatus may help to resolve this issue.

The success of a fish species in a stressful environment may depend primarily

upon the proportion of the population that survives the adverse conditions, with even short-

term increases in tolerance significant (Matthews and Styron, 1981). This hypothesis

appears to be relevant to the results of the present study. Cyprinodon variegatus is well

adapted to a varying salinity environment. Its metabolic rate, Pc, and ability to efficiently

osmoregulate is unaffected by changes in salinity over the typical range encountered, even

when salinity is changed very rapidly. Furthermore, C. variegatus appears to tolerate

extremes in salinity by decreasing energy expenditures, waiting for conditions to improve,




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