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First published online March 16, 2007
Journal of Experimental Biology 210, 1245-1254 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02709
Temperature and acid–base balance in the American lobster Homarus americanus
1 Department of Neuroscience, University of Virginia, Charlottesville, VA
22908, USA
2 Division of Biostatistics and Epidemiology, University of Virginia,
Charlottesville, VA 22908, USA
3 Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric
Sciences, University of Miami, FL 33149, USA
* Author for correspondence (e-mail: mkw3k{at}virginia.edu)
Accepted 4 January 2007
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Summary |
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Key words: acid–base balance, lobster, pH, temperature, hemolymph
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Introduction |
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). The breadth of this temperature range poses an
interesting physiological challenge for the lobster because as poikilotherms
they are unable to regulate their own body temperature. The body temperature
of the lobster closely matches the external temperature of the seawater
(Worden et al., 2006),
therefore changes in seawater temperature also warm and cool lobster
physiological systems. As demonstrated for other marine arthropods
(Florey and Hoyle, 1976;
Stenseng et al., 2005;
Stillman and Somero, 2000;
Young et al., 2006),
temperature can be a potent modulator of the excitability of muscles and
nerves because it alters the activity of ion channels and pumps in excitable
membranes. The heart rate, stroke volume and heartbeat kinetics in H.
americanus are all temperature dependent within the thermal range the
lobster encounters in the wild (Worden et
al., 2006). Moreover, the water temperature to which lobsters are
acclimated determines the upper and lower thermal limits of lobster cardiac
performance (Camacho et al.,
2006).
Understanding the effects of temperature change and thermal stress on
lobster physiology is particularly important given that a recent period of
unusually warm water temperatures in Long Island Sound correlated with lobster
mortality rates sufficiently high to collapse the commercial H.
americanus fishery in that region
(Pearce and Balcom, 2005).
Furthermore, understanding how the signaling properties of the H.
americanus nervous system are affected by the environmental conditions in
which this species lives is also important given the long history of this
species as a model system for studying the excitability of neural circuits,
the dynamics of synaptic plasticity and the neural control of behavior (e.g.
Battelle and Kravitz, 1978;
Bucher et al., 2005;
Edwards and Kravitz, 1997;
Harris-Warrick and Kravitz,
1984; Heinrich et al.,
1999; Huber et al.,
1997; Kravitz,
1988; Kravitz et al.,
1983; Kravitz et al.,
1963; Livingstone et al.,
1980; Mahadevan et al.,
2004; Prinz et al.,
2005; Richards et al.,
1999).
The overall goal of this study is to determine how thermal change and
thermal stress might affect the properties of lobster hemolymph that are
important for neural and muscle physiology in vivo. Hemolymph is the
blood that bathes the internal organs, muscles and synapses of the lobster to
supply gasses, nutrients and metabolites to the neurons and muscles
(Martin and Hose, 1995). The
ionic composition of the hemolymph is critically important for physiological
function because it determines the transmembrane ion gradients that provide
the driving force for sodium, calcium and potassium flux across neural and
muscle membranes. Moreover, in other decapod crustaceans extracellular pH
varies with temperature (Truchot,
1978), and the acid–base status of the hemolymph might
regulate neural and muscle excitability.
Although the effects of temperature on hemolymph properties have been
investigated in several species of marine crabs
(Truchot, 1978;
Wood and Cameron, 1985), the
effects of temperature on the hemolymph of the lobster H. americanus
are relatively unexplored. Recent observations that hemolymph of H.
americanus acidifies by 0.2 pH units when lobsters acclimated to 16°C
seawater are exposed to a temperature of 23°C for 3 weeks have been
interpreted to suggest that prolonged thermal stress disrupts acid–base
homeostasis in lobsters (Dove et al.,
2005). However, in other decapod crustaceans and ectothermic
vertebrate species the pH of the blood passively follows temperature to
maintain a constant ratio of [OH–]/[H+] according
to the rule of constant relative alkalinity
(Reeves, 1977;
Truchot, 1978). In these
species variations in pH as a function of thermal change are not interpreted
as physiological anomalies attributable to thermal stress. Furthermore,
because Dove and colleagues measured pH only at the end of the 3-week study
period it is not clear whether the acidification they observed had occurred
over a timescale of minutes, as demonstrated in crabs
(Truchot, 1978), rather than
over days or weeks.
To examine how water temperatures in the physiological range of 2–25°C might affect the acid–base properties of lobster hemolymph that bathes the excitable membranes of muscle and nerve, we investigated the effects of temperature change on the hemolymph of lobsters in the laboratory and in the wild. Our results demonstrate that temperature change alters lobster acid–base status both on long-term (weeks) and short-term (min) timescales. An abrupt temperature increase triggers a metabolic acidosis that is compensated by a temperature-dependent increase in ventilation rate. In addition, thermal change alters hemolymph pH within a physiological range that modulates cardiac activity in vitro, suggesting that the temperature dependence of cardiac performance in vivo is mediated in part by thermal changes in acid–base status.
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Materials and methods |
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pH was immediately measured using a pH meter (Accumet BASIC, Fisher
Scientific) with a combination pH electrode with silver/silver chloride
references (Fisher Scientific). Measurements were made non-anaerobically with
reference to certified buffers at the same temperature. Hemolymph samples were
stored overnight on ice before assaying total CO2 levels.
Preliminary experiments (N=3) on hemolymph from lobsters acclimated
to a temperature of 12°C demonstrated that storing samples overnight on
ice did not affect total CO2 measurements (11.40±2.59
compared with 11.43±1.79; P=0.94 in a paired t-test).
CO2 was measured using a Corning 965 carbon dioxide analyzer
(Corning, NY, USA). Levels of bicarbonate, carbonate and partial pressure of
CO2 (PCO2) were calculated from total
CO2 levels and pH using the Henderson-Hasselbach equation, the
CO2Sys software (Lewis,
1996) and seawater pKI and pKII constants
appropriate for the experimental temperature.
Hemolymph samples were drawn both from lobsters acclimated to artificial
seawater in laboratory aquaria and from lobsters acclimated to the ambient
seasonal temperatures of natural seawater in February 2006 (5°C) at the
Marine Biological Laboratory in Woods Hole, MA, USA and in July 2006
(16°C) at the Mount Desert Island Biological Laboratory in Salisbury Cove,
ME, USA. The majority of samples drawn from laboratory lobsters were
postbranchial hemolymph sampled from the pericardial sinus. In lobsters in
Maine and Woods Hole (and occasionally in laboratory lobsters) prebranchial
hemolymph was sampled from the infrabranchial sinus at the base of the walking
legs. To verify whether prebranchial and postbranchial hemolymph would be
comparable we compared hemolymph properties of pre- and postbranchial
hemolymph from single individuals. In agreement with previous reports
(Booth et al., 1984;
Cameron and Batterton, 1978;
Rose et al., 1998;
Truchot, 1978) there were no
significant differences in pH or total CO2 in prebranchial and
postbranchial samples in quiescent animals [For pH: P=0.21
(N=8) at 12°C, P=0.83 (N=9) at 22°C. For
total CO2: P=0.11 (N=4) at 12°C,
P=0.92 (N=3) at 22°C; all values were tested with a
paired t-test]. As discussed by Truchot, the similarity between
prebranchial and postbrancial hemolymph values of pH and CO2 can be
attributed both to the low oxygen-carrying capacity of crustacean hemolymph
and the high buffering capacity of hemolymph proteins
(Truchot, 1978).
Recordings of cardiac activity in vitro were performed as
described previously (Worden et al.,
2006). Briefly, hemolymph enters the single chamber of the lobster
heart through paired ostia and is pumped out through seven arteries. Isolated
hearts continue to beat because of rhythmic neural output from the cardiac
ganglion, located on the dorsal inner wall of the heart. Following isolation
of the heart, the sternal artery was cannulated and perfused with
temperature-controlled lobster saline at 3.5 ml min–1 to
maintain stretch. The antennal arteries were tied off with 6.0 surgical silk
and attached to a tension transducer (model FT-03; Grass Instruments, Quincy,
MA, USA) and amplifier (CyberAmp model 320; Axon Instruments, Union City, CA,
USA) to record contractions of the heart during each heartbeat. All
physiological signals were recorded on VCR and digitized by an analog to
digital converter using pClamp software (Digidata1200 A-D converter and pClamp
software from Axon Instruments-Molecular Devices, Union City, CA, USA).
Chronic electrodes were implanted to record heart rate and ventilation rate
by measuring changes in impedance resulting from movements of the heart and
the gill balers (scaphnogathites), respectively. The procedure for
implantation of chronic electrodes for recording lobster cardiac activity
in vivo has been described previously
(Worden et al., 2006).
Briefly, two wires insulated to within 0.5 cm of their tips were inserted
through small holes drilled 3 cm apart in the dorsal carapace overlying the
heart and glued in place. To record ventilation a second set of electrodes was
implanted on one side of the ventrolateral thorax in the region overlying the
gills. All animals were allowed a 48 h recovery period before physiological
experiments began. Control experiments verified that implantation of the
electrodes did not alter the pH of the hemolymph.
To measure heart and ventilation rates individual lobsters were placed in a temperature-controlled chamber and acclimated to a temperature of 2°C for at least 30 min before the water was warmed at a rate of approximately 0.75°C min–1 to a maximal value of 30°C. Heart and ventilation rates were measured in the same experiments by alternating recording periods of 30 s for each parameter as a function of temperature. Signals from the electrodes were input to an impedance converter (UFI model 2991), digitized by pClamp software and stored on VCR tapes. In contrast to the heart, which beat rhythmically at all temperatures, the movements of the gill balers were interrupted occasionally by periods when ventilation spontaneously ceased. If ventilation halted during the recording period, the ventilation rate was calculated over a period of at least 15 s where ventilation occurred. Trials in which ventilation could not be recorded were not included in calculations of mean ventilation rates (<2% of observations). Lobsters were cold acclimated by housing them in aquaria at a temperature of 4°C for 3 weeks or more. Warm acclimated lobsters were housed at a temperature of 20°C for a period of at least 2 weeks before data were recorded.
Repeated-measures models (Crowder and
Hand, 1990) were used for the analyses of experiments involving
multiple measurements to investigate specific hypotheses concerning
comparisons between groups or to specific temperatures. F-tests were used to
compare the values for cardiac parameters at different temperatures. Linear
spline models were fit to analyze the relationship between the temperature and
ventilation rate as well as heart rate. Analyses were performed using SAS
software (The SAS Institute, Cary, NC, USA) version 9.1 module `PROC
MIXED'.
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Results |
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pH/T coefficient of –0.011 pH
°C–1. Values of hemolymph pH measured in lobsters
acclimated to natural seawater at ambient seasonal temperatures were in good
agreement (open symbols). Lobsters acclimated to warmer temperatures also had
significantly higher heart rates (see Fig.
1, inset) and relatively high levels of locomotor and startle
activity (data not shown) compared with cold acclimated lobsters.
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). To test
whether storing hemolymph might affect pH values we compared pH in the same
samples before and after overnight storage on ice or at –80°C. In 20
samples drawn over a range of acclimation temperatures, the value of the pH
dropped significantly [from 7.83±0.16 (mean ± s.e.m.) to
7.77±0.129; two-sample paired t-test, P=0.02] after
storage on ice overnight. In a similar experiment in which samples
(N=11) were stored at –80°C for 12 days the pH value also
dropped significantly (from 7.72±0.22 to 7.601±0.14; two-sample
paired t-test, P=0.03). Slopes of linear fits to plots of
the change in hemolymph pH relative to the original pH were –0.391 for
samples stored on ice and –0.511 for samples stored at –80°C.
Both values are significantly different from zero (P=0.0093 and
P=0.0026, respectively), demonstrating that samples that were
originally the most alkaline showed the greatest degree of acidification as a
consequence of storage. Therefore, subsequent experiments were designed such
that pH was always measured immediately after withdrawal of the hemolymph, to
avoid potential artifacts associated with storage of the samples.
Other parameters of acid–base status in the lobster also differ as a function of the temperature to which the lobsters are acclimated. Fig. 2 shows the total CO2 measured in the hemolymph of lobsters acclimated to different temperatures as well as the concentrations of bicarbonate, carbonate and PCO2 in hemolymph calculated from the pH and total CO2 values of the samples. Total CO2 depends on water temperature, with the highest values measured at 12°C and significantly lower values at the extremes of 4°C and 20°C (Fig. 2A). Bicarbonate levels, like total CO2 levels, were significantly higher at 12°C than they were at 4 or 20°C (Fig. 2B). Carbonate levels did not change between 4 and 12°C but were significantly lower at 20°C (Fig. 2C). Levels of PCO2 doubled between 4 and 12°C, but did not change significantly at higher temperatures (Fig. 2D).
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The time course of acid–base changes in response to acute temperature change
A recent study reporting a significant drop in hemolymph pH in lobsters
exposed to 3 weeks of warm (23°C) temperatures attributed the acidosis to
`prolonged thermal stress' (Dove et al.,
2005). To test whether hemolymph pH acidifies over several weeks
of acclimation over a range of temperatures, we repeatedly sampled hemolymph
pH in populations of lobsters undergoing 3 weeks of acclimation to water
temperatures of 4, 12 and 20°C. Hemolymph pH did not vary significantly
over time at acclimation temperatures of 4 and 12°C (P values for
linear fits were P=0.28 and P=0.09, respectively),
suggesting that pH remains relatively stable at colder acclimation
temperatures. However, pH values measured in lobsters housed at 20°C
tended to become more acidic over the 3 weeks of acclimation, at a rate of
0.0075 pH units day–1 (P=0.004). At this rate,
calculations of the change in hemolymph pH in lobsters over 3 weeks of
exposure to 20°C would predict acidification by an average of
approximately 0.16 pH units.
To test how quickly hemolymph pH might change during acute temperature shifts we measured pH repeatedly in a single lobster as the temperature of the surrounding seawater increased from 2 to 12°C over a 35-min time period. Fig. 3 illustrates the results from one of four experiments of this type. Hemolymph pH acidified rapidly as temperature warmed, dropping from 8.3 to 8.1 over a 3-min period before leveling off at 7.85 (Fig. 3A). Over the same time course levels of hemolymph PCO2 approximately doubled (Fig. 3B). These results qualitatively demonstrate that acute thermal change can strongly and rapidly alter the acid–base status of the hemolymph.
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Quantitative measurements of the time course of changes in hemolymph acid–base status measured in a population of lobsters subjected to an abrupt temperature increase from 4 to 20°C are shown in Fig. 4. Hemolymph pH acidified by more than 0.3 pH units within the first 10 min and then remained stable for 6 h before recovering to control values at 24 h (Fig. 4A). The corresponding decreases in the concentrations of total CO2 and HCO –3 over 24 h were not statistically significant (Fig. 4B,C). However, values of PCO2 (Fig. 4D) more than doubled over the first 10 min, remained stable at 2 h and recovered completely at 6 h. By 24 h, PCO2 had decreased significantly compared with control values. The Davenport diagram illustrating these changes in acid–base status (Fig. 5) shows that the initial transient acidosis and subsequent recovery of hemolymph pH is not accompanied by significant changes in total CO2.
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Respiratory rate increases as a function of temperature
Our observation that hemolymph pH, total CO2 and
PCO2 levels are temperature dependent raises the
possibility that the lobster's physiological response to thermal change might
include changes in respiration. To test this directly we measured the
temperature dependence of ventilation rates in individual lobsters and made
simultaneous measurements of heart rates for comparison. In agreement with
previous reports (Camacho et al.,
2006; Worden et al.,
2006), lobster heart rates increase over the temperature range
between 2 and 20°C but decrease at higher temperatures
(Fig. 7A). In addition, heart
rates are significantly higher in warm acclimated lobsters exposed to
temperatures of 22°C and above. In the same lobsters the rate of
ventilation also increased as a function of temperature
(Fig. 7B), with warm acclimated
lobsters exhibiting significantly higher respiratory rates at temperatures
>10°C in comparison with cold acclimated lobsters.
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). However, it
is important to note that because mean rates of ventilation were calculated by
excluding periods when ventilation ceased (see Materials and methods), the
plots in Fig. 7B overestimate
the ventilation rates in cold acclimated lobsters and therefore underestimate
the differences between ventilation rates in cold- and warm-acclimated
lobsters. |
Discussion |
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),
cardiac function (Camacho et al.,
2006; Worden et al.,
2006), growth and reproduction
(Ennis, 1995;
Waddy et al., 1995), and
locomotion into lobster traps (Drinkwater
et al., 2006). As reported for other crustacean species, we find
the pH of the hemolymph of H. americanus varies inversely with
temperature. The value for the coefficient pH/T
(–0.011 pH °C–1) in H. americanus is in
good agreement with that measured in other marine decapods (reviewed by
Truchot, 1983), as well as
with the coefficient describing the variations of the neutral point of pure
water with temperature, suggesting that lobster hemolymph follows the rule of
relative alkalinity. Over the temperature range from 4 to 20°C hemolymph
pH values in the lobster decrease from a mean value of 7.9 to nearly 7.7.
These values are similar to those measured over the same thermal range in the
crabs Carcinus maenas (Truchot,
1978) and over the thermal range 10–30°C in the blue
crab Callinectes (Wood and
Cameron, 1985) and at a temperature of 14–15°C in the
lobster Homarus vulgaris (a species also known as Homarus
gammarus) (McMahon et al.,
1978).
However, previous studies of the properties of the hemolymph in H.
americanus reported more acidic values for pH: 7.45–7.61 for
lobsters in different seasons (Cole,
1940), 7.6 for lobsters at 15°C
(Stewart et al., 1966), and
values in the range 7.2–7.4 for lobsters maintained at 16 or 23°C
(Dove et al., 2005). We
suspect that some of these pH values might be artificially depressed because
we have observed that hemolymph acidifies with storage (see Results), and
authors of previous studies (Dove et al.,
2005; Stewart et al.,
1966) stored hemolymph before measuring pH. Moreover, earlier
authors did not comment on whether their lobsters struggled during the
sampling procedure. We noted that pH values were more acidic in lobsters that
struggled during sampling (data not shown), in agreement with previous reports
that pH of the hemolymph in H. vulgaris acidifies in response to air
exposure and struggling during handling
(McMahon et al., 1978) and
that the acid–base chemistry of H. americanus hemolymph varies
as a function of exercise, handling and disturbance
(McMahon, 1995;
Rose et al., 1998). Overall,
these results suggest the importance of sampling from quiescent animals,
minimizing handling procedures and assaying pH immediately after hemolymph
withdrawal in order to obtain the most accurate measures.
Interestingly, many previous neurophysiological studies have employed a
saline developed specifically for H. americanus in which pH is
buffered to 7.4 (e.g. Bykhovskaia et al.,
1999; Bykhovskaia et al.,
2004; Golan et al.,
1994; Golan et al.,
1996; Goy and Kravitz,
1989; Grossman and Kendig,
1990; Kravitz et al.,
1980; Vorob'eva et al.,
1999; Worden et al.,
2006; Worden et al.,
1997; Worden and Camacho,
2006; Worden et al.,
1995). The composition of this saline is based on a `perfusing
solution' that Cole developed more than 60 years ago by comparing a series of
solutions with varying concentrations of sodium, potassium, calcium, magnesium
and sulfate to determine which was the optimal mixture for maintaining the
strength and frequency of the lobster heartbeat in vitro at 17°C
(Cole, 1941). The pH of all the
solutions was buffered to 7.3–7.5, slightly lower than the author's
previous measures of hemolymph pH values of 7.45–7.61 in intact lobsters
(Cole, 1940). A value of 7.4 is
more acidic than any we measured in this study, even in samples from lobsters
housed at the warmest acclimation temperature. Whether the pH of the
traditional lobster saline is appropriate physiologically should be of
particular concern in the design of experiments performed at cold
temperatures, in which hemolymph in the intact animal is most alkaline and the
discrepancy between hemolymph pH and the traditional Homarus saline
pH is the greatest. Cold (
5°C) temperature protocols have been used in
studies of lobster neuromuscular transmission as a method of improving quantal
resolution by decreasing the synchrony of quantal neurotransmitter release
(Bykhovskaia et al., 1999;
Worden et al., 1997) and
enhancing the strength of inhibitory synaptic potentials
(Worden and Camacho,
2006).
Our demonstration that shifting pH within the physiological range
7.4–8.1 alters both the frequency and the strength of the lobster
heartbeat (Fig. 6) suggests
that thermally triggered changes in hemolymph pH could modulate cardiac
physiology in vivo. Previous studies of the temperature dependence of
lobster cardiac performance have demonstrated that temperature change directly
modulates the heartbeat strength, frequency and kinetics of isolated hearts
bathed in saline (at pH 7.4) in vitro, whereas heart rates measured
in vivo are similarly temperature dependent but faster
(Worden et al., 2006). The
higher heart rates observed in vivo have been ascribed to the
presence of neural and hormonal inputs in the intact animal that are absent in
the isolated heart. However, the results of the present study suggest another
possibility: intact lobsters exposed to increases in seawater temperature will
experience a fall in hemolymph pH in addition to warming of the hemolymph and
internal tissues, and both effects tend to increase heart rate. Additional
experiments will be required to determine whether pH might affect the
neurophysiological properties of neurocardiac synapses on the heart or the
process of excitation–contraction of lobster cardiac muscle. Precedence
for the idea that extracellular pH modulates muscle membrane currents has been
described for crayfish skeletal muscle
(Pasternack et al., 1992).
Finally, Dove et al. observed a depression in hemolymph pH by –0.2 pH
units in lobsters moved from 16°C to 23°C for a period of 3 weeks and
attributed the acidosis to the `prolonged thermal stress'
(Dove et al., 2005). Although
our results confirm that the pH of lobster hemolymph decreases slowly by
approximately –0.16 units over 3 weeks of acclimation to warm (20°C)
water, we also observed that hemolymph pH changed by 0.2 to 0.3 pH units
within minutes of a temperature increase from 2 to 12°C or from 4 to
20°C (see Figs 3 and
4, respectively). These results
demonstrate that thermal stimuli that are neither prolonged nor sufficiently
warm to be physiologically stressful can trigger significant changes in
hemolymph pH. A temperature change from 4 to 12°C (see
Fig. 3), for example, is within
the cooler half of the entire temperature range lobsters inhabit in their
natural habitat and yet it depresses hemolymph pH within minutes. We interpret
the temperature-dependent changes in lobster hemolymph pH as a passive
response to temperature that approximates to that of water (the rule of
relative constant alkalinity), rather than as a true acidosis.
Temperature and acid–base balance in crustaceans
Although the temperature dependence of hemolymph pH in H.
americanus is similar to that previously reported for the crab C.
meanus, a species that shares its geographical and thermal habitat, our
data also suggest that these two species differ in terms of the temperature
dependence of total CO2. Crabs acclimated to different water
temperatures show a monotonic decrease in total CO2 as temperature
increases from 5 to 25°C (Truchot,
1978). By contrast, in temperature-acclimated lobsters total
CO2 peaks at 12°C and is lower at cold (4°C) and warm
(20°C) temperature extremes (see Fig.
2), an effect than can be attributed primarily to the temperature
dependence of the lobster hemolymph bicarbonate concentration. In addition,
PCO2 in C. maenas increases linearly as a
function of temperature, doubling between 5 and 25°C. By contrast, in
lobster PCO2 doubles between 4 and 12°C and then
decreases by approximately 20% at 20°C. Overall, the primary differences
between temperature-acclimated lobster and crabs in terms of the temperature
dependence of total CO2, HCO –3 and
PCO2 appear at the coldest temperatures. Compared with the
mid-range temperature of 12°C, levels of CO2 and HCO
–3 at 4°C are depressed in lobster but
elevated in the crab.
Another difference between these two species is that their acid–base
status undergoes different changes in response to abrupt temperature
increases. Both C. maenas and H. americanus initially
respond to a temperature increase with hemolymph acidosis and an increase in
blood PCO2 that occurs within the first 10 min and is
sustained over the next 6 h. By 24 h both parameters recover to near control
values in the lobster (see Fig.
4). By contrast, neither parameter returns to control values in
the crab; pH remains relatively stable and acidotic whereas
PCO2 remains stable and elevated by more than 50% [see
fig. 2 of Truchot
(Truchot, 1978)]. Truchot
concluded that crab hemolymph maintains a constant acid–base state as
defined by the relative alkalinity. Whereas lobsters acclimated to temperature
over days to weeks also follow the rule of relative alkalinity (see
Fig. 1), an abrupt temperature
increase from 4 to 20°C produces changes in lobster hemolymph
acid–base status over 24 h that are consistent with a respiratory
compensated metabolic acidosis (see Fig.
5). Respiratory compensated metabolic acidosis has previously been
observed in the crab C. magister following strenuous exercise
(McDonald et al., 1979), but
not in the lobster H. americanus, where exercise triggers a
predominantly respiratory acidosis (Rose
et al., 1998). The results of the present study suggest that the
acute and transient decrease in hemolymph pH that occurs when lobsters are
subjected to an abrupt temperature elevation will be followed by a gradual
decrease in pH over the subsequent days and weeks of acclimation to warm
temperature.
It is unclear as to what accounts for the differences between our
observations on H. americanus and earlier reports describing
acid–base regulation in the crab. Although it is possible that there are
true species-specific differences in respiratory physiology between these
species, it may also be the case that differences in experimental approaches
can explain the apparent discrepancies in the results. For example, Truchot
reported that gill ventilation does not increase as a function of temperature
in C. maenas, based on an indirect method in which he calculated
water convection requirements from oxygen consumption records recorded
intermittently over a period of days and only after (not during) a temperature
change (Truchot, 1983). By
contrast, we directly and continually observed ventilation rates in H.
americanus both during and after a temperature change. Further
experiments will be required to resolve the interesting issue of whether there
are true differences between the respiratory physiology of lobsters and crabs,
and how these differences might relate to the temperature dependence of the
behavioral ecology of each species.
Our observation that ventilation rates increase significantly in laboratory
lobsters as temperature warms (Fig.
7B) are in agreement with previous observations that lobster
ventilation rates increase in the wild as the ocean waters seasonally warm
(Mercaldoallen and Thurberg,
1987). In the present study, lobsters acclimated to warm
(20°C) temperatures exhibited especially strong temperature-dependent
increases in ventilation: at temperatures of 20–26°C ventilation
rates in warm-acclimated lobsters were 2.5- to 7-fold higher than those
measured in cold (4°C)-acclimated lobsters. Warm acclimation also
increases heart rates at warm (20–26°C) temperatures, although the
magnitude of this increase is much smaller, in the order of 50–60% (see
Fig. 7A), in agreement with
previous results (Camacho et al.,
2006). Overall, these observations suggest (1) that thermal
acclimation alters the neural output of the circuits that drive the beating of
the heart and the movement of the gill balers, and (2) that the circuitry
driving respiration is more sensitive to warm acclimation than the circuitry
driving the heart. The factors that determine the upper limit of thermal
tolerance in lobster are unknown but are likely to be related to the inability
of ventilatory and circulatory systems to supply sufficient oxygen (or other
metabolites) to the tissues, as shown for the spider crab Maja
squinado (Frederich and Portner,
2000).
Overall, the results of this study suggest that several factors play a role
in determining the temperature dependence of lobster hemolymph pH. From 12 to
20°C pH/T can be attributed to the decrease in [HCO
–3] and increase in metabolism at high
temperatures, as shown by the increase in heart rate (see
Fig. 1, inset). However, from 4
to 12°C pH/T can be attributed to the increase in
PCO2, with this change explained by the classic closed
system of the hemolymph (Reeves,
1977). Wood and Cameron observed a similar phenomenon in
Callinectes sapidus and noted that it is not clear why this should
occur in a water breather, because CO2 is generally excreted easily
across the gills (Wood and Cameron,
1985). However, because PCO2 is relatively low
in crustacean blood, even relatively small changes in PCO2
can have a large effect on pH, as demonstrated for fish blood in which
measured PCO2 levels are in the same range
(Perry and Wood, 1989).
In summary, thermal acclimation and thermal change alter not only the
acid–base properties of lobster hemolymph but the properties of the
neural circuits driving lobster ventilation and cardiac activity. In response
to abrupt temperature increases lobsters undergo a respiratory compensated
metabolic acidosis of sufficient magnitude that the fall in hemolymph pH can
modulate the strength and frequency of the heartbeat. These observations
suggest that thermally triggered changes in hemolymph acid–base status
can act to modulate lobster cardiac function, and perhaps other physiological
systems as well. In their native habitat, lobsters experience thermal change
as water currents flowing along the ocean floor transiently engulf them, as
storms churn the ocean waters, as tides ebb and flow and as the seasons
progress. Given that the environmental stress of high seawater temperatures
has been linked both to the prevalence and spatial distribution of lobster
shell disease (Glenn and Pugh,
2005) and to unusually high levels of lobster mortality and low
levels of abundance of H. americanus in Southern New England and Long
Island Sound waters in recent years
(Howell et al., 2005),
understanding the thermosensitivity and thermal tolerance of this commercially
valuable species becomes especially important. Interestingly, lobsters
acclimated to warm (20–22°C) temperatures appear especially
physiologically efficient at delivering oxygen to the tissues and clearing
PCO2 from the hemolymph, suggesting that they can
partially compensate for thermal stress that might otherwise endanger the
health and survival of this species.
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Acknowledgments |
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References |
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5, except as follows: heart rate in cold-acclimated animals
at 30°C (N=1), heart rate in warm-acclimated animals at 26°C
(N=4) and 30°C (N=2), ventilation in cold-acclimated
animals at 6°C (N=3), at 22°C (N=4), at 24°C
(N=4), at 26°C (N=1) and at 28°C (N=1), and
ventilation in warm-acclimated animals at 2°C (N=4), at 28°C
(N=4) and at 30°C (N=2).