Influence of local adaptation and interstock hybridization on the cardiovascular performance of largemouth bass Micropterus salmoides
Department of Natural Resources and Environmental Sciences, University of Illinois, and Center for Aquatic Ecology, Illinois Natural History Survey, 607 East Peabody Drive, Champaign, IL 61820, USA
* Author for correspondence at current address: Centre for Applied Conservation Research, Department of Forest Sciences, University of British Columbia, Forest Sciences Center, 2424 Main Mall, Vancouver, British Columbia, Canada, V6T 1Z4 (e-mail: scooke{at}interchange.ubc.ca)
Accepted 15 March 2005
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Summary |
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Key words: cardiac output, heart rate, local adaptation, outbreeding, translocation, largemouth bass, Micropterus salmoides
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Introduction |
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To date, few studies have examined biological differences among fish stocks
delineated from the results of molecular genetic analyses, and even fewer have
examined the physiological variation among putative stocks or the consequences
of mixing them. Theoretically, one would predict that physiological variables
would differ among locally adapted and genetically distinct populations, and
that mixing individuals from different populations would result in alterations
in these variables that could provide a mechanistic explanation for reductions
in fitness. Interestingly, there have been few attempts to evaluate
population-specific differences in cardiovascular performance, or the
cardiovascular consequences of hybridizing fish from different populations.
This is surprising since cardiovascular performance is intimately linked to
the energetics and physical performance of fish
(Thorarensen et al., 1996) and
serves as a sensitive and logical technique for assessing intraspecific
variation (Gamperl and Farrell,
2004
). Furthermore, there is a growing interest in intraspecific
variation in fish cardiovascular function
(Gamperl and Farrell, 2004
),
and more broadly in the physiological diversity of animals
(Spicer and Gaston, 1999
).
In this study, we used two genetically distinct stocks of largemouth bass Micropterus salmoides Lacépède that were collected from different geographic regions in the upper midwestern United States to: (1) assess whether cardiovascular function differs in bass from geographic locations with different climatic conditions and (2) quantify the impact that interstock hybridization has on this important physiological characteristic. For both comparisons, we monitored the cardiovascular response of individuals to exhaustive exercise at temperatures of 10 and 20°C.
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Materials and methods |
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During fall 1997 adult largemouth bass that were fixed for alternative homozygous genotypes at a single locus (MDH-B) were selected from each of two of the proposed stocks: (1) IL: from Central Illinois: Lake Shelbyville in the Kaskaskia River Drainage within the Mississippi River Basin; Genotype=MDH-B2B2; (2) WI: from Southeastern Wisconsin: Big Cedar Lake in the Lake Michigan Drainage within the Great Lakes Basin; Genotype=MDH-B1B1.
The WI fish came from a less temperate environment with mean annual temperatures (1971-2000) of 7.5°C and 63.4 days with maximum temperatures <0°C. The IL fish experienced mean annual temperatures (1971-2000) of 11.3°C and 33.9 days with maximum temperatures <0°C. In the spring of 1998, we stocked adult fish (10 males and 12 females, each with the appropriate homozygous genotype) from one or the other stock into each of eight 0.04 ha clay-lined, earthen ponds at the Illinois Natural History Survey Aquatic Research Field Laboratory in Champaign, IL, USA to produce four distinct, genetically tagged experimental stocks: each of the two pure parental (P1) stocks (ILxIL and WIxWI), as well as both reciprocal F1 interstock hybrids (WIxIL and ILxWI). In the fall of 1998, production ponds were drained. Fingerlings from each stock were given differential fin clips for external identification and then stocked into a series of ponds in a set of common garden experiments designed to assess their relative survival and growth, as well as their relative fitness (lifetime reproductive success) after they matured. Fish fed on natural invertebrate forage, as well as fathead minnows Pimephales promelas and juvenile bluegill Lepomis macrochirus produced in the ponds. The artificial ponds were substantially smaller than the lentic systems where fish were originally captured; however, the ponds contained substrates and vegetation typical of natural Midwestern systems and were subject to seasonal variation in temperature and photoperiod.
Cardiovascular assessments
Twice during fall 2000 (i.e. when ambient water temperatures corresponded
to 10 and 20°C), some individuals from each stock were seined from the
ponds and held in raceways for 1 week prior to experimentation. Although water
temperatures were relatively stable for a period of approximately 2 weeks
prior to experimentation, they did vary up to 3°C on a diel basis. During
the experiments, water temperatures were controlled so that they varied by no
more than 1°C. All experiments were conducted between 10:00 h and 16:00 h.
Fish were exposed to the natural photoperiods of Champaign, Illinois during
residency in pond and raceway environments, as well as during experimentation.
As a result, fish were field-acclimated to conditions in central Illinois.
Experiments were conducted between 18 September and 4 December, 2000.
Detailed descriptions of the surgical procedure are provided elsewhere
(Schreer et al., 2001;
Cooke et al., 2003b
). Briefly,
each fish was anaesthetized prior to surgery with 60 p.p.m. clove oil
(emulsified with ethanol, 9:1 ethanol: clove oil) for approximately 8 min, at
which point the fish had lost equilibrium and was non-responsive. Once
anaesthetized, each fish was placed on its side on a wetted sponge. The
anaesthetized state was maintained during surgery by irrigating the gills with
water containing a maintenance concentration of anesthetic (30 p.p.m. clove
oil). Connective tissue surrounding the aorta was carefully removed, and a
flexible silicone cuff-type Doppler flow probe (sizes from 0.9 to 1.2 mm)
subminiature 20 MHz piezoelectric transducer: Iowa Doppler Products, Iowa
City, IA, USA) was placed around the aorta. The cuff was secured around the
vessel using a single suture, and the lead wire leading to the probe was then
sutured to the body of the fish at several locations.
Following surgery, individual fish were placed immediately into a 70 liter
tank (50 cmx50 cm) and monitored closely until they had regained
equilibrium. Fish were allowed to recover from surgery and to acclimate to the
tank for at least 18 h (Cooke et al.,
2003b). A darkened area covering approximately 30% of the tank
provided cover and ensured that the fish were not disturbed by general
laboratory activity. The experimental tanks were continuously supplied with
pond water at either 10 or 20°C. To elicit exercise, fish were chased
around the tank by hand (Cooke et al.,
2003b
) until they reached exhaustion; a standard technique in fish
physiology (Kieffer, 2000
). At
this point the fish would no longer swim and would lose equilibrium. Cardiac
variables were recorded continuously for at least 1 h prior to the exercise
period (the resting period), during the exercise period, and for at least 6 h
post-exercise (the recovery period).
Following experimentation, fish were killed with an overdose of anesthetic
(180 p.p.m. clove oil), and a postmortem calibration was conducted to
convert Doppler shift (in V) to actual blood flow (ml min-1;
Cooke et al., 2003b). The
bulbous arteriosus was catheterized with tubing (PE 120) and a constant
infusion pump (Harvard Apparatus, South Natick, MA, USA) was used to perfuse
anticoagulated blood (2 g sodium oxalate + 0.4 g sodium chloride + 10 ml
distilled H2O l-1 pig's blood) through the aorta and
partial gill arches to maintain output pressure. This procedure permitted the
calibration of the probes over a range of flow rates encompassing the rates
recorded during the trials. Reference flow rates were analyzed with linear
least-squares regression (mean r2=0.971). The ventricles
were patted dry and weighed to the nearest 0.001 g and were mass corrected for
the size of the fish to represent relative ventricular mass (RVM). To examine
the temperature dependence of stock-specific resting cardiac variables, we
calculated Q10 rates
(Schmidt-Nielsen, 1997
).
Data collection and analysis
A flowmeter (545C-4 Directional Pulsed Doppler Flowmeter: Bioengineering,
The University of Iowa, Iowa City, IA, USA) and a digital strip-chart recorder
(LabVIEW, Version 4.0.1, National Instruments Corporation, Austin, TX, USA)
were used to record real time data on cardiac output. Heart rate
(fH) was determined by counting the number of heart beats
(flow trace peaks) over a 60 s period and stroke volume
(VS) was calculated as cardiac output
()/fH.
To determine recovery times, traces for
, fH and
VS, adjusted to resting (100%), were plotted for each fish
and evaluated visually. A fish was considered to be recovered when values
returned to resting and became stable (within 10 % of resting values;
Schreer et al., 2001
;
Cooke et al., 2003b
). Maximal
disturbance was determined as the greatest change in a cardiac variable
(either positive, >100% basal or negative, <100% basal) during the
recovery period. Data were visually assessed for normality using quantile
plots and homogeneity of variance using residual plots (SYSTAT, V8.0, SAS
Institute). The premise of all analyses was to test two null hypotheses: (1)
that there were no differences in cardiovascular performance among molecularly
defined P1 stocks and (2) there were no differences in performance
of F1 hybrids compared to the P1 stocks. All tests were
conducted using two-way analysis of variance with stock being the main effect
and water temperature the secondary effect (JMPIN, V4.01, SAS Institute). We
used planned contrasts to examine where specific differences of interest
occurred. All values reported are means ±
S.E.M. Tests were considered significant at
P=0.05.
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Results |
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Resting cardiac function
Cardiac output and heart rate values were significantly lower at 10 than
20°C for all four stocks, whereas stroke volume, differed significantly
between 10 and 20°C only for the ILxWI stock
(Table 2,
Fig. 1). Resting cardiac output
for the ILxIL stock was consistently lower than for the other three
stocks. The only departure from this pattern was that at 20°C, resting
cardiac output rates were similar between both P1 stocks. Heart
rate followed a similar pattern to cardiac output, with the ILxIL fish
exhibiting the lowest resting heart rates; however, fewer stock-specific
differences were noted (Fig.
1). Resting stroke volume values did not vary significantly among
stocks (Table 2, Fig. 1). Q10 values
for resting cardiac output and heart rate ranged from 1.33 to 1.65, and were
highest for ILxIL fish (Fig.
1). Resting stroke volume Q10 values ranged from 0.89
to 1.01, with values being highest (i.e. close to 1) for both P1
stocks (Fig. 1).
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Cardiac recovery
Cardiac recovery times for ILxWI fish varied by temperature for heart
rate and cardiac output, but in the opposite direction to what was observed
for WIxWI fish (Fig. 3).
At 10°C, cardiac output recovered similarly quickly for both P1
stocks, but significantly more slowly, for both hybrids. At 20°C, cardiac
output for the ILxIL stock recovered more rapidly than all other stocks.
The same temperature-specific patterns of recovery observed for cardiac output
were also observed for heart rate (Tables
1,
2,
Fig. 3). Stroke volume at
10°C recovered most rapidly for both P1 stocks, although the
WIxWI recovery rate did not differ from the WIxIL stock
(Table 2,
Fig. 3). At 20°C, the
ILxIL stock recovered more rapidly than the WIxIL and WIxWI
stock.
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Discussion |
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Our data illustrate that when translocated to novel environments, there are
substantial differences in the cardiovascular performance of these largemouth
bass relative to locally adapted individuals in the recipient population.
Furthermore, when these two stocks were mixed, the interstock hybrids
performed either similarly to the Wisconsin stock or more poorly than both
P1 stocks. These data suggest that interstock hybrids did not
experience heterosis (i.e. hybrid vigor;
Emlen, 1991;
Thornhill, 1993
) and instead
exhibited performance indicative of a brake down of co-adapted gene complexes
(Templeton, 1986
;
Hallerman, 2003
) even at the
F1 generation. These data emphasize the fact that cardiovascular
performance characteristics exist in locally adapted populations and that
outbreeding occurs when fish from different populations are mixed. In
Illinois, locally adapted bass generally exhibited the lowest resting cardiac
activity, the lowest maximal cardiac activity in response to exhaustive
exercise, and recovered almost 25-35% faster than other stocks examined.
Interestingly, at the lower temperature (10°C), the northern P1
stock from Wisconsin recovered in the same time period as did the more
southern P1 stock (ILxIL). At the warmer temperature
(20°C), however, the northern stock took longer to recover than the
southern stock. Data presented here provide clearer evidence than has been
previously reported for largemouth bass using swimming performance and
respirometry (i.e. Cooke et al.,
2001
) that the translocation of fish and their subsequent
hybridization with locally adapted stocks has physiological consequences that
may have direct fitness implications, since cardiovascular performance is a
determinant of aerobic swimming ability
(Keen and Farrell, 1994
;
Kolok and Farrell, 1994
).
When we evaluated resting cardiac activity, cardiac output and heart rate
values for ILxIL fish were consistent with previous studies on
largemouth bass (Cooke et al.,
2003b) and were approximately 30% less than both of the interstock
hybrids. The elevated resting cardiac output and heart rate of the WIxWI
fish and in particular, the interstock hybrids, could result in greater
myocardial energetic costs, but this would be minimal in the overall energetic
budget as myocardial metabolism is generally less than 4.5% of total metabolic
rate (Farrell and Jones,
1992
). The maximal values for cardiac output and heart rate were
also frequently higher for the interstock hybrids than the P1
stocks, potentially yielding additional energy costs. However, without further
analyses of oxygen uptake, transport and use, it is not possible to determine
if the bioenergetic consequences of these cardiovascular performance
differences are ecologically meaningful. Despite having higher resting rates
and maximal rates, the scope for cardiac output and heart rate were similar
among all four stocks. The interstock hybrids, therefore, maintained scope,
but likely did so with a higher cost because they were operating at higher
resting rates. Largemouth bass (Cooke et al.,
2003a
,b
)
and other centrarchid fishes (e.g. smallmouth bass Micropterus
dolomieu, Schreer et al.,
2001
; black crappie Pomoxis nigromaculatus,
Cooke et al., 2003a
) are
frequency modulators, responding to performance challenges typically by
increasing heart rate and decreasing stroke volume. In this study, the pattern
of largemouth bass being frequency modulators was maintained in the
translocated stock (WIxWI) as well as the interstock hybrids and was
similar to that of the ILxIL fish.
When exposed to a performance challenge such as exhaustive exercise,
recovery times provide information on the duration that individual fish are
forced to deal with the challenge, precluding them from allocating resources
to other functions. In our study, recovery times were consistently about
25-35% longer for both interstock hybrids than for the Illinois stock.
Previous work on largemouth bass revealed that cardiac recovery time was
remarkably consistent across temperatures when exposed to 150 s of exercise
and 30 s of air (135 min for
and
fH and
110 min for VS;
Cooke et al., 2003b
). In the
current study, the largemouth bass cardiac recovery times for translocated and
interstock hybrids (
110 min for
and fH and
70 min for VS) were
more variable than those of Cooke et al.
(2003b
). Although these were
more rapid recovery times than the previous research (i.e.
Cooke et al., 2003b
), in the
current study fish were not exposed to air, which is an additional stressor.
Furthermore, when contrasted with the relatively rapid recovery of the locally
adapted Illinois stock, the recovery times of the Wisconsin stock and
interstock hybrids are indicative of major physiological differences in how
they respond to exercise. During this period of lengthened recovery,
interstock hybrids could exhibit reduced digestion
(Randall and Daxboeck, 1982
)
and food intake (Smart, 1981
;
Barton and Schreck, 1987
),
negatively affecting acquisition of new energy resources. Furthermore, because
cardiovascular performance is strongly correlated with metabolic rate, when
the fish are at an elevated stage on their scope, fish may have less scope for
activity available to escape predators or deal with other stressors (Priede,
1977
,
1985
). Although cardiac
performance is a factor in determining oxygen consumption and metabolic rate,
there are also other factors that we did not measure here that could
exacerbate or mitigate the consequences of intra-specific variation in
cardiovascular performance.
Evidence suggesting that inbreeding and outbreeding depression may be
manifested as alterations in cardiovascular performance in higher organisms
comes from selective breeding experiments with mice Mus domesticus
(e.g. Mattson, 2001) or
observations of high rates of cardiac abnormalities in some inbred organisms
such as Florida panther Felis concolor coryi
(Roelke et al., 1993
). To our
knowledge, however, no information on the cardiovascular consequences of
inbreeding or outbreeding currently exist for teleost fish
(Waldman and McKinnan, 1993
).
The only research that may be relevant is the assessment of cardiovascular
performance in transgenic and domesticated fish. Triploid brown trout
Salmo trutta appeared to have reduced factorial metabolic scope
relative to diploid fish (Altimiras et al.,
2002
), while domesticated rainbow trout reared in a hatchery did
not exhibit appreciable differences in cardiac morphometrics or performance
relative to wild fish (Gamperl and
Farrell, 2004
). While these studies did evaluate the
cardiovascular consequences of selective breeding, they did not, however,
evaluate the effects of interstock hybridization. The approach used in our
study differs in that it provides a possible proximal mechanism for reductions
in fitness that can result from such hybridization. Because there were minimal
differences in cardiac morphometrics among the treatment groups we examined,
the variation in cardiac performance may represent disruption in
humoral/neurological regulation and/or efficiency of enzymatic and other
cellular processes, consistent with the breakdown of co-adapted gene complexes
(Templeton, 1986
).
In addition to providing insight into the fundamental physiological
diversity and intraspecific cardiovascular performance of fish, this study
also has implications for conservation scientists. Our findings suggest that
molecular genetic techniques are able to delineate populations that have
sufficiently different physiologies to warrant separate management and
conservation activities (e.g. Nielsen,
1995). The current study supported previous findings (i.e.
fitness, Philipp and Claussen,
1995
; swimming performance,
Cooke et al., 2001
) in that the
northern stock of largemouth bass (WIxWI) did not perform as well in
Illinois as the Illinois stock. For largemouth bass, therefore, local
adaptations in cardiovascular performance exist among populations that occur
within a relatively small geographic region so translocations should me
minimized. Finally, our results illustrate the negative consequences of mixing
fish from different locally adapted populations. Future efforts focusing on
fish from the F2 generation will provide additional insight into
intra-specific cardiovascular performance and the potential long-term
consequences of fish translocation practices.
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Acknowledgments |
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References |
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