I 1:1
EFFECTS OF SALINITY AND TEMPERATURE ON THE RESPIRATORY
PHYSIOLOGY OF THE DUNGENESS CRAB, CANCER MAGISTER,
DURING DEVELOPMENT
by
ANNE CHRISTINE BROWN
A DISSERTATION
Presented to the Department of Biology
and the Graduate School of the University of Oregon
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
March 1991
-iii
An Abstract of the Dissertation of
Anne Christine Brown for the degree of Doctor of Philosophy
in the Department of Biology to be taken March 1991
Title: EFFECTS OF SALINITY AND TEMPERATURE ON THE RESPIRATORY
PHYSIOLOGY OF THE DUNGENESS CRAB, CANCER MAGISTER,
DURING DEVELOPMENT
Ap~roved:
Cancer magister, the Dungeness crab, occurs in
different habitats during its life cycle, habitats which
vary widely in the magnitude of salinity and temperature
changes. Cancer magister hemocyanin also changes in
structure and oxygenation properties during development. The
following question was considered in this thesis: what are
the effects of environmental salinity and temperature on
metabolic rates, ionic and osmotic regulation and hemocyanin
oxygen affinity in Q. magister during development.
Metabolic rates and hemolYmph ionic and osmotic
concentrations were measured in the megalopa, 1st juvenile,
5th juvenile and adult crab eight hours after acute exposure
to 100% seawater (=32 ppt), 75% seawater and 50% seawater at
both 10°C and 20°C. The oxygen binding properties of the
whole hemolymph from these stages in 100% seawater at 10°C
iv
was determined. The effects of calcium and magnesium on the
oxygen affinity of purified hemocyanin from different stages
were also determined.
In 100% seawater, routine metabolic rates of the four
stages scale with body mass over the size range, 0.05 gm to
500 gm. The Q10 (10°C to 20°C) for the megalopa is higher in
75% seawater and 50% seawater than in 100% seawater. For the
1st juvenile, 5th juvenile and adult the Q10 values (10°C to
20°C) are independent of salinity. The megalopa, 1st
juvenile and 5th juvenile are weaker regulators of hemolymph
chloride, sodium and osmotic concentrations than the adult.
The megalopa and adult, unlike the 1st juvenile and 5th
juvenile, strongly regulate hemolymph calcium in reduced
salinity. In 100% seawater hemolymph magnesium is
significantly higher in the megalopa, 1st juvenile and 5th
juvenile than in the adult. The oxygen affinities of whole
hemolymph from the four stages are indistinguishable when
adjusted for endogenous L-lactate concentrations; the Bohr
coefficients are not significantly different among stages.
The effect of magnesium on oxygen affinity of purified adult
hemocyanin is influenced by proton concentration; the effect
of calcium is independent of proton concentration. In 100%
seawater, endogenous inorganic ion concentrations in the
whole hemolymph of the various stages reduce the intrinsic
stage specific differences in hemocyanin oxygen affinity.
vVITA
NAME OF AUTHOR: Anne Christine Brown
PLACE OF BIRTH: Denver, Colorado
DATE OF BIRTH: October 4, 1962
GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED:
University of Oregon
Bowdoin College
DEGREES AWARDED:
Doctor of Philosophy, 1991, university of Oregon
Bachelor of Arts, 1984, Bowdoin College
AREAS OF SPECIAL INTEREST:
comparative Physiology
Invertebrate Zoology
PROFESSIONAL EXPERIENCE:
Graduate Teaching Fellow, Department of Biology,
University of Oregon, Eugene and Charleston, 1985-1991
American Heart Association Summer Research Fellow,
Department of Biology, Bowdoin College, Brunswick,
Maine, 1985
Teaching Fellow, Department of Biology, Bowdoin College,
Brunswick, Maine, 1984-1985
Research Assistant, Department of Chemistry, Bowdoin
College, Brunswick, Maine, 1984
vi
AWARDS AND HONORS:
Lerner-Gray Fund for Marine Research, 1989
American Heart Association Fellowship, 1985
PUBLICATIONS:
Brown, A. C., Terwilliger, N. B. and Terwilliger, R. C.
(1988). Effects of salinity on osmotic concentration
and oxygen binding of Cancer magister hemolYmph. IUBS,
2nd International Congress of Comparative Physiology
and Biochemistry.
Brown, A. C. and Terwilliger, N. B. (1989). Developmental
changes in osmotic and ionic regulation in the
Dungeness crab, Cancer magister. Am. Zool. 29, 156A.
Brown, A. C. and Terwilliger, N. B. (1990). Developmental
changes in the oxygen binding of whole hemolymph in the
Dungeness crab, Cancer magister. Am. Zool. 30, 93A.
vii
ACKNOWLEDGEMENTS
I wish to express my deepest appreciation for the
guidance, encouragement and support of my advisor, Dr. Nora B.
Terwilliger and my mentor, the late Dr. Robert C. Terwilliger.
This investigation was supported in part by a grant from the
National Science Foundation, number DCB 89-08362, to Dr. Nora
B. Terwilliger, and a grant from the Lerner-Gray Fund for
Marine Research, to the author.
viii
DEDICATION
To the memory of Dr. Robert C. Terwilliger: teacher,
scholar and friend
Chapter
ix
TABLE OF CONTENTS
Page
I. INTRODUCTION . 1
Estuarine and Ocean Habitats............. 1
Responses to Environmental Change........ 2
Hemocyanin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Cancer magister.......................... 10
II. OXYGEN CONSUMPTION.•..••.•..•.............. 14
Introduction............................. 14
Materials and Methods.................... 17
Results 24
Discussion............ .. . 35
III. IONIC AND OSMOTIC REGULATION••....•........ 45
Introduction............................. 45
Materials and Method..................... 47
Results. . . . . . . . . . . . . .. . . . . . . . . . . . . . . 50
Discussion. .. . . .. . . . . . .. . . ... . . . . . . . . . 72
IV. OXYGEN BINDING PROPERTIES OF WHOLE
HEMOLYMPH AND PURIFIED HEMOCyANIN . 80
Introduction............................. 80
Materials and Methods.................... 82
Results.. . . . .. . . .... . .. . ... . . . . . . 89
Discussion.. .. . .. .. . .. .. . .. . . . . . . 129
V. INTEGRATION OF OXYGEN CONSUMPTION, ION
REGULATION AND HEMOCYANIN FUNCTION . 140
Introduction 140
Calculations 141
Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
VI. CONCLUS ION ...•••.•.•..•...•.......•........ 165
REFERENCES. . . . . • . . . • • • . . • • . . • . • . • . . . . . . . . . . . . . . . . 172
Figure
2.
3.
4.
5.
6.
1.
2.
3.
4.
5.
LIST OF FIGURES
CHAPTER II
Schematic of the Timing of Measurement
of Oxygen Consumption and
Aeration Intervals .
weight Specific Rate of Oxygen consumption
of~. magister Megalopae .
Weight specific Rate of Oxygen Consumption
of~. magister 1st Juveniles .
Weight specific Rate of Oxygen Consumption
of~. magister 5th Juveniles .
Weight Specific Rate of Oxygen Consumption
of~. magister Adults .
Relationship Between log Weight specific
Rate of oxygen Consumption and log of
Wet Weight in~. magister .
CHAPTER III
Hemolymph Osmolality of~. magister .
Hemolymph Chloride Ion Concentration
of ~. magister .
Hemolymph Sodium Ion Activity
of ~. magister .
Hemolymph Potassium Ion Activity
of ,Q. magister .
Hemolymph Magnesium Ion Concentration
of ~. magister .
Page
21
26
28
30
32
36
51
55
58
62
66
x
6. Hemolymph Calcium Ion Activity
of .Q. magister .
CHAPTER IV
xi
69
1. The Oxygen Affinity of ~. magister Whole
Hemolymph with Endogenous L-lactate and
Inorganic Ions............................ 92
2. The cooperativity of ~. magister Whole
Hemolymph with Endogenous L-lactate and
Inorganic Ions............................ 94
3. The Oxygen Affinity of ~. magister Whole
Hemolymph with log Po Values Adjusted
to 0.05 mmol/L L-lac~ate.................. 96
4. The Effect of Magnesium on Oxygen Affinity
of ~. magister Fresh Adult 258 Hemocyanin. 98
5. The Relationship Between log Pso andMagnesium Concentration for ~. magister
Fresh Adult 258 Hemocyanin at pH 7.8 101
6. The Effect of Magnesium on the Cooperativity
of ~. magister Fresh Adult 258 Hemocyanin. 103
7. The Effect of Magnesium on the Oxygen
Affinity of ~. magister Fresh and Frozen
258 Hemocyanin............................ 106
8. The Effect of Magnesium on the Cooperativity
of ~. magister Fresh and Frozen 258
Hemocyanin.. . .. ... .. ... .. ... .. ... ... . . . . . . 108
9. The Effect of Magnesium on the Oxygen
Affinity of ~. magister Frozen Adult 258
Hemocyanin and Frozen 1st Juvenile 258
Hemocyanin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
10. The Effect of Magnesium on the Cooperativity
of ~. magister Frozen Adult 258 Hemocyanin
and Frozen 1st Juvenile Hemocyanin •....... 112
11. The Effect of Calcium on the Oxygen
Affinity of ~. magister Frozen Adult 258
Hemocyanin .....•.................... .0. . . • . 115
xii
12. The Relationship Between log Pso and logCalcium Concentration for ~. magister
Frozen Adult 25S Hemocyanin at pH 7.8 ..... 117
13. The Effect of Calcium on the Cooperativity
of ~. magister Frozen Adult 25S
Hemocyanin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 119
14. The Effect of Calcium on the Oxygen Affinity
of ~. magister Frozen Adult 25S Hemocyanin
and Frozen 1st Juvenile 25S Hemocyanin .... 122
15. The Effect of Calcium on the Cooperativity
of ~. magister Frozen Adult 25S Hemocyanin
and Frozen 1st Juvenile 25S Hemocyanin .... 124
16. The Effect of stage Specific Salines on
the Oxygen Affinity of ~. magister Fresh
Adult 25S Hemocyanin and Fresh 1st
Juvenile 25S Hemocyanin 127
17. The Effect of stage Specific Saline on the
cooperativity of ~. magister Fresh Adult
25S Hemocyanin and Fresh 1st Juvenile 25S
Hemocyanin. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 130
CHAPTER V
1. Estimated Cardiac output for Adult
,Q.magister................................. 143
2. Estimated Cardiac output for ~. magister
Megalopa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
3. Estimated Cardiac output for ~. magister
1st Juvenile.............................. 151
4. Estimated Cardiac output for ~. magister
5th Juvenile.................... . . . . . . . . . . 153
5. The Relationship of log QH and log Wfor Four Life Stages of~. magister 155
xiii
LIST OF TABLES
Table
1.
2.
1.
2.
CHAPTER II
Weight Specific Metabolic Rates and Q10
Values for Four Life stages of ~. magister.
Regression Equations for log Q10 vs. log W.....
CHAPTER III
Composition of Seawater Treatments .
Hemolymph Osmolality of Megalopae, 1st
Juvenile, 5th Juvenile
and Ad~lt~. magister .
Page
34
35
48
53
3. Hemolymph Chloride Concentration of Megalopa,
1st Juvenile, 5th Juvenile and Adult
.Q. magister................................ 57
4. Hemolymph Sodium Ion Activity of Megalopa,
1st Juvenile, 5th Juvenile and Adult
.Q. magister................................ 60
5. Hemolymph Potassium Ion Activity of Megalopa
1st Juvenile, 5th Juvenile and Adult
~. magister................................ 64
6. Hemolymph Magnesium Concentration of Megalopa
1st Juvenile, 5th Juvenile and Adult
g. magister................................ 68
7. Hemolymph Calcium Ion Activity of Megalopa
1st Juvenile, 5th Juvenile and Adult
~. magister . 71
xiv
CHAPTER IV
1. Formulae for stage Specific Salines for
100% Seawater/10°C Conditions.............. 85
2. Formulae for Saline containing 0, 16, 32 and
100 mmol/L Magnesium.................. ..... 87
3. Formulae for Saline containing 0, 16, 32 and
100 mmol/L Calcium..................... . . . . 88
4. Hemocyanin Concentrations and Oxygen carrying
Capacities of Four Stages of g. magister... 90
5. Regression Equations for log Pso vs. pH forAdult g. magister Fresh 25S Hemocyanin
in Saline with Varying Magnesium
Concentration 100
6. Regression Equations for log Pso vs. pH forAdult Frozen 25S Hemocyanin from g.
magister in Salines with Varying
Calcium Concentration...................... 114
7. Cooperativity of Frozen Adult 25S Hemocyanin
at Different Calcium Ion Concentrations .... 121
8. Regression Equations for log P~ vs. pH for
g. magister Fresh 1st Juvenlle and
Fresh Adult 25S Hemocyanins in
stage Specific Salines 126
CHAPTER V
1. Cardiac Output of Four Life Stages of
~. magister................................ 146
2. Regression Equations for log QH vs. log W..... 147
1CHAPTER I
INTRODUCTION
The various processes by which organisms adapt to their
environment have fascinated biologists for a long time.
Contemporary studies of adaptational biology, spanning the
range from molecules to organisms, are reviewed by Hochachka
and Somero (1984) and by Prosser (1986). Adaptation to the
environment may involve changes in gene expression,
physiological processes and behavior. These responses can be
caused by environmental cues or as part of a genetically
determined developmental process which coincides with
changes in mode of existence or habitat.
Estuarine and Ocean Habitats
Chemical and physical parameters in estuarine waters
are highly variable. There are spatially and temporally
predictable changes in the chemical and physical environment
in the estuary related to tidal, diurnal and seasonal
patterns and to the physical structure of the estuary
(Newell, 1976). Morris and Taylor (1983), for example,
measured salinity, temperature, pH and partial pressure of
oxygen (p02) in a temperate intertidal area. Large diurnal
fluctuations in all of these physical and chemical
2parameters were observed. In contrast to highly variable
estuarine areas the ocean is a more stable habitat with
regard to temperature, salinity, p02 and pH.
Responses to Environmental Changes
General
There are many ways in which planktonic and benthic
animals within the estuary cope with changes in
environmental conditions, including behavioral,
morphological and physiological mechanisms. Behaviorally,
motile organisms can move to evade unfavorable conditions:
i.e. seasonal migrations, diurnal and tidally linked
activity patterns and selection of suitable microhabitats.
Reynolds and Casterlin (1979) found that Homarus americanus
is capable of behavioral thermoregulation in the lab. Non-
motile organisms such as bivalves, snails and tube dwelling
worms, can reduce or cease activity for varying lengths of
time to avoid or reduce the effects of deleterious
environmental conditions. These organisms can also take
advantage of their relatively impermeable shells and tubes
to seal themselves in when they are exposed to stressful
conditions.
Physiologically, organisms have two options for dealing
with environmental changes: regulating their internal
conditions or conforming to whatever is changing around
tnem. Estuarine invertebrates vary greatly in their ability
3to either regulate or tolerate changes in ambient salinity.
Regulation of internal ionic and osmotic concentrations can
be energetically costly, requiring the active pumping of
ions. However, passively letting the internal ionic and
osmotic concentrations vary may exceed the tolerance of the
molecular and cellular processes in the animal.
Estuarine invertebrates must conform to ambient
temperature and are unable to regulate their body
temperature except by behavioral mechanisms, such as moving
to and selecting particular microhabitats. It is possible
for a mobile animal to move to a less stressful place but
the effectiveness of this is limited by how far and how fast
the animal can move. By conforming to environmental
temperature changes, the animal's metabolic systems may
become so stressed that the ability to transport sufficient
oxygen and maintain physiologi~al processes is surpassed.
Different stages in the life cycle of a species are in
many respects like separate types of animals, especially in
those species with distinctly different larval types. There
are many crustacean species with benthic adults and
planktonic larval stages. The different stages may encounter
very different magnitudes of change in environmental
\
conditions. Survival through the larval stages has long been
considered a critical point for many marine invertebrates.
In this light, many researchers have examined the effects of
various salinities and temperatures on the survival, growth,
4development time and metabolic rates of the larvae of
numerous species of crustaceans (reviewed in sastry, 1983).
Oceanic planktonic larvae, although many are mobile and
migrate vertically, tend to move within a large and
relatively uniform water mass. In this situation they tend
not to be exposed to large or sudden changes in either
salinity or temperature. Planktonic larvae may encounter
changes in salinity and temperature as they move within the
water column in the estuary. Estuaries typically have an
outward flowing freshwater lens at the surface and an inward
flowing salt wedge at the bottom. Benthic estuarine animals
are exposed to large and rapid changes in salinity and
temperature with each change of the tides.
Crustaceans
There are a number of investigations of the effects of
environmental change on many aspects of the biology of
estuarine and intertidal animals (reviewed in Newell, 1979).
A group which has been the focus of a great deal of research
is the decapod Crustacea. Decapods are relatively large
animals and a number of species occur in high abundance in
estuaries. Many estuarine decapods are of great commercial
value as well as being ecologically important species as
predators and scavengers.
Two physical parameters of particular importance to
estuarine organisms are salinity and temperature. Changes in
5environmental temperature often have a direct effect on
oxygen uptake in crustaceans (reviewed in Vernberg, 1983). A
decrease in environmental temperature generally reduces the
rate of oxygen consumption; conversely, increases in
temperature cause an increase in metabolic rate. Changes in
environmental salinity affect crustacean ionic and osmotic
regulation (reviewed in Mantel and Farmer, 1983). Some
species, hyperosmoregulators, are able to strongly regulate
hemolymph osmolality above the ambient level in reduced
salinity. Those species which maintain hemolymph osmolality
below ambient levels are hypoosmoregulators. There are also
species which are able to maintain hemolymph osmolality
above ambient levels but as ambient salinity changes the
hemolymph changes in parallel; these are
hyperosmoconformers. Salinity changes also affect
respiratory physiology in terms of oxygen uptake (oxygen
consumption of the whole animal) via such processes as
ventilation, gas exchange, permeability, oxygen transport by
the respiratory protein and oxygen utilization by the
tissues (reviewed in Kinne, 1964; Cameron and Mangum, 1983;
Wheatly, 1988). Kinne (1964) described four types of
metabolic response to changes in environmental salinity. In
type 1 response, the metabolic rate increases in salinity
less than the normal range and/or decreases in salinity
higher than normal. In type 2 response, the metabolic rate
increases in salinities above or below the normal salinity
6range. In type 3 response, the rate decreases in salinities
above or below the normal salinity range, and in type 4
response, the metabolic rate is not affected by changes in
salinity. Temperature and salinity may have interactive
effects in a variety of ways, influencing the range of
tolerance to salinity and temperature, as well as survival,
growth, development and metabolic rates (reviewed in Kinne,
1964; Vernberg, 1983). Typically, salinities below normal
decrease the tolerance to increased temperature, and above
normal salinities increase temperature tolerance (Kinne,
1964).
Hemocyanin
As described above the estuary is a stressful and
variable environment. The ability to maintain adequate
oxygen transport under many different environmental and
physiological conditions is of primary importance for
survival in any animal. The oxygen transport molecule
utilized by the decapod crustaceans is the copper containing
respiratory protein, hemocyanin. This extracellular protein
circulates in the hemolymph. Both hemolymph and hemocyanin
are at an important interface between the organism and the
environment and are also important in the animal's internal
transport of metabolites. The structure of the arthropod
hemocyanin molecule and the reversible binding of oxygen to
hemocyanin have been studied in detail (see Bonaventura and
7Bonaventura, 1980; Mangum, 1980; Van Holde and Miller, 1982;
McMahon, 1985; Markl, 1986 for reviews). Arthropod
hemocyanins have molecular weights ranging from 450
kilodaltons (kD) to 3,200 kD. The smallest polymeric unit
found in the hemolymph is a hexamer with a molecular weight
of 450 kD. The hexamers are comprised of 75 kD subunits;
each subunit contains two copper atoms and combines
reversibly with one molecule of oxygen. The hexamers may
aggregate into mUltiples of 2, 4, 6 and 8.
certain subunits have been shown to play distinct
structural roles within the polymeric molecules. Some are
important in linking the hexamers and others appear to be
important within each hexamer (Decker et al., 1989).
The hemocyanin of adult brachyuran crabs is typically
composed of a two-hexamer aggregation with a molecular
weight of approximately 900 kD made up of a species specific
heterogeneous combination of subunits. The sedimentation
coefficient of the two-hexamer is approximately 25S. A small
amount of single hexamer, 16S, hemocyanin molecules is
usually present in the hemolymph of adult crabs.
Interspecific comparison of adult crab hemocyanins
reveals a complex pattern of hemocyanin subunit composition
(Markl, 1986; Markl et ale 1986). Evidence has been
presented that the differences in subunit composition in
crab hemocyanin may playa role in oxygen binding function.
Subunits from Cancer magister hemocyanin have been
8artificially reaggregated into 25S molecules which have
different oxygen binding properties than the native 25S
hemocyanin (Graham, 1983). In Callinectes sapidus oxygen
affinity of hemocyanin from estuarine and ocean populations
is different and this functional difference appears to
correlate with differences in subunit composition (Mangum
and Rainer, 1988).
Not only is there variation in subunits between
crustacean species, there are also ontogenic changes in
hemocyanin subunit composition in those species that have
been studied, Cancer magister (Terwilliger and Terwilliger,
1982; Terwilliger et al., 1986), Cancer productus (Wache et
al., 1988), Hyas araneus and Carcinus maenas (Markl, 1986)
and Homarus americanus (Olson et al., 1988). The changes in
~. magister hemocyanin will be discussed in more detail in
the next section.
Studies of the effects of hemolymph organic and
inorganic factors and of salinity and temperature on the
oxygen equilibrium properties of hemocyanin from a number of
adult crustaceans have been reviewed recently (Mangum, 1980,
1983; Van Holde and Miller, 1982; McMahon, 1985). Hemocyanin
oxygen affinity and cooperativity are dependent on pH;
within the physiological pH range there is a normal Bohr
shift (Mangum, 1983). The magnitude of the Bohr shift varies
between species. Oxygen affinity increases as temperature
decreases (Johansen et al., 1970; Truchot, 1973, 1975; Mauro
9and Mangum, 1982; Burnett et al., 1988; Morris and Bridges,
1989). Environmental salinity usually affects crustacean
hemolYmph salinity which in turn alters hemocyanin function
in the hemolYmph. Oxygen affinity decreases at low salinity
(Truchot, 1973, 1975; Weiland and Mangum, 1975; Mangum and
Towle, 1977) and/or increases after the crustacean
acclimates to higher salinity (Taylor et al., 1985). This
response is variable between species and acclimation times.
These salinity effects are often mediated by changes in
specific ion concentrations, especially calcium and
magnesium, in the hemolymph (Taylor et al., 1985; Morris et
al. 1988). Calcium and magnesium have been shown to have a
strong effect on the oxygen equilibrium of the hemocyanin
from several crustacean species (Larimer and Riggs, 1964;
Truchot, 1975). There are also several effectors or
modulators of hemocyanin oxygen equilibria which are organic
molecules. Truchot (1980) identified L-Iactate as a factor
increasing oxygen affinity in Carcinus maenas. L-Iactate has
subsequently been identified as a factor in a variety of
crustaceans (Booth et al., 1982; Graham et al., 1983;
Mangum, 1983b; Bridges et al., 1984; Taylor et al., 1985;
Bridges and Morris, 1986; Morris and Bridges, 1989). Urate
has also been identified as an effector of oxygen binding of
hemocyanin from the crayfish Austropotamobius pallipes
(Morris et al., 1985: Morris et al., 1986). Morris and
McMahon (1989 a,b) describe a potentiating effect of
10
dopamine on oxygen affinity of hemocyanin from Cancer
magister.
There are to date only two published studies of larval
hemocyanin function (Terwilliger et al., 1986; Olson et al.,
1989). The physiological significance and timing of the
ontogenic structural and functional changes in crustacean
hemocyanin are unknown.
Cancer magister
Cancer magister, the Dungeness crab, is a species which
uses different portions of the estuary as well as coastal
waters throughout its life cycle. The embryos, which are
attached to the pleopods of the female, hatch in December
through April in coastal Oregon waters. The newly hatched
larvae go through five zoeal stages, all of which are
planktonic in ocean waters. The final larval stage is the
megalopa, which is also planktonic. The megalopa is an
extremely active swimmer. From mid-April through the
beginning of July megalopae enter the coastal and estuarine
waters of Oregon. The precise mechanism of transport from
oceanic to coastal waters is not clear (Lough, 1975). After
the megalopae appear on-shore, they soon metamorphose into
1st instar juvenile crabs and join the benthic community
within the bay and in the nearshore areas. The juvenile
crabs molt frequently through their first summer and are
found in large numbers on the tideflats. Cancer magister
11
reaches sexual maturity at 2-3 years, approximately 10 cm
carapace width (MacKay, 1942; Butler, 1961; wild and Tasto,
1983). The adult crabs are found mainly in the deeper
channels in the estuary and in the nearshore coastal waters.
Due to their distribution and utilization of different
habitat types, the megalopae, juveniles and adults of Cancer
magister are exposed to different magnitudes of change in
environmental salinity and temperature. Since the megalopae
are planktonic and tend to move with the water mass in which
they enter the bay, the changes in temperature and salinity
to which they are exposed are not likely to be extreme. In
contrast, the late spring and early summer low tides in Coos
Bay occur in the morning and rapid solar heating of the
tideflats occurs. salinity on the tideflats is also low when
the tide is out because the lens of fresh water on the
surface passes down the flats as the tide recedes. Therefore
the juveniles on the tideflats are exposed to extreme
fluctuations in salinity and temperature. The adult crabs
inhabit the subtidal portions of the bay which vary little
in temperature over the tide cycle and vary far less in
salinity than the tideflats in summer months. In the winter
months the salinity of the channels changes widely with the
tide because of the increased fresh water input from rain.
Along with the changes in mode of existence, habitat
and concomitant variations in environmental conditions
outlined in the preceding paragraphs there are differences
12
in the respiratory protein, hemocyanin, during the life
cycle of ~. magister (Terwilliger and Terwilliger, 1982).
Adult ~. magister hemocyanin is predominately in the form of
258 (two-hexamer) assemblages of 58 subunits with a trace of
168 (single hexamer) molecules. The larval and juvenile
hemocyanin is predominately in the 168 form. The larval and
juvenile hemocyanin is lacking one of the subunit types
found in the adult. The stoichiometry of the other subunits
changes as the crabs develop from the juvenile to the adult.
There are also differences in hemocyanin function in
purified samples from these different stages (Terwilliger et
al., 1986). The oxygen affinity of the juvenile is less than
that of the adult, under the same experimental conditions.
Both the juvenile and adult hemocyanin have essentially the
same sensitivity to L-lactate and pH (Terwilliger et al.,
1986). Lactate is an end product of anaerobiosis in ~.
magister and has been shown to affect hemocyanin oxygen
binding (Graham et al., 1983). Cancer magister hemocyanin
has also been shown to be sensitive to dopamine (Morris and
McMahon, 1989a,b).
Many aspects of the physiology and ecology of ~.
magister have been studied in depth, including respiratory
physiology of the adult (Johansen et al., 1970; McDonald et
al., 1980), the structure and function of adult hemocyanin
(Larson et al., 1981; Ellerton et al., 1970; Wajcman et al.
1977; Graham et al., 1983), ionic and osmotic regulatory
13
abilities of the adult (Jones, 1941; Alspach, 1972;
Engelhardt and Dehnel, 1973; Hunter and Rudy, 1975) and
larval transport (Lough, 1975, 1976). Larvae have been
reared in the lab and optimum salinity and temperature
conditions for development and growth have been determined
(Reed, 1969), however the ontogeny of ionic and osmotic
regulation is unknown. In addition, the respiratory response
of adult Q. magister to changes in environmental salinity
and temperature are unknown.
The Dungeness crab, Cancer magister, is an ideal animal
in which to study the ontogeny of response to environmental
change at the whole animal and at the molecular and
physiological levels. Adult Q. magister are large and
abundant in the Coos River estuary. The reproductive cycle
and life cycle of the species is well documented (MacKay,
1942; Butler, 1961; wild and Tasto, 1983) and larval and
juvenile stages are generally available in the field in a
predictable seasonal pattern.
The purpose of the present study is to examine the
effects of short term, tidal changes (6-8 hr) of
environmental salinity and temperature on metabolic rates,
ionic and osmotic regulation and hemocyanin oxygen affinity
in Q. magister during development. This study is unique in
that not just the whole animal response of different
developmental stages are examined but also some internal and
molecular responses are elucidated.
14
CHAPTER II
OXYGEN CONSUMPTION
Introduction
studying the rate of oxygen consumption under different
environmental conditions provides insight into both the
physiological capacities and limitations of a species,
physiological limitations which may restrict the
distribution of a species to particular habitats or
geographical ranges. Measuring the rate of oxygen
consumption of an organism is a method of determining its
metabolic rate. Other methods include calorimetry, the
measurement of total heat production of the organism, and
calculations of the difference in energy in the food
ingested and the energy value of the excreta (primarily
feces and urine). For organisms which are primarily
respiring aerobically, the measurement of oxygen consumption
is a suitable estimation of metabolic rate.
Temperature and salinity are both dominant
environmental factors affecting metabolism in estuarine
invertebrates. salinity and temperature change with every
tidal cycle in the estuary.
There are data available on metabolic response to
salinity and/or temperature for adults of several species of
15
decapod crustaceans. These include Hemigrapsus nudus and
Hemigrapsus oregonensis (Dehnel, 1960), Panopeus herbstii
(Dimock and Groves, 1975), Carcinus maenas (Taylor, 1977),
Callinectes sapidus (Findley et al., 1978), Cancer magister
(Prentice and Schneider, 1979), Cambarus acuminatus (Pruitt
and Dimock, 1979), Palaemon elegans (Morris and Taylor,
1985) and Palaemonetes antennarius (Dalla Via, 1987). This
topic has also been reviewed by Scholander et ale (1953),
Kinne (1964) and Vernberg (1983). Generally, oxygen
consumption increases with increasing temperature. Q10
values for many crustacean species range from around 1.0 to
as high as 4.0. A Q10 of 1.0 indicates thermal
insensitivity, whereas a Q10 of 4.0 means that the rate of
oxygen consumption quadruples with a ten degree increase in
temperature. Q10 values tend to be lower at lower
temperatures, but may also vary with acclimation
temperature. Kinne (1964) outlines four different metabolic
responses to salinity: type 1, metabolic rate increases in
salinity less than the normal range and/or decreases in
salinity higher than normal; type 2, the metabolic rate
increases in salinities above or below the normal salinity
range; type 3, the rate decreases in salinities above or
below the normal salinity range, and type 4, the metabolic
rate is not affected by changes in salinity.
Data on the effects of salinity and/or temperature on
oxygen consumption rates of early life stages of decapod
16
crustaceans are also available, including studies on Uca
spp. (Vernberg and Costlow, 1966), Emerita talpoida and
Libinia emarginata (Schatzlein and Costlow, 1978), Cancer
irroratus (Sastry and McCarthy, 1973; Sastry, 1978, 1979),
Cancer borealis (Sastry and McCarthy, 1973), Cancer
productus and Panulirus interruptus (Belman and Childress,
1973), Pagurus criniticornis (Vernberg et al., 1981),
Macrobrachium holthuisi (Moreira et al., 1980) and Emerita
brasiliensis (Moreira et al., 1981). The temperature and
salinity sensitivity of metabolic rates of different
developmental stages may vary greatly. Changes in
sensitivity to temperature and salinity between the life
stages in a given species tend to correspond to such
ecological factors as changes in habitat utilization or
seasonal shifts in environmental salinity and/or
temperature.
In areas with a semi-diurnal tide the duration of
exposure of estuarin~ organisms to potentially stressful
environmental conditions as a result of the tides may be as
long as 6-8 hours. Cancer magister megalopae, juveniles and
adults inhabit different portions of the estuary and are
therefore exposed to different combinations and extremes of
salinity and temperature.
The goal of this study was to determine the metabolic
responses of several life stages of ~. magister over the
range of salinity and temperature conditions each life stage
17
encounters. Determining the oxygen consumption rates under
naturally occurring combinations of salinity and temperature
for different instars, including the adult crab, will enable
us to better define ontogenic changes in physiological
capacities which may relate to habitat selection and
utilization throughout the life history of the species.
The rate of oxygen consumption of ~. magister
megalopae, 1st juveniles, 5th juveniles and adults were
measured over a period of a tidal cycle, eight hours, after
acute exposure to 100%, 75% and 50% seawater at 10°C and
20°C.
Materials and Methods
Animals
Cancer magister megalopae were collected from the
surface waters near the mouth of the Coos River estuary with
a dip net. Since the megalopae were usually in a premolt
condition and would molt within 48-72 hours of being
captured, they were used as soon as possible. During the
brief period the megalopae were maintained in the lab they,
were kept in running unfiltered aerated seawater and given
no food. Seawater was pumped from near the mouth of the Coos
River where salinity varies in the range of 30-33 ppt, and
temperature varies from 9-15°C. The high salinities and high
temperatures occur in the drier season, usually May-October.
Low salinities and temperatures are typical of the rainier
18
season, usually November-April.
Juvenile crabs were reared from field caught megalopae
in 10 gallon aquaria with running seawater and aeration.
They were fed shelled mussels, pieces of fish or squid 3 to
5 times a week. Food was not given for 24 hours prior to
experiments to ensure that the animals were in a post-
absorptive state and to reduce fouling of the respirometers.
Molt stage determination was based on time since most recent
molt and hardness of the carapace. Only individuals jUdged
to be in intermolt were used.
Adult male ~. magister larger than 12 cm in carapace
width were collected in the Coos River estuary next to the
deep water ship channel near the river mouth. Adult crabs
were maintained in large (1000 L.) holding tanks with
running seawater and aeration. The crabs were fed with
shelled mussels, pieces of fish or squid 2 to 3 time a week.
Food was withheld for 24-48 hours prior to experiments as
with the juveniles.
All stages were maintained in holding facilities
exposed to natural light/dark cycles and ambient Coos Bay
seawater salinity and temperature.
Measurement of Environmental Conditions
Measurements of salinity and temperature on the
tideflats and in the deeper channels near the tideflats were
made at different stages of the tide to determine the ranges
19
of salinity and temperature to use in laboratory
experiments. Temperature was measured with a field
thermometer (Fisher scientific). Water samples were brought
back to the lab and the osmolality determined using a Wescor
5500 vapor pressure osmometer. Throughout this thesis 100%
seawater refers to Coos Bay seawater at 32 ppt or -950
mOsmjkg.
Oxygen consumption measurements
Whole animal oxygen consumption was measured in closed
respirometers. Respirometers of 80 and 100 ml were used for
the megalopae and 1st juveniles. Respirometers of 250 and
400 ml were used for the 5th juveniles and 5.5 L
respirometers were used for the adult crabs. Seawater of the
appropriate salinity, 100%, 75% and 50% seawater, was made
using filtered seawater diluted with distilled water.
Salinity measurements were made with a refractometer
(American optical Co.). The desired temperature, 10°C or
20°C, was maintained for the entire measurement period by
immersing the respirometers in a thermostatted recirculating
water bath. The rate of decrease of oxygen concentration in
the sealed respirometer was measured with either a YSI
(Yellow Springs Instruments) Model 5739 oxygen probe and a
YSI Model 57 dissolved oxygen meter or a YSI Model 5420A
oxygen probe (stirring boot removed) connected to a YSI
Model 54 dissolved oxygen meter.
20
Animals were transferred from the holding aquaria into
the respirometers immediately prior to the experiments.
Animals remained in the respirometers for the entire eight
hour measurement period. All measurements were made during
natural daylight hours. The megalopa and 1st juvenile
metabolic rates were measured for periods of one hour
followed by one hour intervals to reoxygenate the water by
bUbbling air through the respirometers, except for the 5th _
7 th hours when there was a two hour aeration interval (Fig.
1A) .
The 5th juveniles were given air breaks within each one
hour measurement period in order to make sure that the
oxygen level in the respirometer was not limiting. within
each one hour period there were three separate ten minute
measurement periods interspersed with 15 minute intervals
during which air was bubbled through the respirometers (Fig.
1B). This pattern was repeated for each one hour measurement
period throughout the eight hour span, i.e. 0-1 hr, 2-3 hr,
4-5 hr and 7-8 hr as in Figure 1A.
The oxyge~ consumption of the adult was measured for
the first and last twenty minutes within each one hour
measurement period with a twenty minute aeration interval
(Fig. 1C). As with the megalopa, 1st juvenile and 5th
juvenile, oxygen consumption was measured for the period 0-1
hr, 2-3 hr, 4-5 hr and 7-8 hr (as in Fig. 1A) after acute
exposure to the desired salinity and temperature
21
Figure 1. Schematic of the timing of measurement of oxygen
consumption and aeration intervals.
(A) Measurement periods from 0 to 8 hr.
(B) Measurement periods within each one hour
period for 5th juveniles. (C) Measurement periods
within each one hour period for adults.
o
A
1 2 3 4 5
Duration. of. exposure {hr)
I
aIr
6
22
8
I
mr
I
BI.r
o
e
o
c.
10
20
25 35
Time (min)
•m.r
Time (min) 40
50 60
00
23
combinations.
Within each hour of oxygen consumption measurement, the
rates measured during the shorter subdivisions of the hour
were pooled to give the average rate of oxygen consumption
over that hour for that animal.
Wet weights for each individual were determined at the
end of each eight hour experiment. They were held face down
in order to drain as much water as possible from the
branchial chambers and blotted with paper towels until they
were no longer wet. Megalopae in the weight range of 0.033
gm to 0.055 gm (3 to 4 mm carapace width), 1st juveniles
from 0.065 gm to 0.125 gm (6 to 8 mm carapace width), 5th
juveniles from 2.53 gm to 5.43 gm (25 to 32 mm carapace
width) and adults from 278.7 gm to 495.4 gm (120 to 144 mm
carapace width) were used.
Data analysis
The effects of salinity and temperature on the
different stages were tested by analysis of covariance
(ANCOVA). MUltiple comparison of means were made using the
Tukey-Kramer method to determine the minimum significant
difference (MSD). statistical significance was accepted at
P < 0.05. statistical analyses were done using SYSTAT
version 4.1 (SYSTAT, Inc.)
24
Results
Measurement of field conditions of salinity and
temperature in estuarine areas where the different
developmental stages of ~. magister were abundant proved
useful in setting the range of these parameters for
laboratory studies. It was found that the mudflat
environment of the juveniles in the summer is exposed to
changes in temperature from 100 e when the tide is high to
25°e when the tide has receeded and the mudflats are
exposed. At the same time salinity drops from 32 ppt (100%
seawater) to 16 ppt (50% seawater) as the freshwater lens on
the surface passes down the flats. The channels where the
adults are found are much more stable with regard to summer
temperature and salinity. The winter range of salinity at
the bottom of the estuary varies nearly as widely as
salinity on the mudflats in the summer and winter.
Figures 2 through 5 show the weight specific oxygen
consumption rates of the four stages plotted against time
for the eight hour exposure to each of the combinations of
salinity and temperature. The Y-axis scale is different in
each figure. There is no significant change in the rate of
oxygen consumption during the eight hours of exposure to any
one combination of salinity and temperature for the megalopa
(Fig. 2) or 1st juvenile (Fig. 3). There is a small and
statistically insignificant decrease in rate of oxygen
consumption over time for the 5th juveniles (Fig. 4) and
25
adults (Fig. 5). This change in oxygen consumption is most
likely related to initial excitement and handling stress
from the transfer of the crabs from the aquaria to the
respirometer. To avoid any complications from this handling
stress effect all further comparisons of rates of oxygen
consumption are made between values for the final hour in
the eight hours of exposure.
The weight specific rates of oxygen consumption for
the final hour of exposure are given in Table 1. There is a
strong interactive effect of temperature and salinity on the
rate of oxygen consumption of the megalopa (Fig. 2). At
10DC, there is no significant effect of salinity on the rate
of oxygen consumption of the megalopa. At 20 DC, however, the
rate of oxygen consumption rises and is greater in 75% and
50% seawater than in 100% seawater. The interaction of
salinity and temperature is also apparent in the Q10 values
(Table 1) at the different salinities; the Q10 values are
higher at lower salinity for the megalopa.
The rates of oxygen consumption of the 1st juvenile
(Fig. 5), 5th juvenile (Fig. 6) and adult (Fig. 7) are not
affected by salinity at either 10DC or 20DC. There is an
effect of temperature on the rate of oxygen consumption in
these stages. The rate more than doubles (Ql0 > 2) with a
ten degree increase in temperature for both the 1st juvenile
and the adult (Table 1). The 5th juvenile is less sensitive
(Ql0 < 2) to the increase in temperature from 10DC to 20 DC.
26
Figure 2. Weight specific rate of oxygen consumption of Q.
magister megalopae. ~100% seawater at 10°C;
~ 100% seawater at 20°C; rs:=s::=sJ 75% seawater at
10°C; ts=:.:SI75% seawater at 20°C; fZZZj50% seawater
at 10°C; e:::=z:J 50% seawater at 20°C.
o
I
0 N
c Il
0 C.N
r-+
0
=:)
0
-+,
~
fTl Ix
-0 (.J1
0
(f)
C
l
(t)
~
:Y
l(j)
'-../
'-J
I
OJ
Rote of Oxygen Consumption
(ml 02/ gm/ hr)
0 0 ~ ~ N N 0J
. .
0 (Jl 0 (Jl 0 (Jl 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
I I I I I
xxxxxxxxx::m--;
././././././././././:;;t}-i
"'-"'-""'-"'-" ""-~
x::B-ix x x x x X
/ / ./ ./ ./ ./ ./ ./ ./ ./ ./
,"'-
"'- "'- "'- "'- "'- "'- "'- "'- "'-
xxxxxxxxx~
,/////.//////A+l
"'- "'- "'- "'- "'- "'- "'- "'- "'- "'- SB-i
x x x x x x x
/' / / / / / / / / / / /E--I
"'- "'- "'-
"
"'- "'- "'-
, ,
"-
xxxxxxxxx]B-I
///////////2&1
"'-"'-"'-"'-"'-" "'-"'-"'-::sIH
x x x x x x X
1/ / ./ / / / / / / / / /
-13--l
"'- "'- "'- "'- "'- "'- "'-
" "
"'- "'-
xxxxxxxx
//////////..&3-1
"''''''-'''-'''-'''-'''-'''-'''-'''-l3-I
x x x x x X x
/ / / ./ / / / / / ./ / /B-i
"'- "- "'- "'- "- "'- "'- "'- "'-
'"
28
Figure 3. Weight specific rate of oxygen consumption of £.
magister 1st juveniles. ~ 100% seawater at 10°C;
~ 100% seawater at 20°C; IT:SJ 75% seawater at
10°C; lS:::3l75% seawater at 20°C; lLZZJ 50% seawater
at 10°C; rz::::::::L3 50% seawater at 20°C.
"- "- "- "- "- "- "- "- "- "-
/ / / / / / / / / /
X X X X X X X X X X X
~"-"-'''-'''-''-
t-fE//'/'////
xxxx
I-a:'- "- "- "- "- , "- "- "- "-
r /' /' ,- ,- ,- ,- / / / /
X X X X X X X X X X X
~,,,- "- "- "- "- "-
~//,-//
~xxxx
"- "- "-
,
"- "-
, , , ,
/ / / /' /' /' /' /' /' /'
X X X X X X X X X X X
~"-, "- "- "- "- "-
t-tzl//////'
J<.. J<.. X J<.. J<.
I-t:::1"- "- , , "- "- "- "- "- "- "- "-
/' /' /' /' /' /' /' /' /' /' /' /'
x x x x x x X X X x x
1---5:"-"-'''-'''-''-
/'/'/'//'/
tiExxx
I I I I , I I I I
0 0 0 O. 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 00 (0 -.;;j- N 0 CD <..0 -.;;j- N 0
. . . . .
N
-- -- -- -- --
0 0 0 0 0
(JL1 jw6 jlo IW)
uO!tdwnsuoJ u85AXQ }O 8tO~
29
CD
I
f"".
~
(J)
I..-
....c
'-.-/
Q)
I..-
::J
(J)
0
LO Q..
I Xw
-.;;j-
'+-
0
c
0
+-'
. r-0 0l..-
I ::J
N 0
I
o
30
Figure 4. Weight specific rate of oxygen consumption of ~.
magister 5th juveniles. ~100% seawater at 10°C;
~100% seawater at 20°C; ~75% seawater at
10°C; ~75% seawater at 20°C; ~50% seawater
at 10°C; ~50% seawater at 20°C.
, , , , , , ,
I-----E:V / / / / / /
x X X x x X
""'"
//////
xxxxxxx
, , , , , ,
/ / / / / ,
X X X X X X X
"" '"
,.
/,;'/ ,;,;,;
XX X X X xxx,
, , , , , , , ,
.; / .; .; .; .; .;
x x x x x x x x
""'"///////
xxxxxxxxxx
, , , , , , , , ,
,; ,; ,; ,; ,; ,; / ,; ,; /
X X X X X X X X X X
""""""
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XXXXXXXXXXXX
I I
I I
31
CD
I
'"
",--.....
Ul
L
..c
'-/
Q)
L
~
Cf)
If) 0
I 0..x
~ W
4-
0
C
0
n +-'
I 0L
C'\J ~
0
I
o
000 0
0
I"} N ~ 0
.. .
000 0
(J~ jw5 jlo IW)
uor~dwnsuoJ' u86AXQ JO 8fD~
32
Figure 5. Weight specific rate of oxygen consumption of Q.
magister adults. ~ 100% seawater at 10°C; .
K:::ZJ 100% seawater at 20°Ci ~ 75% seawater at
10°C; ~75% seawater at 20°Ci (l7lJ50% seawater
at 10°C; IZ==zJ 50% seawater at 20°C.
" " " " " " " "/ / / / / / / /' /'
x x x x x x x
~''''''''''
/////
xxXXXX)<
, ,
" " " "
,
"/' /' /' /' ./ /' /' './ /'
x x x x x x x x
""""""/f///////'
xxxx xx
, , , , ,
.....
,
"/ / /' /' / / / /
X X X X X X X X X
''''''''''''''''''
/////./
xxxxxxxXX
" "
, , , , , , , , ,
/ / / / / / / /
"
/ /
X X X X X X X X X X X X
"",","","""
~//'/'//'/'.//'////////
XXXXXXXXXXXX
I I I I I , I I I
33
ex)
I
"
......----.
(j)
L
£
"-../
<l)
L
::J
CJ)
0
L{) 0..
I X
~ W
'+-
0
C
0
~
n 0L
I ::J
N 0
I
o
0 0 0 0 0 0 0 0 0 0 0
0 m ex) f"'- lO L{) ~ to N ~ 0
~ 0 0 0 0 0 0 0 0 0 0
. . . . .
0 0 0 0 0 0 0 0 0 0 0
(JLf /w6 /ZO IW)
uO!tdwnsuoJ u85AXO 1'0 a-lDt!
34
Table 1. Weight specific metabolic rates and QlO values for
four life stages of £. magister
STAGE SEAWATER 10 0 e 20 0 e
Q02 n Q02 n QlO
MEGALOPA 100% 0.990 10 1. 676 9 1. 69
±0.106 ±0.171
75% 1. 060 10 2.380 10 2.25
±0.051 ±0.076
50% 1.057 10 2.156 9 2.04
±0.060 ±0.222
1st JUVENILE 100% 0.386 12 1. 582 10 4.10
±0.067 ±0.079
75% 0.497 10 1.483 10 2.98
±0.025 ±0.078
50% 0.538 10 1. 442 10 2.68
±0.038 ±0.113
5th JUVENILE 100% 0.102 8 0.157 6 1. 54
±0.026 ±0.010
75% 0.082 8 0.146 8 1. 78
±0.013 ±0.010
50% 0.093 6 0.152 8 1. 63
±0.015 ±0.013
ADULT 100% 0.028 4 0.060 4 2.14
±0.004 ±0.010
75% 0.023 4 0.065 4 2.83
±0.005 ±0.007
50% 0.026 3 0.057 4 2.19
±0.003 ±0.006
Note. Weight specific rate of oxygen consumption, Q02'
ml 02/gm/hr, mean ± S.E.
35
Examining all four stages in different salinities at
either 10°C or 20°C, the regressions of the log of weight
specific metabolic rate (Q02) scales linearly with the log
of weight (W) ( Table 2 and Fig 6).
Table 2. Regression equations for log Q02 vs. log W for ~.
magister (0.033 gm megalopae to 495.4 gm adults) at
combinations of salinity and temperature
log Q02 = -0.383 (log W) - 0.741 (R2 : 0.767), 100% SW, 10°C
log Q02 = -0.440 (log W) - 0.691 (R2 : 0.921) , 75% SW, 10°C
log Q02 = -0.433 (log W) - 0.670 (R2 : 0.937) , 50% SW, 10°C
log Q02 = -0.416 (log W) - 0.326 (R2 : 0.935) , 100% SW, 20°C
log Q02 = -0.435 (log W) - 0.317 (R2 : 0.921) , 75% SW, 20°C
log Q02 = -0.440 (log W) - 0.345 (R2 : 0.922) , 50% SW, 20°C.
The slopes of the regressions given in Table 2 are not
significantly different. The regression constants (y-
intercepts) are different between 10°C and 20°C.
Discussion
A large number of factors, such as nutritional state,
culture conditions, molt stage, age, size, activity and
rhythms associated with tidal, diurnal, lunar and seasonal
cycles, as well as salinity and temperature, can affect the
metabolic rates of crustaceans. It is therefore important to
define the experimental conditions and the goals of the
experiment before making comparisons with other experiments
or other species. For example, Findley et ale (1973)
reported the rate of oxygen consumption was less for adult
36
Figure 6. Relationship between log weight specific rate of
oxygen consumption and log of wet weight in ~.
magister. (a) 100% seawater, 10°C; (b) 75%
seawater, 10°C; (c) 50% seawater, 10°C; (d) 100%
seawater, 20°C; (e) 75% seawater, 20°C; (f) 50%
seawater, 20°C.
o
----------------- ._--------- C0
o
N
o
a
.
r"
I
I
I
10
,
---l .
I I I ,N
LD 0 LD 0 LD 0'
0 0 0 ,.- ,.- (\J
I I I I
Uo!~dWnSU08 Ue6AXO 8!J!88dS +~6!8M 60l
37
38
Callinectes sapidus exposed to cyclic changes in salinity
compared to the rate of oxygen consumption for those in
constant salinity exposure at a similar range of salinities.
The experiments for the present study were carried out for
eight hours after acute exposure. For this reason comparison
to studies using cyclic or acclimated temperature and
salinity designs must be made jUdiciously.
The rates of oxygen consumption reported in the present
study would be defined as routine metabolic rates since the
animals were able to move freely within the respirometers.
The megalopae swam nearly continuously, and there were brief
periods of locomotor activity in the other stages. In
general, reported metabolic rates for crustaceans are
routine metabolic rates since the measurements are on
essentially unrestrained animals, in respirometers or crabs
fitted with masks and free to move within a given area.
Comparisons of ~. magister metabolic rates with the
megalopae and juveniles of other species can not be directly
made because ~. magister at these stages are extremely large
relative to other species for which data is available. For
the extremely small larval stages of other species the
regression coefficient of log Q02 vs. log W is nearly -1.0
(Vernberg et al., 1981) indicating a very different
relationship between metabolic rate and size than for the
larger megalopae of ~. magister.
The rates of oxygen consumption reported for
39
unrestrained adult ~. magister at 8 to 10°C, range from
0.025 to 0.050 ml 02/gm/hr (Johansen et al., 1970; McDonald
et al., 1980), and correspond very closely to the values
reported in the present study (Table 1). comparisons between
adults of different species of the same magnitude in size
reveal similar rates of oxygen consumption to those measured
in the present study. Aldrich (1975) and Aldrich and
McMullan (1979) report oxygen consumption rates of 0.007 to
0.018 ml 02/gm/hr for Cancer pagurus in the weight range of
336 to 640 gm at 10°C. Callinectes sapidus at 20°C consumes
oxygen at a rate of 0.034 ml 02/gm/hr in seawater at 30 ppt
seawater while oxygen consumption is significantly higher at
lower salinity, 0.053 and 0.052 ml 02/gm/hr in 20 ppt
seawater and 10 ppt seawater respectively (Findley et al.,
1978). Callinectes sapidus therefore exhibits a type 1
response to salinity, as defined by Kinne (1964). In
contrast to ~. sapidus, the rate of oxygen consumption of
adult ~. magister is independent of salinity at both 10°C
and 20°C and thus can be categorized as a type 4 response to
salinity according to Kinne (1964). The 1st juveniles and
5th juveniles also fit the type 4 response to salinity at
both 10°C and 20°C. The rate of oxygen consumption of the
megalopae, however, is affected by salinity at 20°C in a
type 1 fashion, an increase in metabolic rate at subnormal
salinities.
Typically, the rate of oxygen consumption of animals is
40
proportional to body mass. Larger animals consume a greater
amount of oxygen per unit time. Per unit body mass, however,
the larger animal consumes less oxygen per unit time than a
smaller animal. Regression coefficients of the log of weight
specific oxygen consumption (Q02) versus log weight (W)
reported in the literature for crustaceans span a fairly
broad range of values. Dehnel (1960) states that the
regression coefficient depends on a number of factors
including those previously mentioned at the beginning of
this discussion and on the thermal and salinity history/
acclimation of the animals used. Dehnel (1960) gave
regression coefficients ranging from -0.685 to -0.333 for
Hemigrapsus nudus and H. oregonensis under a variety of
salinity and temperature conditions. WeYmouth et al. (1944)
reported a coefficient of -0.2 for Pugettia productai
Roberts (1957) reported a coefficient for Pachygrapsus
crassipes of -0.336. In the current study, the regression
coefficients (Table 2) of the least squares regression of
log Q02 versus log Ware in the range of -0.383 and -0.440
and are similar to values reported for other crabs. The
weight range of crabs used in the present study spans a much
broader range than any of the other reports.
Gutermuth and Armstrong (1989) examined the extent to
which oxygen consumption is temperature dependent in
juvenile g. magister. Juveniles from estuarine and coast~.l
areas were compared. The average summer estuarine water
41
temperature is higher than the average summer coastal water
temperature. They report that 0+ year class juveniles and 1+
year class juveniles respond differently to temperature in
the range encountered in coastal and estuarine waters in the
summer months. The 0+ juveniles are more sensitive to
temperature in the low part of the temperature range studied
than the 1+ juveniles. Gutermuth and Armstrong (1989) relate
this to a faster growth rate of estuarine crabs compared to
coastal crabs: the higher metabolic rate of the juveniles in
the warm temperature areas (i.e. the estuary) mean they can
assimilate nutrients faster and take advantage of the food
rich estuary. Gutermuth and Armstrong (1989) have pooled
together several different instars in the juvenile
development of this spe~ies by only distinguishing between
year classes.
The results of the current study are not directly
comparable to the results reported by Gutermuth and
Armstrong (1989) for a number of reasons. First, the
individual instars are.pooled into year class groups by
Gutermuth and Armstrong. Secondly, the crabs were acclimated
to the experimental temperatures for several days prior to
the experiments. Thirdly, the rate of oxygen consumption is
reported per unit dry weight. Nonetheless, using the factor
of dry weight equaling 20% of wet weight given by Schatzlein
and Costlow (1978), the rate of oxygen consumption at lODe,
presumably in 100% seawater, of 0+ crabs, 0.145 ml 02/gm/hr
42
(Gutermuth and Armstrong, 1989) and 1st juveniles or 5th
juveniles at 100 e in 100% seawater, 0.386 or 0.102 ml
02/gm/hr respectively (present study), are similar.
The differences in metabolic response between stages in
the current study indicate some interesting patterns related
to differences in mode of existence and habitat of the
different stages of ~. magister. The remarkable change in
response to salinity and temperature at metamorphosis from
megalopa to 1st juvenile coincides with the transition from
oceanic megalopae to estuarine 1st juveniles. Upon entering
the bay, from the relatively constant oceanic waters, the
megalopae rapidly metamorphose into 1st juveniles. The
metabolism of the 1st juveniles at high temperature is less
sensititve to salinity and they may therefore be better
suited to the more changable estuarine waters than the
megalopae. Secondly, the high Q10 of the 1st juvenile (Table
1), especially in 100% seawater, would potentially enable
this rapidly growing stage to readily assimilate food from
the nutrient rich mudflats as suggested by Gutermuth and
Armstrong (1989). Thirdly, the low Q10 for 5th juvenile is
interesting in relationship to long duration of 5th juvenile
from late summer through the winter to the following spring.
The 5th juvenile is therefore exposed to a far broader range
of environmental conditions than the other stages examined.
A reduced sensitivity to temperature over the seasonal
extremes may be important in enabling these small crabs to
43
spend a portion of their time hidden on the mudflats and
therefore protected from most large aquatic predators.
~he pattern of change in metabolic response described
for ~. magister can be compared with patterns observed for
smaller larvae of other species. Belman and Childress (1973)
conclude that the temperature sensitivities of the early
larval stages of both Panulirus interruptus and Cancer
productus correlate with the normal temperature range at
which the larvae are usually found in the ocean. Vernberg et
al. (1981) suggested that the respiratory responses to
temperature of Pagurus criniticornis larvae are probably
related to habitat and environment changes during
development. Sastry and McCarthy (1973) discuss differences
in thermal tolerance and changes in respiratory rates during
development of the larvae of the sympatric species, Cancer
irroratus and ~. borealis. They conclude that the
differences in the physiological requirements and capacities
result in the temporal succession of these two species
within the same area.
Given the information available regarding metabolic
response to environmental parameters and how these
capabilities change related to transitions from larval to
adult habitats, further questions involving examination of
other ontogenic changes in physiological and molecular
processes present a broad potential for further study. At
the physiological level, the ontogeny of ionic and osmotic
44
regulatory ability is the focus of Chapter III. As noted by
Wheatly (1988) an increase in the rate of oxygen consumption
due to salinity stress may not be due solely to an increase
in energy expended in ionic and osmotic regulation, but may
be partly due to increases in ventilation, perfusion and
changes in other processes related to gas exchange and
oxygen transport in the hemolymph. Accordingly, at the
molecular level, the oxygen binding properties of the whole
hemolymph and of hemocyanin from different stages is
examined in Chapter IV.
45
CHAPTER III
IONIC AND OSMOTIC REGULATION
Introduction
Estuarine invertebrates vary greatly in their
abilities to regulate hemolymph ion concentrations when
exposed to changes in ambient salinity. Among the Crustacea,
the species with the strongest regulatory abilities are
generally the ones living in the most changable environments
(Krogh, 1939; Lockwood, 1962; Prosser, 1973). There is a
long history and a huge number of studies concerning the
effects of changes in environmental salinity on the internal
osmolality of adult decapod crustaceans (for reviews see
Krogh, 1939; Beadle, 1957; Lockwood, 1962; Mantel and
Farmer, 1983). Comparable information about larval and
juvenile crustacean osmoregulation, however, is quite small
in comparison, and there is very little data available
regarding specific ion regulation in larval and juvenile
stages of decapods. (for review see Gilles and Pequeux,
1983) .
In the early 1970's Alspach (1972), Engelhardt and
Dehnel (1973) and Hunter and Rudy (1975) all studied the
osmotic and ionic regulatory abilities of the adult male
46
Dungeness crab, Cancer magister. They measured hemolymph
osmolality and ionic concentrations when the animals had
reached equilibrium, 72-96 hr after acute exposure to
altered seawater. They found that Q. magister is a weak
hyperosmoregulator at salinities lower than that of ocean
seawater. They also found that Q. magister adults are able
to strongly hyperregulate calcium and hyporegulate
magnesium. In reduced salinity the hemolymph concentrations
of sodium, potassium and chloride are all higher than the
seawater concentration but are not as strongly
hyperregulated as calcium. The ontogeny of osmotic and ionic
regulation in Q. magister was not investigated.
studies of osmoregulation in decapod larvae are
limited. Those studies that have investigated the
osmoregulatory abilities of larval and post larval animals
have found several differnt patterns. (See Charmantier et.
al., 1988) In some cases there is no change in
osmoregulatory ability with developmental stage, e.g.
Sesarma reticulatum (Foskett, 1977). In Macrobrachium
petersi (Read, 1984) the beginning of the adult type
osmoregulation pattern is evident in the first larval stage.
In Uca sUbcylindrica (Rabalais and Cameron, 1985), Homarus
americanus and Penaeus japonicus (Charmantier et al., 1988)
there is a marked change from larval to adult osmoregulatory
patterns. These studies examined osmoregulation, but not the
regulation of specific ions, in the hemolymph of larval or
47
post-larval stages.
The purpose of this study was to investigate the short-
term (8 hr) osmotic and ionic regulatory abilities of
selected stages in the life-cycle of ~. magister.
Materials and Methods
Animals
The different life stages of ~. magister were caught
and maintained as previously described in Chapter II.
Protocol and Sampling
Megalopae, 1st instar juveniles (6-8 rom carapace width)
5th instar juveniles (25-33 mm carapace width) and adults
(larger than 120 rom carapce width) were exposed to seawater
of different temperatures and salinities for eight hours.
Hemolymph samples were then taken for subsequent ionic and
osmotic analysis. Experimental seawaters included 100%
seawater (32 ppt seawater from the mouth of Coos Cay), 75%
seawater and 50% seawater (Coos Bay seawater diluted with
glass distilled water) (Table 1) at either 10°C or 20°C.
One gallon glass aquaria were used for the experimental
chambers. Adults were kept one to an aquarium for the
duration of the experiments. About 250 megalopae or 1st
juveniles and two or three 5th juveniles were placed in each
aquarium.
48
Table 1. Composition of seawater treatments
TREATMENT
100% SW 75% SW 50% SW
Osmolality 958 773 488
Sodium 402 302 199
Chloride 535 413 268
Potassium 5.7 4.3 2.6
Calcium 7.7 6.3 4.5
Magnesium 55.2 43.0 28.0
Note. Osmolality in mOsm/kg and ion concentrations in
mmol/L.
Hemolymph samples were taken from the megalopae by
puncturing the heart with a glass micro-capillary pipette.
The juveniles and adults were bled by puncturing the
arthrodial membrane at the base of a walking leg. The 1st
juveniles were bled with micro-capillary pipettes and the
5th juveniles and adults were bled with needle and syringe.
The hemolymph samples from all individuals in each aquarium
were pooled in order to collect a large enough volume for
the ionic and osmotic analyses. In Tables 2-7 and Figures 1-
6, n refers to the number of analyses of separate pooled
samples. Samples were immediately frozen (-73°C) and stored
for ionic and osmotic analysis. Water samples from each
aquarium were also collected and frozen.
Osmotic and Ionic Analysis
Osmotic pressure of water and hemolymph samples was
49
measured using a Wescor 5500 vapor pressure osmometer.
Chloride concentration in water and hemolymph was measured
using a Buchler-Cotlove chloridometer. Hemolymph sample size
was 10ul for megalopae and 1st juveniles and 40ul for 5th
juveniles and adults. Magnesium concentration was measured
colorimetrically after the method of Sky-Peck (1964).
Samples (10ul) were deproteinized with 5% trichloroacetic
acid and reacted with thiazole yellow in the presence of
excess base. The absorbance at 540 nm was measured with a
Beckman DU-70 spectrophotometer. Sodium, calcium and
potassium concentrations were measured with ion specific
electrodes. The reference electrode in all cases was an
Orion 90-02 double junction reference electrode with NH4CI
as the outer chamber filling solution and a AgCI saturated
inner chamber filling solution. All measurements were made
using a Radiometer Ion 83 ion meter in mV mode. Sodium
concentration for adult hemolymph and water samples was
measured using a Radiometer G502 sodium Selectrode. A sodium
microelectrode (MI-420 from Microelectrodes, Inc.) was used
to measure sodium concentration in megalopa and juvenile
hemolymph and water samples. All samples were diluted 1:100
inO.1 molar Ca(N03 )2 to adjust the ionic strength of the
samples. Calcium concentration was measured using a
Radiometer F2112 calcium Selectrode. The ionic strength of
water and hemolymph samples was adjusted by diluting 1:100
with 0.3 molar KCI. Potassium concentration was measured
50
with an Orion 93-19 potassium electrode. Samples were
diluted 1:100 in 0.1 molar NaCI. Prior to analysis of
samples, calibration for measurement of each ion species was
done with salt solutions of known concentration spanning the
expected range of values.
Data analysis
Results are expressed as mean ± S.E., n is the number
of observations. Three-way analysis of variance (ANOVA) was
used to test for significance among treatments
(developmental stage, salinity and temperature). SUbsequent
mUltiple comparisons of means were performed using the
Tukey-Kramer method. statistical significance was accepted
at P < 0.05. statistical analyses were done using SYSTAT
version 4.1 (SYSTAT, Inc.).
Results
In the following presentation of results, all
measurements were made on animals that had been exposed for
8 hr to the specified temperatures and salinities.
In 100% seawater, the megalopa, 1st juvenile, 5th
juvenile and adult are isosmotic with the ambient seawater
(Fig. 1 and Table 2). In 75% seawater the hemolymph
osmolalities of all four stages are significantly lower than
in 100% seawater, yet they are all hyperosmotic relative to
75% seawater. In 50% seawater the hemolymph osmolalities of
51
Figure 1. Hemolymph osmolality of~. magister. Mean ± S. E ..
Salinity treatments are indicated below the x-
axis. c::Jseawater, K:X:1JI0°C, ts:=SJ20°C. H,
megalopa; IJ, 1st juvenile; 5J, 5th juvenile; A,
adult.
M 1J 5J A
75%
r--...
CJ1
.:::L
~
o
E
E
"--"
~
-+-J
o
o
E
(J)
o
1000 T
900t n
800-!- II
700!11
600 j. II
500! II
4001 II
300! II
200t II
100 -!- II
o M 1J 5J A
100%
~
1'\
M 1J 5J A
50%
OJ
t\,)
TABLE 2. Hemolymph osmolality of megalopa, 1st juvenile,
5th juvenile and adult ~. magister
53
STAGE TREATMENT
100% SEAWATER 75% SEAWATER 50% SEAWATER
100 e 200 e 100 e 200 e 100 e 20 0 e
MEGALOPA 970.7 997.0 852.0 836 695 642
±l. 7 ±4.3 ±3.3 837 640
n = 3 3 3 2 1 2
1st 986.6 955 794.3 no 605.3 590
JUVENILE ±1.9 946 ±1.2 data ±1.2 586
n = 3 2 3 3 2
5th 927.3 937.0 810.3 738.7 717 622.7
JUVENILE ±27.8 ±4.5 ±17.6 ±20.3 650 ±6.2
n = 3 3 3 3 2 3
ADULT 970.9 948.1 925.6 865.8 815.4 748.8
±12.7 ±22.0 ±12.9 ±13.6 ±19.1 ±12.4
n = 8 8 8 8 8 8
Note. Osmolality in mOsm/kg, mean ± S. E..
54
all four stages are significantly lower than in 75% seawater
and all are significantly hyperosmotic compared to the
seawater. The 1st juvenile is least able to maintain
hemolymph osmolality in dilute seawater compared to the
other stages examined. Water temperature affects the
osmoregulatory abilities of the crabs. The hemolymph
osmolality of the adult and 5th juvenile are significantly
lower at 20 De than at lODe in both 75% and 50% seawater,
while the megalopa hemolymph osmolality is less at 20 De than
at lODe (p<0.05) in 50% seawater.
The hemolymph chloride concentration in all stages in
100% seawater is hypoionic compared to ambient water (Fig 2
and Table 3). In 75% seawater the adult becomes nearly
isoionic compared to the water but has significantly lower
hemolymph chloride concentration at 20De than at lODe. In
50% seawater the adult hemolymph chloride concentration is
hyperionic and is lower at 20De than at lODe. The hemolymph
chloride concentration of the megalopa and of the 5th
juvenile are also lower at 20 De than at lODe in 75%
seawater. In 50% seawater the megalopa and 1st juvenile
hemolymph chloride concentrations are the same as the
ambient water chloride. In 50% seawater the hemolymph
chloride concentration of the5th juvenile, however, is
significantly higher than that of the megalopa and 1st
juvenile and is lower than that of the adult.
Hemolymph sodium concentration (fig. 3 and table 4) in
55
Figure 2. Hemolymph chloride ion concentration of ~.
magister. Mean ± S. E.. Salinity treatments are
indicated below the x-axis. c==Jseawater,
rx=:.:x::lI10°C, E:3J20°C. M, megalopa; 1J, 1st
juvenile; 5J, 5th juvenile; A, adult.
t-H/ ./ ./ ./ ./ ./ ./ / /
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1-8:/ / / / / / / /
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~./ ./ ./ ./ ./ / /
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I ./ ./ ./ / / / /
xxxxxxxxxxxxxxx
I
f-H/ / / / / ./ ./ ./ ./ ./ ./
~XXXXXXXXXXxxxxxxxxxxxxxx
~/ / / / ./ ./ ./ ./ ./ ./
~xxxxxxxxxxxxxxxxxxxxx
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l+l / / / / ./ ./ ./ ./ ./ ./
~xxxxxxxxxxxxxxxxxxxxxx
I
Hl ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./
~xxxxxxxxxxxxxxxxxxxxxxxxx
~ / / ./ ./ ./ ./ / / / ./ ./ / /
xxxxxxx xxxxxxxxxxxxx xx
f / / / / / / / / / / / / /
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I-H./ ./ ./ ./ ./ ./ ./ ./ ./ / / / /
~xxxxxxxxxxxxxxxxxxxxxxxxx
I
I I I I I I I I
I I I I I I I I I I
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~
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o
56
000
LD 0 LD
L() L() ~
o 0 0 0 000
o L() 0 L() 0 L() 0
~ n n N N ~ ~
o 0
L()
(l/IOWW) UOq.DJtU8JUOJ UOI 8P!JO IL1J
57
TABLE 3. Hemolymph chloride concentration of megalopa, 1st
juvenile, 5th juvenile and adult ~. magister
STAGE TREATMENT
100% SEAWATER 75% SEAWATER 50% SEAWATER
lODe 20De lODe 20De lODe 20De
MEGALOPA 456.7 466.7 406.7 380 270.0 240.0
±4.7 ±9.4 ±4.7 370 240.0
n = 3 3 3 2 1 2
1st 480.0 450.0 383.3 no 260.0 260.0
JUVENILE ±14.1 450.0 ±4.7 data ±8.2 270.0
n = 3 2 3 3 2
5th 457.5 453.5 378.1 346.9 294.7 301.1
JUVENILE ±1.1 ±3.9 ±9.3 ±13.4 306.8 ±11.9
n = 3 3 3 3 2 3
ADULT 452.6 423.5 438.0 401.4 389.8 322.3
±7.3 ±12.4 ±14.7 ±21.3 ±16.1 ±20.6
n = 8 8 8 8 8 8
Note. Chloride ion concentration in mmol/L, mean + S. E•.
58
Figure 3. Hemolymph sodium ion activity of Q. magister.
Mean ± S. E.. salinity treatments are indicated
below the x-axis. c==Jseawater, ~10°C,
~20°C. M, megalopa; 1J, 1st juvenile; 5J, 5th
juvenile; A, adult.
tf[/ / / / / / /
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I-R"/ / / / / /
~)(YYYYYYYY){YY
Ii 7 7 7 / 7
IX X )< X X X X )< )< )< )<
I / / / / /
yyyyyXX)()(){){
r
~ / / / / / / / / / /
~XXXXX)(XXXVVXVXXXX
f-R"/ 7 / /' / /' 7
H;KYYY"X YYYYYYYY
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IIIYYx,><:YYVYVYYYYVV
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~ / / / / / / / / / 7
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HI / / / / / / / / /
~X)()()()(xxxvvvv){)(YYX){){
1/ / / / / / / / /
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000
L() 0 L()
L() Ul ..q-
000 0
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~ n n N
000
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L()
TABLE 4. Hemolymph sodium ion activity of megalopa, 1st
juvenile, 5th juvenile and adult Q. magister
60
STAGE TREATMENT
100% SEAWATER 75% SEAWATER 50% SEAWATER
100 e 20 0 e 100 e 200 e 100 e 200 e
MEGALOPA 302.7 341.1 270.1 257.5 193.5 186.3
±13.7 ±3.5 ±3.6 252.7
n = 3 3 3 2 1 1
1st 327.3 334.6 278.4 no 193.9 187.2
JUVENILE ±6.0 334.6 ±3.3 data ±1.6 183.7
n = 3 2 3 3 2
5st 353.6 331.1 289.2 264.9 229.9 224.0
JUVENILE ±15.4 ±5.6 ±9.2 ±12.1 241.1 ±8.0
n = 3 3 3 3 2 3
ADULT 349.2 343.5 315.2 355.8 290.3 266.5
±9.4 ±12.8 ±17.9 ±13.8 ±15.1 ±16.0
n = 8 8 8 8 8 8
Note. Sodium ion activity in mmol/L, mean + S. E..
61
all four stages shows essentially the same pattern as
hemolymph chloride. In 100% seawater all four stages are
hypoionic with respect to ambient seawater sodium. In 75%
seawater the megalopa, 1st juvenile and 5th juvenile
hemolymph sodium concentrations are significantly less than
in the 100% seawater treatment, while the adult hemolymph
sodium concentration is not significantly changed from the
100% seawater treatment. In 50% seawater the megalopa and
1st juvenile are isotonic to ambient sodium. The 5th
juvenile hemolymph sodium concentration is significantly
higher than the megalopa and 1st juvenile and less than the
adult in 50% seawater. There is no effect of temperature on
the hemolymph sodium concentration in any of the stages.
The hemolymph potassium concentration in all four
stages in 100% seawater is not significantly different from
ambient potassium concentration (fig 4 and table 5). In 75%
seawater the adult hemolymph potassium concentration is not
different from ambient potassium concentration but is
significantly less than hemolymph potassium in 100%
seawwater treatment. The 1st juvenile and 5th juvenile in
75% seawater have a significantly lower hemolymph potassium
concentration than in 100% seawater. The megalopa shows no
significant decrease in hemolymph potassium concentration in
either 75% or 50% seawater compared to 100% seawater. The
adult, 1st juvenile and 5th juvenile hemolymph potassium
concentrations are not significantly different in 50%
62
Figure 4. Hemolymph
magister.
indicated
IK:X::Xl 1 a°e I
juvenile;
potassium ion activity of ~.
Mean ± S. E.. Salinity treatments
below the x-axis. c::J seawater I
L::SJ 2 aOe. M, megalopa; 1J I 1st
5J , 5th juvenile; A, adult.
are
I.....,
j
r-B/ / / / /
~xxxxxxxxxxxxx
/ / / /
IX )( X X X X X X x: X
I / / / / / /
ax x x x x x x x x x
IH / / / / / / /
xxxxxxxxxxxxxxxxx
I
f-H/ / / / / / / /
~XXXXXXXXXXXXXX
f-8/ / / / / /
IIfXXXXXXXXX XX
~XXXXXXXXXxx)<
r-H/ / / / / / / / /
MHXXXXXXXXXXXXXXX
I
r-H/ / / / / / /
~XXXXXXXXXXXXXXXXXXXXX
t---EV / / / / / / /
xXXXXXXXXXXX
t-f::i/ / / / / / / / /
~XXXXXXXXXXXXXXXXXX
III / / / / / / / / /
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I
,I ,\ ,I ,I ,I ,I ,\ ,\ ,I
'I '\ 'I 'I 'I 'I 'I 'I 1
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63
o N o
64
TABLE 5. Hemolymph potassium ion activity of megalopa, 1st
juvenile, 5th juvenile and adult £. magister
STAGE TREATMENT
100% SEAWATER 75% SEAWATER 50% SEAWATER
10°C 20°C 10°C 20°C 10°C 20°C
MEGALOPA 5.36 6.15 5.62 5.22 5.44 4.44
±0.08 ±0.07 5.94 ±0.41 4.53
n = 3 3 2 3 1 2
1st 5.92 5.94 4.04 no 3.20 4.08
JUVENILE ±0.17 6.35 ±0.25 data ±0.03 4.08
n = 3 2 3 3 2
5th 4.67 6.84 3.74 3.84 3.12 3.23 .
JUVENILE ±0.60 ±2.01 ±0.07 ±0.29 3.18 ±0.73
n = 3 3 3 3 2 3
ADULT 7.07 5.49 4.69 4.53 4.40 2.70
±0.58 ±0.32 ±0.51 ±0.14 ±0.78 ±0.16
n = 8 8 8 8 8 8
Note. Potassium ion activity in mmol/L, mean + S. E..
65
seawater than in 75% seawater. There is no effect of
temperature on hemolymph potassium concentration.
Magnesium concentration is strongly hyporegulated in
adult hemolymph in all salinity treatments (fig 5 and table
6). In 100% seawater the megalopa, 1st juvenile and 5th
juvenile hemolymph magnesium concentrations are
significantly higher than the adult hemolymph magnesium
concentration. The megalopa, 1st juvenile and 5th juvenile
hemolymph magnesium concentrations are significantly lower
in 75% and 50% seawater than in 100% seawater. In 50%
seawater there is no difference in the hemolymph magnesium
concentrations between the four stages. In 100% seawater the
5th juvenile has significantly lower hemolymph magnesium in
10°C than in 20°C. The 1st juvenile also appears to have a
lower concentration at 10°C than at 20°C, however this is
not significant at P<0.05.
In 100% seawater the hemolymph calcium concentrations
in all four stages are not significantly different from the
ambient water calcium concentration (fig 6 and table 7). The
hemolymph calcium concentrations of the megalopa and adult
do not change significantly with salinity. The 1st juvenile
and 5th juvenile have significantly lower hemolymph calcium
concentrat~ons in 75% and 50% seawater than in 100%
seawater; the 1st juvenile and 5th juvenile hemolymph
calcium concentrations are also lower than the values in the
megalopa and adult in 75% and 50% seawater. Overall there
66
Figure 5. Hemolymph
magister.
indicated
tK:D 10°C,
juvenile;
magnesium ion concentration of ~.
Mean ± S. E.. Salinity treatments
below the x-axis. c::Jseawater,
1S:::::Sl20°C. M, megalopa; 1J, 1st
5J, 5th juvenile; A, adult.
are
l-H/ / /
~XXXXXX
M:Y / / /
xxxxx
I / / / /
I+KXXXXXX
I / / /
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N
o o
(l/IOWW) UO!lDJ lU8:)UOJ UOI wn!sau50V\J
68
TABLE 6. Hemolymph magnesium concentration of megalopa, 1st
juvenile, 5th juvenile and adult ~. magister
STAGE TREATMENT
100% SEAWATER 75% SEAWATER 50% SEAWATER
10°C 20°C 10°C 20°C 10°C 20°C
MEGALOPA 33.0 32.8 18.1 21.7 16.3 14.3
30.9 31.9 20.2 25.3 15.3 14.2
n = 2 2 2 2 2 2
1st 20.1 30.2 23.7 no 16.3 15.9
JUVENILE 31.0 32.1 24.7 data 15.1 15.8
n = 2 2 2 2 2
5th 23.2 35.2 18.6 18.4 17.7 17.1
JUVENILE ±1.1 ±8.0 ±2.9 ±1.2 11. 3 ±2.6
n = 3 3 3 3 2 3
ADULT 14.0 14.5 12.8 12.1 14.8 14.7
±1.6 ±2.0 ±2.1 ±2.8 ±3.5 ±3.5
n = 8 8 8 8 8 8
Note. Magnesium ion concentration in mmol/L, mean ± S. E..
69
Figure 6. Hemolymph
magister.
indicated
I:[X]J 10°C I
juvenile;
calcium ion acivity of ~.
Mean ± S. E.. Salinity treatments
below the x-axis. c::Jseawater ,
IS::3l 20°C. M, megalopai 1J lIst
5J , 5th juvenile; A, adult.
are
1-8/ / / / / / / / /
~xxxxxxxxxxxxxxxxxxxx
It! / / / / / / /
~x x x x xx xxx
I-fi/ / / / / /
IXXX x x x'><'><)< x x x
f-/i/ /' /' / / / / / / /
IXXXXXXXxxxxxxxxxxx
,
1-[/ /' /' /' /' /' /' /' / /
~ xXXXXXXXXXXXXxxxxXxxxxx
I/f / / / / / / /
~xxxxxxxxxxxxxxx x
~xxxxxxxxxxxxxxx
t-H/' / / / / / / / /' / /'
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I
~ /' / / /' / /' / /
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t-H/ / / / /' / / / /
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fit / / / / / / / / / /
~xxxxxxxxxxxxxxxxxxx
rfl/ / / / / / / / / / /
~xxxxxxxxxxxxxxxxx
I
,I,
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70
TABLE 7. Hemolymph calcium ion activity of megalopa, 1st
juvenile, 5th juvenile and adult e. magister
71
STAGE TREATMENT
100% SEAWATER 75% SEAWATER 50% SEAWATER
10°C 20°C 10°C 20°C 10°C 20°C
MEGALOPA 7.39 8.69 7.79 8.85 6.84 7.89
±0.45 ±0.27 ±0.23 ±0.30 ±0.24
n = 3 3 3 3 1 3
1st 7.45 7.84 6.25 no 4.68 4.85
JUVENILE ±0.08 7.70 ±0.08 4.68 5.12
n = 3 2 3 2 2
5st 8.48 7.38 7.04 5.71 6.32 5.31
JUVENILE ±0.59 ±0.32 ±0.35 ±0.08 5.53 ±0.16
n = 3 3 3 3 2 3
ADULT 9.24 7.01 9.59 7.90 7.85 6.83
±0.56 ±1.07 ±1.13 ±0.33 ±0.77 ±0.79
n = 8 8 8 8 8 8
Note. Calcium ion activity in mmol/L, mean ± s. E..
72
is no significant effect of temperature on hemolymph calcium
concentration.
Discussion
This study shows that different developmental stages in
the life cycle of ~. magister have different patterns of
regulation of hemolymph osmolality and ionic concentrations
in reduced salinity over the 8 hr time period of a tidal
cycle.
The results for ionic and osmotic regulation in adult
~. magister in the present study essentially coincide with
the findings of Jones (1941), Alspach (1972), Engelhardt and
Dehnel (1973) and Hunter and Rudy (1975) with some
differences due to experimental purpose and design. All of
the previous investigations found that the hemolymph of ~.
magister is weakly hyperosmoregulated in water less
concentrated than normal ocean seawater. The earlier
studies all examined hemolymph concentrations after a state
of osmotic and ionic equilibrium had been reached,
approximately 72-96 hr. The values for hemolymph osmolality
and ionic concentrations in the present study were obtained
after an exposure time which is physiologically and
ecologically important for these animals, the duration af a
tide cycle. The general trends for osmotic and ionic
regulation reported for long term exposure (Jones, 1941;
Alspach, 1972; Engelhardt and Dehnel, 1973; Hunter and Rudy,
73
1975) are apparent, however, after the 8 hr exposure time
used in the present study. The adult hemolYmph is
hyperosmotic compared to the experimental seawater
osmolalities, chloride, sodium and potassium are somewhat
hyperregulated in reduced salinity, calcium is strongly
hyperregulated and magnesium is very strongly hyporegulated.
Studies on the larvae of a number of species of decapod
crustaceans indicate that most are capable of maintaining
hemolYmph osmolality above that of the ambient water, either
by hyperosmoconforming or by weakly hyperosmoregulating (See
Charmantier et. al. 1988). Hyperosmoconformers maintain
hemolYmph osmolality above that of the ambient water, but as
the ambient osmolality changes the hemolymph osmolality
changes to the same extent. Hyperosmoregulators maintain
hemolymph osmolality higher than the ambient water and as
the ambient osmolality decreases the hemolymph becomes more
hyperosmotic compared to the external water. Charmantier et
al. (1988) note that metamorphosis often marks a profound
change in osmoregulation from larval to adult type patterns,
i.e., Homarus americanus, Penaeus japonicus (Charmantier et
al., 1988)and Uca subcylindrica (Rabalais and Cameron,
1985). In the present study there is also a marked change in
osmoregulation at metamorphosis from megalopa to 1st
juvenile in ~. magister. In this case, however, the juvenile
is less able to regulate over the short 8 hr than are the
adult and the megalopa. The direction of change in ~.
74
magister does not parallel the pattern seen for tl.
americanus, £. japonicus and ~. sUbcylindrica, since the
megalopa, not the juvenile osmoregulates more like the
adult, but the phenomenon of change at metamorphosis is
consistent.
The hyperosmoregulation observed in the megalopa may be
a result of its premolt state. Kalber and Costlow (1966,
1968) report that just prior to molting the hemolymph
osmolality of the larvae of Rhithropanopeus harrisii and of
the land crab Cardisoma guanhumi increases. They hypothesize
that the increase in hemolymph osmolality is necessary for
the uptake of water at the molt and SUbsequent increase in
size of the animal. However Foskett (1977) found no such
increase in hemolymph osmolality in the larvae of Sesarma
reticulatum prior to molting.
This is the first report of the ontogenic changes in
specific ion regulation in brachyuran crabs. The data show
that there are differences in ion regulation between larval,
juvenile and adult ~. magister. The 1st and 5th juveniles
studied here may be starting to show the pattern of ion
regulation seen in the adult but may not yet be able to
regulate to the extent of t~e adult. There is certainly a
shift at metamorphosis in the ion regulatory pattern which
is not evident by simply looking at the data on hemolymph
osmolality. The ion regulation in the 5th juvenile is more
like the adult than the 1st juvenile is like the adult.
75
Hunter and Rudy (1975) reported that smaller crabs (-30 gm)
have a greater ability to hyperosmoregulate than larger
crabs. They correlate this ability with a larger gill
surface area to body volume ratio in the smaller animals
(Gray, 1957), thereby affording a greater relative area for
salt transport. The present study using crabs of
approximately 0.1 gm (1st juvenile) and up to 5.0 gm (5th
juvenile) indicates that the osmoregulatory ability is
clearly much less than that of the adult. Also important in
these considerations is the proportion of area on the gill
associated with salt transport and the efficiency of that
salt transport. Felder et al. (1986) have shown differences
in Na+/K+ ATPase activity in the different prehatch stages
of Callianassa jamaicense var. louisianensis, and have shown
the presence of salt transport type tissue on the
brancheostegites of the zoeae. It is possible that the
different larval and juvenile stages of ~. magister have
different Na+/K+ ATPase activity levels as well as different
relative proportions of salt transporting tissues.
There are aspects of specific ion regulation in the
present study which are particularly striking. First, there
are high hemolymph magnesium concentrations in megalopa, 1st
juvenile and 5th juvenile in 100% seawater, when all of the
other ions show no differences between stages in 100%
seawater. Second, there is strong hyperregulation of calcium
in megalopa and adult hemolymph compared to weak regulation
76
of calcium in the two juvenile stages studied.
Adults of all species of crustaceans that have been
examined maintain hemolymph magnesium well below the
magnesium concentration of the ambient water, except when
they are in extremely dilute water. Engelhardt and Dehnel
(1973) stated that 'hyporegulation of magnesium is the most
universal feature of ionic regulation in crustacean blood.'
Adult ~. magister excrete magnesium in urine formed in the
antennaI gland. The urine to hemolymph ratio of magnesium is
nearly 4:1 in 100% seawater (Hunter and Rudy, 1975;
Holliday, 1980). The antennaI gland may not be fully
developed and functional inthe early stages of crustaceans
(Waite, 1899; Conte, 1984). This may account for the high
hemolymph magnesium in megalopa and juvenile crabs.
Hyporegulation of magnesium has often been discussed
either in the light of maintaining high levels of activity
or in regard to a greater extent of terrestriality.
According to Robertson (1960) the decapod species with
hemolymph magnesium concentrations less than 50% that of
seawater are more active than those with higher hemolymph
magnesium concentrations. In fact, high magnesium
concentrations are often used to anaesthetize marine
invertebrates. Gross (1964) discusses magnesium regulation
at length in relation to the extent of terrestriality of
various species of crabs. Mantel and Farmer (1983) note that
grapsids and other species of semi-terrestrial and
77
terrestrial decapods all have low hemolymph magnesium
concentrations. Neither of these hypotheses, activity level
or terrestriality, proves to be helpful in understanding the
role of high magnesium in the hemolymph of larval and
juvenile Q. 'magister. The totally aquatic megalopae are
extremely active animals, capable of swimming very rapidly
for extended periods. The juvenile crabs, although they are
somewhat exposed on the mudflats at low tide, are never
completely emersed and are in no sense amphibious. Their
small size enables them to remain in the thin layer of water
in the algae on the flats when the tide is out. The
juveniles are also more active than the adults.
The relatively high magnesium concentrations in the
megalopa and juvenile crabs may influence the hemocyanin
oxygen binding properties in the hemolymph of these animals.
Miller and Van Holde (1974) and Truchot (1975) among others
have shown that magnesium concentration can affect the
oxygen affinity and the cooperativity of some crustacean
hemocyanins.
In addition to the stage specific magnesium levels the
strong regulation of calcium in the megalopa and adult is
remarkable. Calcium regulation in Q. magister adults has
been previously reported (Alspach, 1972; Engelhardt and
Dehnel, 1973; Hunter and RUdy, 1975). The strong regulation
of calcium by the megalopa and weak regulation by the
juveniles is noteworthy. The observed hyperregulation of
78
calcium in ~. magister megalopa could be the result of
calcium resorbed from the carapace. Greenaway (1985),
however, in a review of calcium balance during molting
states theat the majority of the calcium sequestered in the
hemolymph of crustaceans during premolt is likely to be in
the form of bound or complexed calcium and that free or
ionized calciium remains nearly constant in the hemolymph
through premolt. The technique used to measure calcium in
this study provided a measurement of ion activity, not total
calcium in the hemolymph, therefore it would seem reasonable
to assume that the high level of free calcium in megalopa
hemolymph is not a result of the animals being in premolt.
The functional significance of the high hemolymph calcium is
not known. As with magnesium, calcium has been shown to have
a strong effect on both the oxygen affinity and the
cooperativity of hemocyanin from a variety of crustacean
species (Larimer and Riggs, 1964; Miller and Van Holde,
1974; Truchot, 1975). The maintainance of high calcium
levels in the megalopae may have a strong effect on the
oxygen transport properties of the hemocyanin in the
hemolymph.
Charmantier et ale (1988) propose a correlation between
hyperregulation in larval and post-larval stages of Homarus
americanus and Penaeus japonicus and the likelihood of those
stages encountering low salinity environments. This
hypothesis does not appear to hold as a possible explanation
79
for the pattern of osmoregulation seen for the different
stages of ~. magister studied here. The megalopa and adult
are the best able to hyperosmoregulate over the 8 hr of
exposure to low salinity. The megalopa is the stage least
likely to be exposed to regular, large changes in salinity,
either due to tides or rainfall. The adult crabs are found
in water as low as 50% seawater and the ability to
hyperregulate hemolymph osmolality and ion concentrations
alleviates the stress of cell volume regulation. The
juvenile stages found on large numbers on the mudflats are
only slightly hyperosmotic in low salinity. In the case of
juvenile ~. magister it would appear that at the cellular
level there is a high level of tolerance for changes in
osmotic and ionic concentration. It seems that the juvenile
crabs must have some mechanisn for regulating cell volume in
the face of large and rapid changes in hemolymph conditions.
As mentioned above, calcium and magnesium may be
involved in altering the oxygen binding function of
hemocyanin. In the next chapter the experiments designed to
test this hypothesis are discussed.