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First published online November 17, 2006
Journal of Experimental Biology 209, 4663-4675 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02581
Lipid remodeling in wild and selectively bred hard clams at low temperatures in relation to genetic and physiological parameters
1 Institut de Recherche sur les Zones Côtières, 232B rue de
l'Église, Shippagan, Nouveau-Brunswick, E8S 1J2, Canada
2 Institut des Sciences de la Mer, 310 allée des Ursulines, Rimouski,
Québec, G5L 3A1, Canada
3 Department of Fisheries and Oceans, Science Branch, Gulf Fisheries Centre,
PO Box 5030, Moncton, New Brunswick, E1C 9B6, Canada
* Author for correspondence (e-mail: fpernet{at}umcs.ca)
Accepted 4 October 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: lipid composition, sterol, fatty acid, homeoviscous adaptation, bivalve, winter acclimatization
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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).
A decrease in temperature usually induces an increase in the overall packing
order of membrane phospholipids (i.e. a decrease in membrane fluidity) and
elevated temperatures decrease membrane order. Large changes in membrane order
can lead to membrane dysfunction. Poikilotherms inhabiting eurythermal
environments usually counteract this temperature effect by remodeling membrane
lipids, a process known as homeoviscous adaptation
(Sinensky, 1974;
Hazel, 1995). Remodeling
membrane lipids during thermal acclimation or acclimatization commonly
involves changes in phospholipid headgroups, acyl-chain composition and
cholesterol content (Hazel and Williams,
1990). These biochemical responses render the membrane more or
less fluid, which is believed to be a compensation for the direct effect of
temperature change on membrane structure. Many intertidal organisms commonly
withstand variations in temperature of 20-30°C on a daily basis and
encounter even wider thermal ranges on a seasonal basis. Several studies have
shown that marine bivalves are able to regulate their membrane fluidity in
response to temperature changes. For example, the mussel Mytilus
californianus exhibits strong seasonal variations in membrane fluidity
that are consistent with homeoviscous adaptation
(Williams and Somero, 1996). A
recent study showed that membrane fluidity in the gills of the sea scallop
Placopecten magellanicus is positively correlated with the level of
20:5n-3 and negatively correlated with acclimation temperature, which may
represent a mechanism to maintain membrane function at low temperatures in
this species (Hall et al.,
2002).
Unexplained overwintering losses of juvenile first-year hard clams
Mercenaria mercenaria in different years and at different grow-out
sites have been reported in Atlantic Canada (T. Landry, personal
communications). It has also been reported that the selectively bred hard clam
M. mercenaria var. notata, a variant of the native hard clam
distinguished by brown bands on the shell surface, which usually shows
improved growth performance (Chanley,
1961), suffer higher overwintering mortalities than the wild
species (Bricelj et al., 2006).
This evidence indicates that the two varieties of hard clams might show
different degrees of adaptation to low temperatures. However, little is known
about the physiological and biochemical responses of this species to low
temperatures, especially in juveniles, as most prior studies have focused on
the physiology of adults.
The hard clam is an infaunal suspension-feeding bivalve occurring in
shallow and turbid estuarine environments and is widely distributed along the
east coast of North America from the baie des Chaleurs (Gulf of St Lawrence,
Canada) to the Florida Keys. In Canada, M. mercenaria is mainly found
from the baie des Chaleurs in New Brunswick to Cape Breton Island in Nova
Scotia at both the intertidal and subtidal depths. This eurythermal species
can tolerate temperature between 0 and 30°C with optimum growth at
20°C (Grizzle et al.,
2001). Because of the difficulties in producing fast-growing hard
clams using native Canadian stocks, the PEI Department of Fisheries,
Aquaculture and Environment set up trials to determine if M.
mercenaria var. notata could be utilized successfully in eastern
Canada. Commercial hatcheries in the US used the variety notata in a
selective breeding program, apparently with no strict application of
quantitative genetics (Hadley et al.,
1991), to distinguish it from wild stock and to develop a more
rapidly growing animal (Chanley,
1961). In 1997, a sample of 60 adult hard clams from the
Aquaculture Research Corporation in Dennis, Massachusetts, USA was imported to
the Ellerslie Shellfish Hatchery in PEI. The F1 generation was
placed at six grow-out sites to assess the performance of the variety
notata in the field for 5 years and was then recovered to produce the
F2 in 2003.
In our study, wild (F1) and selectively bred hard clam juveniles
from the Ellerslie hatchery were placed in the field at their northern
distribution limit (Neguac, NB, Canada) from August 2003 to May 2004. Over the
course of this study, animals were exposed to a gradual lowering in
temperature from 
24 to 0°C over 4 months and maintained at
overwintering temperatures <0°C for 3.5 months; they were regularly
sampled for lipid analysis. Our present investigation focuses on the changes
in lipid class and fatty acid composition in wild and selectively bred hard
clams in relation to their metabolic requirements and genetic characteristics
as evaluated by the degree of heterozygosity. Several studies have shown
negative correlations between heterozygote deficiency and survival in marine
bivalves exposed to stressful environmental conditions, and this relationship
was attributed to higher metabolic requirements for homozygous individuals.
For example, mussel stocks susceptible to summer mortality had a lower degree
of heterozygosity and showed higher oxygen consumption rates than mussels from
resistant stocks, thus suggesting that the energy expenditure for maintenance
was higher for mussels from susceptible stocks
(Tremblay et al., 1998;
Myrand et al., 2002). These
results are in agreement with several other studies on molluscs that indicated
that more heterozygous individuals show more efficient protein synthesis and a
higher scope for growth compared to more homozygous individuals
(Hawkins et al., 1989), which
results in higher growth and survival rates
(Zouros and Foltz, 1987;
Koehn, 1991;
Mitton, 1993). There are
similarities between our study and previous work done on mussels: there is
high seasonal mortality, populations are characterized by a heterozygote
deficit, and relationships exist between the genetic make-up and physiology.
The novelty of our study is that we examined a different species in which the
mortality occurs in winter, and the physiological character that we relate to
the genetic difference is lipid biochemistry.
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Materials and methods |
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).
Clams of each variety (mean shell length 11.0±1.2 mm) were
transferred on 15 July 2003 to Neguac, NB, in the Gulf of St Lawrence
(47°N; 65°W), the northern distribution limit of this species. Seed
were set at two tidal locations: individuals close to the littoral zone (i.e.
subtidal site) were submerged during low tide, but individuals near the upper
end of the distribution (i.e. intertidal site) were exposed for 6-8 h between
high tides. Clams were held under predator-proof mesh covers mounted on 1.8
x 1.2 x 0.3 m steel tables at an initial stocking density of
6000 clams m-2 (Kraeuter
et al., 1998). Tables were filled with sediment that had been
sieved to remove predators and gravel. One table was used per combination of
tidal location and variety. To achieve replication, 72 subsections within each
table were delimited and randomly chosen for sampling. On 30 October, before
winter ice set in, a group of clams was overwintered subtidally in the field
while another group was transferred to the laboratory at CZRI until 16 May
2004 to allow sampling during winter. In the laboratory, animals were held in
a 630 l tank continuously supplied with 12 µm sand-filtered seawater. Clams
were fed 10 cells µl-1 Instant Microalgae Shellfish Diet
1800® (Reed mariculture, Campbell, CA, USA). Animals were not fed between
17 December and 28 April, when seawater temperature was <0°C.
Variety characterization
Genetic
Juvenile hard clams were stored at -80°C at CAMGR for later
electrophoretic study of enzymes as described
(Tremblay et al., 1998). A
small piece of muscle was homogenized in an approximately equal volume of
homogenization buffer (0.2 mol l-1 Tris-HCl, pH 8.0, with 30%
sucrose, 1% polyvinyl-polypyrrolidone, 0.1% NAD, 5 mmol l-1
dithiothreitol and 1 mmol l-1 phenylmethylsulfonyl fluoride),
centrifuged at 15 000 g for 30 min at 4°C, and the
supernatant applied to a horizontal cellulose acetate gel
(Hebert and Beaton, 1989).
Eight polymorphic allozymes were analyzed: glucose phosphate isomerase
(GPI*, EC 5.3.1.9), phosphoglucomutase
(PGM*, EC 5.4.2.2), mannose phosphate isomerase
(MPI*, EC 5.3.1.8), pyruvate kinase
(PK*, EC 2.7.1.40), alkaline phosphatase
(ALP*, EC 3.1.3.1), fumarate hydratase
(FUM*, EC 4.2.1.2), isocitrate dehydrogenase
(IDH*, EC 1.1.1.42) and leucine aminopeptidase
(LAP*, EC 3.4.11.1). Alleles were named A, B, etc., in
order of electrophoretic mobility, such that the slowest allele was called A.
For each variety, 144 clams were individually analyzed.
Metabolism
After 21 days of acclimation to laboratory conditions, maximal oxygen
consumption
(
or maximum metabolic rate) was approximated by feeding the clams at a maximal
rate for 4 days by increasing the food ration to 15 x 103
cells ml-1 while minimum oxygen consumption
(
or standard metabolic rate) was measured after starving the same clams for 14
days. Pseudofaeces were observed during measurement of
.
Oxygen uptake was measured for 50 animals of each variety in individual 50 ml
chambers. Clams were kept individually in their metabolic chambers for 60 min
before starting measurement. Empty shells were used as a procedure blank. Six
chambers were used simultaneously, which allowed us to measure five clams and
one blank (empty shell) at a time. Animals that remained closed in the chamber
were excluded from physiological analysis. Oxygen consumption for an
individual clam was determined by sealing the chamber and measuring the
reduction in %O2 with a YSI (5331) polarographic analyzer and
electrode (Yellow Springs, OH, USA). Seawater was well-mixed with a magnetic
stirrer. The output signal was monitored continuously on a chart recorder
until a decrease of at least 20% O2 was reached. After oxygen
uptake, each set of clams was frozen at -80°C for later determination of
dry mass, which was measured after drying at 70°C for 72 h. Respiration
rate was expressed as the rate expected for a standard clam with a dry mass of
1 g by applying the allometric correction (ml-1 g-1 dry
mass h-1) described by Widdows and Jonhson
(Widdows and Jonhson,
1988).
Temperature measurements
Temperature was recorded every hour with 8-bit Minilog-TR data loggers from
Vemco (Shad Bay, Nova Scotia, Canada) at two tidal locations from 11 August
until 30 October 2003. The data loggers were anchored to the sand tables and
placed on the sand. Subtidal temperature monitoring continued in the field
until 16 May 2004. We also monitored water temperatures in the laboratory
holding tank.
Lipid analysis
Sampling
Sampling took place in the field from 15 August until 6 October and then in
the laboratory until 16 May. Clams were sampled simultaneously in the field
and in the laboratory on 16 May to ensure that there was agreement between
results from the field and the laboratory. Every 1-2 months, clams were
removed from five randomly chosen subsections per table for determination of
shell length, whole tissue dry mass, and lipid composition of soft tissues.
Clams were carefully dissected and 300 mg wet mass of tissue,
corresponding to a pool of 5-10 individuals per subsection, was frozen at
-80°C for later determination of dry mass and lipid composition.
Lipid classes
Lipids were extracted following the method of Folch et al.
(Folch et al., 1957), spotted
onto S-III Chromarods (Iatron Laboratories Inc., Tokyo, Japan), and separated
into aliphatic hydrocarbons, sterol and wax esters, ketones, triacylglycerols,
free fatty acids, free fatty alcohol, free sterols, diacylglycerols, acetone
mobile polar lipids and phospholipids
(Parrish, 1999). Chromarods
were scanned by a flame ionization detection system (FID; Iatroscan Mark-VI,
Iatron Laboratories Inc., Tokyo, Japan) and chromatograms were analyzed using
integration software (Peak Simple version 3.2, SRI Inc.).
Fatty acids
Fatty acid methyl esters (FAME) were prepared using 12% BF3 in
CH3OH following the American Oil Chemists' Society method
(AOCS, 1989). FAME were run on
an SRI 8610C gas chromatograph equipped with a DB-Wax fused-silica capillary
column (30 mx0.25 mm ID x0.25 µm film thickness; Supelco,
Bellfonte, PA, USA). Hydrogen was used as the carrier gas (flow velocity: 80
cm s-1 at 145°C). FAME were introduced into a glass liner
(uniliner, drilled, 4 mm, 6.3 78.5 mm; Restek, Bellfonte, PA, USA) maintained
at 300°C. The temperature ramp was 58°C for 4 min at 40 psi (275.79
kPa), followed by an increase of 20°C min-1 to 170°C at 20
psi (137.895 kPa), followed by an increase of 1°C min-1 to
180°C, and finally by an increase of 2°C min-1 to
220°C, where it was held for 5 min. The detector was maintained at
260°C. FAME were identified by comparison of retention times with known
standards (37 component FAME Mix, PUFA-3, BAME and menhaden oil; Supelco
Bellefonte, PA, USA) and quantified with tricosanoic acid (23:0) as an
internal standard. The non-methylene interrupted dienoic fatty acids (22:2
NMI) were clearly identified by the comparison of our chromatograms with those
from Kraffe De Laubarede (Kraffe De
Laubarede, 2003) for the same species. Chromatograms were analyzed
using integration software (Peak Simple version 3.2, SRI Inc).
Statistical analyses
Allelic and genotypic frequencies for the polymorphic loci were obtained
using GENETIX 4.05 (Belkhir et al.,
1998). The fixation index (Fis), which represents the
Mendelian equilibrium and deviation from Hardy-Weinberg equilibrium, was
calculated using GENEPOP 3.3 (Raymond and
Rousset, 1995). Index of genetic differentiation, Fst,
was calculated using methods of Weir and Cockerham
(Weir and Cockerham, 1984) and
significant differences between allelic frequencies were tested using the
Fisher exact test; both of these tests are implemented in GENEPOP 3.3.
Significance levels for statistical tests were adjusted according to the
sequential Bonferroni procedure (Rice,
1989).
A repeated-measures analysis of variance (ANOVA) was conducted to determine differences in oxygen consumption between the two varieties of hard clam, M. mercenaria and the selectively bred M. mercenaria var. notata, under two feeding regimes (high and starvation) in the laboratory. Since maximal oxygen consumption was approximated by feeding the clams at a maximal rate for 4 days and minimum oxygen consumption was measured after starving the same individuals for 14 days, the two measurements of oxygen consumption constituted a repeated measures factor.
Three-way ANOVAs were conducted to determine differences in the phospholipid to sterol ratio and the unsaturation index (average number of double bonds per acyl chain) as a function of time, tidal location and variety. A three-way multiple analysis of variance (MANOVA) was carried out on major polyunsaturated fatty acids (PUFA), namely 22:6n-3, 20:5n-3 and 22:2 NMI, as a function of time, tidal location and variety.
Owing to logistical limitations, only one table was used per combination of
tidal location x variety, which means that the experiment was not
replicated and the interaction between the two experimental factors cannot be
estimated. Therefore, the unit of replication used in these analyses was the
subsection of the table instead of the table itself. In theory, our approach
violated a fundamental assumption of the ANOVA, which is that each measurement
on each table is independent of every other measurement. However, the
independence of data among samples is a biological issue: biological
(behavioral and ecological) processes cause non-independence among replicates
[(Underwood, 1997), p. 179].
Here we supposed that the biochemical response of clams to each other when on
the table would have no serious outcome for statistical tests on data derived
from animals in groups. Indeed, a previous study clearly showed that there was
no effect of clam density on growth or survival of clams maintained at the
initial stocking density used in our study [6000 clams m-2
(Kraeuter et al., 1998)].
Finally, another limitation of our approach is that it did not allow us to
unambiguously separate out the interaction of location x variety from
among-table differences.
Where differences were detected, Tukey's HSD multiple comparison tests were used to determine which means were significantly different. Residuals were screened for normality using the expected normal probability plot and further tested using Shapiro-Wilk. Residuals never showed significant deviation from normality for any variable. Homogeneity of variance-covariance matrices were graphically assessed and further tested using the Levene test for each variable. Analyses were carried out using SPSS 10.0 (SPSS Inc., Chicago, IL, USA).
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2 test was markedly higher for M. mercenaria
var. notata (376.4) compared with the wild variety, where the
Fisher's 2 test was 44.7. Furthermore, it must be kept in mind
that the Fis value of ALP* in the wild variety
included a dominant allele frequency of 0.96, which biases the statistical
test because of the predominant influence of rare alleles
(Hummel et al., 2001). The two
tested varieties of hard clams showed significant differences in their allelic
frequencies for GPI*, PGM*,
MPI* and ALP* (Fisher exact test for
each of these four loci: 2=
; d.f.=16;
P<0.001). The overall Fst value between the two varieties
of clam was 0.032 (P<0.001), which indicates a high level of
genetic differentiation between them.
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The oxygen uptake of the selectively bred M. mercenaria var.
notata showed increases of 22 and 33% in their mass specific rates
compared with those of the wild type after feeding to satiety and after food
deprivation, respectively (Fig.
1, variety effect: P=0.024, F=5.33, d.f.=1;
feeding regime effect: F=171.41, d.f.=1, P<0.001). There
was no interaction between clam variety and feeding regime (F=1.16,
d.f.=1, P=0.286). Therefore, selectively bred hard clams were
characterized by a higher heterozygote deficiency and a higher
than
the wild M. mercenaria.
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0.2°C day-1. The overwintering period,
from 15 December to 5 April, was characterized by stable temperatures of
-0.1±0.3°C in the laboratory and -1.6±0.2°C in the
field. Finally, the third period, from 5 April until the end of the study, was
characterized by a marked temperature increase, up to 8-9°C, which is
typical of spring. Averages and standard deviations in daily temperatures were
similar between intertidal and subtidal locations except at the end of
September, when intertidal temperatures seemed somewhat more variable than
those recorded at the subtidal location
(Fig. 2B).
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Lipid analysis
Lipid classes
Lipid classes detected in juvenile hard clams in this study were sterols,
acetone-mobile polar lipids and phospholipids. Minor amounts of triglycerides
(
0.5%) were occasionally detected and are not discussed further.
Acetone-mobile polar lipids, a lipid class that includes photosynthetic
pigments, glycolipids and monoacylglycerol, contributed 7% of the total
lipids in clam tissues and showed no consistent pattern over time in the
various treatments (data not shown). The phospholipid to sterol ratio,
generally considered as an indicator of membrane fluidity, varied as a
function of time, tidal location and variety
(Fig. 3). Indeed, wild M.
mercenaria held at the intertidal location were characterized by a
1.4-fold increase in their phospholipid to sterol ratio between Augusts and
October, immediately followed by a rapid decrease to initial values in
December before overwintering. M. mercenaria held at the subtidal
location showed a similar pattern of variation but exhibited a rate 2.6 times
higher than their intertidal counterparts (respectively 0.049 and 0.131
d-1). Interestingly, the selectively bred M. mercenaria
var. notata was characterized by a phospholipid to sterol ratio that
was stable with time, irrespective of tidal location.
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70% of the total fatty acids
(Table 2).
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The unsaturation index, which is the number of double bonds per 100 molecules of fatty acids, increased significantly, by 7.2% between October and December before overwintering, and remained elevated until the end of the study period. This increase in the unsaturation index coincided with a reduction in the seawater temperature in the field from 13.7°C to 0°C. The unsaturation index also varied as a function of tidal location (intertidal>subtidal) and variety of hard clam (M. mercenaria var. notata>M. mercenaria; Fig. 4). A stepwise multiple regression model using groups of fatty acids as explanatory variables and the unsaturation index as the response variable showed that the unsaturation index was positively correlated with PUFA (y=5.9 x PUFA-83.5; r2=0.928, N=171, F=2169.9, d.f.=1, P<0.001; Fig. 4). A second regression model using individual PUFA as explanatory variables showed that variations in the unsaturation index were attributable to 22:6n-3 (y=4.18 22:6n-3+228.7; r2=0.892, N=171, F=17485.9, d.f.=1, P<0.001; Fig. 4). Indeed, 22:6n-3 increased significantly by 29.9 and 34.6% in M. mercenaria var. notata and M. mercenaria, respectively, between October and December before overwintering, and remained elevated until the end of the study period, as observed for the unsaturation index. The selectively bred M. mercenaria var. notata showed a 2.7% increase in 22:6n-3 compared to the wild variety.
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23°C to 0°C, and remained constantly high (8.8%) during the
overwintering period (Fig. 6).
The selectively bred M. mercenaria var. notata showed a 4.8%
reduction in 22:2 NMI compared to the wild variety.
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). Although the
responses of cholesterol to temperature remain membrane-specific, some
positive correlations between membrane cholesterol content, as determined by
the cholesterol to phospholipid ratio, and acclimation temperature have been
reported in several tissues of rainbow trout
(Robertson and Hazel, 1995). A
widely accepted notion is that cholesterol stabilizes membranes, i.e. it
increases the order of the surrounding acyl chains in membranes in the fluid
phase (Crockett, 1998).
Therefore, the observed increase in the phospholipid to sterol ratio during
the early stage of decreasing temperatures in M. mercenaria is
consistent with a role for cholesterol in the membrane's homeoviscous
response. It is worth mentioning that the sterol profile of marine bivalves
consists of a complex mixture of C26 to C30 sterols
(cholesterol=C27), with each molecular species being characterized
by a planar ring system with a 3-hydroxyl group and a side chain of varying
length (Knauer et al., 1998).
However, an increase or decrease of one or more carbons in the side chain
length reduces the ordering effect on membranes compared with cholesterol
(Suckling et al., 1979), thus
raising the need for caution in interpreting the modulation of sterols from
marine bivalves in relation to cholesterol in other species.
The unsaturation index increased during the later stage of the temperature
decrease between November and December, principally due to 22:6n-3, and is
primarily responsible for the maintained differences in lipid composition
between fall- and winter-acclimatized clams. Although membrane fluidity was
not measured in our study, it is tempting to speculate that temporal
variations in 22:6n-3 represent an adaptive response of the membranes to
counteract the ordering effect of a decrease in the environmental temperature.
In support of this hypothesis, several studies have shown that molecular
species of phospholipids containing 22:6n-3 are important in controlling
membrane fluidity during cold acclimation of fish
(Dey et al., 1993;
Buda et al., 1994;
Tiku, 1996;
Logue et al., 2000). It is
noteworthy that absolute concentrations of 22:6n-3 increased from 266 to 305
µmol clam-1 between October and December while total fatty acids
decreased from 1679 to 1492 µmol clam-1, thus suggesting that
the machinery of membrane biogenesis favors the incorporation of 22:6n-3 at
low temperatures. A reliance on 22:6n-3 for controlling membrane fluidity in
hard clams could potentially limit their capacity for thermal acclimatization
since bivalves cannot de novo synthesize long-chain PUFA
(DeMoreno et al., 1976).
Therefore, 22:6n-3 needs to be obtained preformed from the diet. Since hard
clams cease feeding at temperatures of <6°C
(Grizzle et al., 2001), the
provision of exogenous 22:6n-3 prior to temperatures falling below 6°C is
probably an important constraint for the long-term acclimatization of hard
clams to low temperatures.
Little is known about the time course of changes in lipid composition
during thermal acclimatization of bivalves. Previous studies on rainbow trout
showed that exposure to low temperatures led to rapid and transient changes in
phospholipid head groups of kidney plasma membranes whereas PUFA increased
gradually during cold acclimation (Hazel
and Landrey, 1988a; Hazel and
Landrey, 1988b). In hard clams, the early transient increase in
the phospholipid to sterol ratio may be viewed as a short-term adjustment to a
lowering of environmental temperatures whereas the later increase in the
unsaturation index (attributable to PUFA) may be viewed as a long-term
adjustment to winter temperature. The rapid response of the phospholipid to
sterol ratio to a temperature decrease probably reflects the ease of use of
sterol in biological membranes. It has been suggested that the use of
cholesterol as a modulator may not require any expenditure of ATP or reduced
cofactors (Crockett, 1998).
Therefore, modulation of the phospholipid to sterol ratio probably provides a
metabolically less expensive mechanism of membrane remodeling than de
novo synthesis of PUFA-rich phospholipids. Alternatively, the influence
of cholesterol on the physical properties of membranes is greater in more
saturated membranes than in more unsaturated ones
(Kusumi et al., 1986).
Therefore, modulation of the phospholipid to sterol ratio may have a more
pronounced effect in clams living in a warm environment, which have relatively
saturated membranes, than in cold-acclimatized clams, which have relatively
unsaturated membranes, thus explaining why the phospholipid to sterol ratio
increases before the unsaturation index.
Juvenile hard clams exhibited an increase in 22:2 NMI during decreasing
temperatures; these fatty acids remained elevated during overwintering.
Although the functional role of 22:2 NMI fatty acids is not well understood,
they are believed to be involved in the regulation of membrane fluidity in
poikilothermic animals (Rabinovich and
Ripatti, 1991). In contrast to 22:6n-3 and other essential PUFA,
which need to be obtained preformed from the diet, NMI fatty acids are
synthesized de novo by bivalves
(Zhukova, 1991). Therefore,
synthesis of 22:2 NMI fatty acids could represent an alternative to the
selective incorporation of 22:6n-3 or other essential PUFA at low
temperatures. This biochemical pathway could be particularly important when
animals cannot acquire essential PUFA from their environment because of low
concentrations in phytoplankton or when feeding has ceased at low
temperatures. In these cases, a higher biosynthesis of the NMI fatty acid in
wild Mercenaria may provide an advantage at low temperatures by
reducing the need for other essential PUFA.
M. mercenaria showed an increase in 20:5n-3 along with decreasing
temperatures but did not maintain the high levels during overwintering. A
recent study showed that 20:5n-3 increased by 45% in gill membranes of
the sea scallop Placopecten magellanicus after 21 days of acclimation
at low temperature, which correlates with an increase in membrane fluidity
(Hall et al., 2002).
Therefore, it was suggested that 20:5n-3 may have contributed to the
maintenance of the sea scallop's membrane fluidity at low temperatures. In our
study, 20:5n-3 was not correlated with the lipid unsaturation index
(r2=0.185, P=0.249), thus indicating that a rise
in 20:5n-3 did not seem to contribute to increasing membrane fluidity in hard
clams. Therefore, 20:5n-3, which is generally considered as a precursor of
biologically active metabolites (Stanley
and Howard, 1998), may have been incorporated for eicosanoid
biosynthesis as a stress response to the early lowering of environmental
temperatures or other unmeasured factors in the field.
Wild M. mercenaria showed a 33% reduction in their standard
compared to the selectively bred M. mercenaria var. notata.
A reduction in the maintenance metabolic rate generally provides an energetic
advantage in cold-adapted organisms by reducing their energetic needs during
overwintering (Peck, 2002;
Petersen et al., 2003), which
suggests that wild M. mercenaria were more adapted to overwintering
conditions than their selectively bred counterparts. Furthermore, there was a
smaller deficiency in heterozygote frequencies in wild clams
(Fis=0.10) compared with selectively bred hard clams, for which the
Fis was 0.44. Several studies have shown negative correlations
between heterozygote deficiency and survival in marine bivalves exposed to
stressful environmental conditions; this relationship was attributed to lower
metabolic requirements for heterozygous individuals
(Hawkins et al., 1989;
Tremblay et al., 1998;
Myrand et al., 2002). For
example, mussel stocks susceptible to summer mortality had a lower degree of
heterozygosity and showed higher oxygen consumption rates than mussels from
resistant stocks, thus suggesting that the energy expenditure for maintenance
was higher for mussels from susceptible stocks
(Tremblay et al., 1998).
Similarly, higher metabolic demands in selectively bred hard clams associated
with a higher heterozygote deficiency probably impose a supplementary stress
that renders these animals more vulnerable to overwintering mortality.
Interestingly, the selective breeding of hard clams for improving growth
performance resulted in an increase in heterozygote deficiency and an increase
in oxygen consumption rates.
A previous study on wild populations of hard clam from the US showed a good
fit to the Hardy-Weinberg equilibrium
(Dillon and Manzi, 1992), thus
suggesting that the marked reduction in heterozygote frequencies in the
variety notata, which originate from Massachusetts, most likely
emerged because of the long-term breeding program and associated loss of
genetic variability in the hatchery in the US and/or in Canada. Another
possibility is that differences in heterozygote frequencies between the two
varieties of clams emerged within the generation sampled, between the time
that the clams were bred in the hatchery and their shipment from the hatchery
to our facility. In this case, the marked heterozygote deficiency
characteristic of the variety notata would be attributable to the
Wahlund effect, inbreeding and/or selection
(Zouros, 1987;
Beaumont, 1991;
Bierne et al., 2000;
Bierne et al., 2003). However,
given the lack of information about the selective breeding program, it is not
possible to further investigate the cause of the decrease in heterozygote
frequencies observed in the selectively bred hard clam.
Wild and selected hard clams differed in their allelic frequencies at four
of the eight loci examined, which means that biochemical and physiological
differences cannot be associated only to heterozygosity. For instance,
selection for particular allozyme alleles was first demonstrated in the blue
mussel, Mytilus edulis, where salinity acted on the
LAP* allele (Koehn et
al., 1980). More recently, the functional significance of
variations in allele frequency at the GPI* locus, a key
enzyme regulating glucose metabolism, was investigated in populations of the
beetle Chrysomela aeneicollis
(Dahlhoff and Rank, 2000).
This study showed that the functional properties of GPI*
allozymes are related to environmental conditions in which each genotype
predominates, thus suggesting that the genotype is associated with local
adaptation to temperature. In our study, biochemical and physiological
differences between varieties of hard clams cannot be related to any
particular genotype at any locus or functional property of allozyme.
Wild M. mercenaria also exhibited a lower unsaturation index than
the selectively bred counterpart M. mercenaria var. notata.
It was previously shown that the unsaturation index of membrane phospholipids
is positively correlated with the basal metabolic rate of the animal
(Hulbert and Else, 1999;
Hulbert and Else, 2005).
Indeed, membrane unsaturation increases the molecular activity of many
membrane-bound proteins and consequently also increases some specific membrane
leak-pump cycles and cellular metabolic activities, which suggest that
membranes could act as pacemakers for overall metabolic activity. Therefore,
the positive relationship between standard
and
the unsaturation index between wild and selectively bred hard clams is in good
agreement with Hulbert's theory of membranes as possible pacemakers of
metabolism. It is tempting to speculate that the lower metabolic requirements
of wild clams characterized by a lower heterozygote deficit could be related
to a lower unsaturation index in their membrane phospholipids. However, there
was only a 4% difference in the unsaturation index between wild and
selectively bred hard clams; this could only have a marginal effect on
membrane fluidity and cellular metabolic activity.
In conclusion, the data presented in this study provide evidence that a
major remodeling of lipids occurred in hard clams exposed to a gradual
lowering in temperature from 24 to 0°C and then acclimatized at
<0°C, as predicted by HVA. Furthermore, we showed that selectively bred
hard clams were characterized by a higher metabolic demand and a marked
deviation from Hardy-Weinberg equilibrium at several loci due to a
heterozygote deficit compared to wild clams, which is believed to impose
additional stress and render these animals more vulnerable to overwintering
mortality. Finally, an intriguing finding is that the lower metabolic
requirements of wild animals coincide with a lower unsaturation index of their
lipids, as predicted by Hulbert's theory of membranes as pacemakers of
metabolism.
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