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Effect of multiple-locus heterozybosity and salinity on clearance rate in a brackish-water clam, Rangia cuneata (Sowerby)
J. Exp. Mar. Biol. Ecol., 1987, Vol. 111, pp. 121-131 Elsevier
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JEM 00926
Effects of multiple-locus heterozygosity and salinity on clearance rate in a brackish-water clam, Rangia cuneata (Sowerby) M. E. Holley and D. W. Foltz Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana, U.S.
(Received 11 August 1986; revision received 17 April 1987; accepted 30 April 1987) Abstract: Previous studies of several species of marine bivalves and gastropods have reported a positive
correlation between growth or size and level of multiple-locus heterozygosity. There is some evidence that the growth advantage of relatively heterozygous individuals is due to a lower rate of standard or routine metabolism, compared with more homozygous individuals, although heterozygosity-dependent differences in feeding rate may also be involved. The present study examined the relationship between clearance rate in three salinity treatments (5,15, and 25x,,) and multiple-locus heterozygosity at nine polymorphic allozyme loci in the clam Rangiu cuneata (Sowerby). Clearance rates were determined by disappearance of an algal suspension from a flowing-water system. Allozyme genotypes were determined using starch-gel electrophoresis. The polymorphic loci examined were those coding for a nonspecific esterase (Est), mannosephosphate isomerase (Mpi), leucine aminopeptidase (Lap), 6-phosphogluconate dehydrogenase (6-Pgd), phosphoglucose isomerase (Pgi), isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), adenylate kinase (Adk), and phosphoglucomutase (Pgm). Weight-corrected clearance rates increased significantly (P < 0.05) with increasing multiple-locus heterozygosity and decreased significantly (P < 0.05) with increasing salinity. These data support the idea that heterozygosity-growth correlations may be due in part to differences in clearance rate. However, further study is needed to understand the exact physiological processes which relate heterozygosity and growth. Key words: Clam; Clearance rate; Heterozygosity; Salinity; Rungib cuneatu
INTRODUCTION
There are now numerous reports of positive correlations between several titnessrelated traits, such as growth, viability and fecundity, and multiple-locus heterozygosity at electrophoretically detected allozyme loci (Mitton & Grant, 1984; Zouros & Foltz, 1987). Much of this evidence is based on research involving marine molluscs, especially members of the Gastropoda and Bivalvia. Recent investigations on bivalves, such as Mytilus edulis (Koehn & Gatfney, 1984), Mulinia lateralis (Garton et al., 1984), and Crassastrea virginica (Singh & Zouros, 1978; Zouros et al., 1980), have demonstrated a positive relationship between multiple-locus heterozygosity and growth rate.
Correspondence address: M.E. Holley, Department University, Baton Rouge, LA 70803, U.S.
of Zoology and Physiology, Louisiana State
0022-0981/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
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M.E. HOLLEY AND D.W. FOLTZ
The growth advantage of heterozygotes could result from any of several physiological mechanisms. In the balanced energy equation (Winberg, 1956), net assimilated energy (or) equals amount of food consumed (C) minus energetic losses through respiration (R), feces (F), and excretory products (U), or Pr = C - (R + F t U). One way for heterozygotes to have a growth advantage is if their standard metabolic rate is lower than that of homozygotes. Koehn & Shumway (1982) found that oxygen consumption in starved oysters (C. virginica) was negatively correlated with heterozygosity, and a similar finding in M. edulis was reported by Diehl et al. (1985). Garton et al. (1984) found that differences in routine metabolic costs in the clam M. later& explained most of the variance in growth rate among heterozygosity classes, after removing the effect of feeding rate differences. However, Rodhouse & Gaffney (1984) found no detectable effect of m~tiple-locus heterozygosity on oxygen consumption in C. v~rgj~~ca, and Diehl et al. (1986) found a si~~c~t ~~~~~veassociation between heterozygosity at five allozyme loci and oxygen consumption in M. edulis starved for 18 days in one of two comparisons. Differences in experimental design among these studies make comparisons among them difficult. A second way for heterozygotes to have a growth advantage over homozygotes is if their feeding rate is higher than that of homozygotes. For example, Garton (1984) found that differences in feeding rate explained most of the difference in scope for growth between relatively homozygous and relatively heterozygous snails (Thais ~ae~ast~~a). Growth in molluscs is more likely to depend on rates of feeding and assimilation than on metabolic costs (Stickle, 1985; Diehl et al., 1986; Stickle & Bayne, 1987). However, except for the studies of Garton (1984) and Garton et al. (1984), there have been no attempts to test the hypothesis that the correlation between growth rate and multiple-locus heterozygosity commonly observed in molluscs is due in part to difference in feeding rate. In bivalves, the advantage of heterozygous individuals over more homozygous ones may be more apparent when the animals are stressed (Koehn & Shumway, 1982; Diehl et al., 1986). For estuarine organisms, one important source of environmental stress is the salinity gradient (Kinne, 1966; Bayne, 1976). The present research was undertaken to study the association of multiple-locus heterozygosity and clearance rate in a brackish-water clam, Rangia cuneata (Sowerby), at different salinities. The hypotheses tested were (1) whether there was a positive association between clearance rate and multiple-locus heterozygosity, and (2) if there is a positive association, is it dependent on salinity. R. cuneatu was chosen for this ~vestigation because (1) it is an estuarine species that is abundant in Louisiana (Hoese, 1973), (2) it is a sessile filter-feeder, and (3) it is extremely euryhaline (Deaton, 1981). To date, there has been no investigation in R. cuneatu of the amount of allozyme variation or of the relationships among clearance rate, salinity, and heterozygosity.
H~TEROZYGOSITYAND CLEARANCERATE IN A CLAM
123
MATERIALSAND METHODS Specimens (N = 210) were collected by dredge from Vermilion Bay, Louisiana, in July 1985 and brought to the laboratory. No data on field salinity at the time of collection were taken. The clams were divided equally among three 30-gal, aerated aquaria containing 5z0 water (Instant Ocean) at a constant temperature of 23 ‘C. The salinity was increased by 2x, per day in two of the aquaria to their final salinities of 15 and 25%,. The clams were then allowed 2 wk to adapt to the final salinities. Clams were fed Tha~assi~sirapseu~o~a~a, reared by the methods of Guillard (1975), at a ration of 0.5 1 of concentrated algae ~750,000 cells/ml, determined via hemac~ometer) per aquarium per day throughout the adaptation and treatment periods. After adaptation, 20 clams were randomly chosen from each salinity for the purpose of estimating flesh dry weight. These clams were oven dried at 60 *C for 72 h, then weighed. Flesh dry weights in grams were then estimated by linear regression for the remaining animals in each salinity treatment, using soft tissue wet weight (in grams) as the dependent variable. Individual clearance rates (CR) were determined using the following formula (Mohlenberg & Riisgard, 1979): CR = FL[ 1 - (C,jCi)], where FL (flow rate) is the volume of water passing through the clearance-rate chamber, C, (excurrent concentration) is the concentration of algae leaving an occupied chamber, and Ci (incurrent concentrations is the concen~ation of algae leaving the control chamber. To make clearance rates independent of flow rates, the following adjustment was used (Hildreth & Crisp, 1976, p. 118): CR’ = (CR x FL)I(FL - CR). Measurements of clearance rates were made over a &day period at the end of the 2-wk acclimation. Clearance rates were determined for each clam by measuring the rate of disappearance of an algal suspension in a flowing-water system using a model ZB Coulter Counter. Clams were placed in clearance rate chambers and allowed 1 h to adapt to the experimental conditions. Then, flow rate was measured and outflow samples were coilected at times 0, 30, and 60 min in the second hour. Algal concentration and flow rate (20,000 cells/ml and 30 ml/mm, respectively) were kept constant during both the adaptation and measurement periods. Clearance rates were homogeneous among days within salinity treatments (P > 0.05) as tested by analysis of variance. Weight-specific adjusted clearance rates (CR’) were calculated from the whole animal rate function using the formula: c;R’ = CR’lWb, where CR’ is the weight-speciIic rate variable in units per gram dry weight of a standard (i.e., constant weight) animal; CR’ is the whole animal rate variable; W is the flesh dry
I24
M.E.HOLLEYANDD.W.FOLTZ
weight of a standard animal; and b is the slope of the regression equation of adjusted clearance rate on estimated flesh dry weight. The slopes of the regression of logic-tram+ formed adjusted clearance rate on estimated flesh dry weight were homogeneous among the three salinity treatments (P > 0.05), so a single pooled slope was fit to the data (b = 0.263). This common-slope model explained 64.3% of the variance in adjusted clearance rates. For this study, the “standard-sized” animals were the mean flesh dry weights per salinity (0.89 g at S%,, 0.67 g at 15%,,,and 0.64 g at 25x,). Samples were prepared for electrophoresis by homogenizing the soft body parts in distilled water, followed by centrifugation at 20,000 rpm for 20 min. The supernatant was used as the enzyme source. Thirteen allozyme systems were examined: esterase (EST), mannosephosphate isomerase (MPI), leucine aminopeptidase (LAP), 6-phosphogluconate dehydrogenase (6-PGD), phosphoglucose isomerase (PGI), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), adenylate kinase (ADK), phosphoglucomutase (PGM), glutamate oxaloacetate transaminase (GOT), indophenol oxidase (IPO), xanthine dehy~ogenase (XDH), and peptidase (PEP). El~trophoresis for all of the allozymes was done in ho~zont~ starch gels, according to the general method of Selander et al. (197 1). EST, MPI, LAP, PGI, PEP, IPO, and GOT were demonstrate on a lithium hydroxide (pH 8.1) buffer system, 6-PGD, IDH, MDH, ADK, and PGM on a t&citrate EDTA (pH 7.0) system, and XDH on a &is-citrate (pH 8.0) system. Nine loci were both polymorphic and scoreable, and were used to determine the number of heterozygous loci per individual: Est, Mpi, Lap, ci-Pgd, Pgm, fdh, Mdh, Adk, and Pgi. Alleles (electromorphs) were designated by lower-case letters, with “a” denoting the fastest-migrating allele, and so on. Allele frequencies, fixation indices (F), and observed heterozygosities (Ho) were estimated by the methods of Hendrick (1983). The remaining statistical procedures were done using the General Linear Models (GLM) and Frequency (FREQ) procedures of the Statistical Analysis System (SAS Institute, 1985).
RESULTS
Of the 148 clams used to initiate the clearance rate experiment, 143 survived the 3 l-day experimental period and the manipulations necessary for determining clearance rates, a mortality of -C4%. Deaths occurred only in the 15 and 25x0 salinity treatments. Mean wet weights and mean estimated flesh dry weights for each treatment are presented in Table I. Wet weights and dry weights decreased si~~c~tly (P c 0.05) with increasing salinity. The number of clams not clearing (i.e., clearance rate of 0) increased si~~c~tly with increasing salinity (G = 36.3,2 df, P -=c0.001); such clams were excluded from further analysis. Excluding animals that did not clear introduced a conservative bias, in that it made the demonstration of a significant difference in clearance rate among treatments more difficult. The mean weight-specific clearance rates were significantly different among treatments by analysis of variance (P < 0.05),
HETEROZYGOSITY
125
AND CLEARANCE RATE IN A CLAM
with rates decreasing with increasing salinity. These differences in clearance rate probably accounted for the difference in wet weight and estimated flesh dry weight observed among the salinity treatments. TABLE I
Sample size (N), mean wet weights, mean estimated flesh dry weights, mean weight-specific clearance rates, and percent animals not clearing for three salinity treatments in R. cuneafa. Mean values are f 1 SE. Salinity (%0)
N
Wet weight (g)
Flesh dry weight (g)
Clearance rate’ (l/h/g)
Percent not clearing (%)
5 15 25
48 48 47
4.43 f 0.11 3.83 f 0.13 3.81 f 0.10
0.89 i: 0.03 0.67 + 0.03 0.64 f 0.02
0.766 f 0.059 0.216 f 0.034 0.084 + 0.018
0.0 16.7 44.7
’ Includes only those animals with clearance rates > 0.0 l/h.
The nine allozyme loci studied were selected from a total of 13 loci examined because they were polymorphic and readily scoreable (the other four loci were excluded from further analysis). The results of tests for fit to Hardy-Weinberg genotypic proportions and estimates of allele frequencies, fixation indices (F), and observed heterozygosities (H,) are presented in Table II. Six of the nine loci were in Hardy-Weinberg equilibrium; genotypic frequency distributions for three loci (Est, Mpi, and Lap) differed significantly from Hardy-Weinberg expectations, with fewer heterozygotes than expected. The number of clams examined for each locus was 143, except that only 139 animals were scored for Est. The four clams not examined for list all had clearance rates of 0, and were not included in the analysis of clearance rates in relation to multiple-locus heterozygosity. Because the 29 animals that did not clear were not significantly more or less heterozygous than the 114 that did clear (P > 0.05), excluding animals with zero clearance rate did not bias the genetic composition of the sample. Also, there were no differences in multiple-locus heterozygosity among the three salinity treatments. The distribution of the 139 individuals across the heterozygosity classes is given in Table III, along with the expected number of individuals in each class. The expected numbers were calculated from the Ho values in Table II, and hence did not assume Hardy-Weinberg equilibrium. The individual weight-specific adjusted clearance rates (CR’) were regressed on multiple-locus heterozygosity (number of heterozygous loci out of nine). Preliminary analysis indicated that the slopes of the regression of clearance rate on heterozygosity were homogeneous among salinity treatments (P > 0.05), although the intercepts were different. Therefore, a single pooled slope (b = 0.053) was tit to the data (Fig. 1). This value was significantly different from 0 (P c 0.05), indicating that heterozygosity at nine allozyme loci was positively associated with weight-specific adjusted clearance rate. However, heterozygosity explained relatively little (2.6%) of the total variance in
126
M. E. HOLLEY AND D. W. FOLTZ TABLEII
Allele frequency estimates ( + 1 SE),fixation index (F), observed heterozygosity (H,), G statistic and degrees of freedom (df) for test of fit to Hardy-Weinberg genotypic proportions for nine polymorphic allozyme loci in R. cuneata. Locus’
Allele frequency estimates
F
HO
G statistic
df
Est
f(a) f(b) f(c) f(d) f(e) f(f)
= = = = = =
0.050 f 0.255 f 0.115 f 0.306 k 0.230 f 0.044 f
0.013 0.026 0.019 0.028 0.025 0.012
0.274
0.561
48.8***
15
Mpi’
f(a) f(b) f(c) f(d) f(e) f(f)
= = = = = =
0.119 f 0.430 f 0.259 f 0.147 * 0.038 f 0.007 +
0.019 0.029 0.026 0.021 0.011 0.005
0.255
0.546
43.8***
10
Lap
f(a) = 0.098 + 0.018 f(b) = 0.832 f 0.022 f(c) = 0.070 * 0.015
0.310
0.194
1s.4***
6-Pgd*
f(a) f(b) f(c) f(d)
= = = =
0.007 f 0.503 f 0.476 f 0.014 k
0.005 0.030 0.030 0.007
- 0.045
0.532
1.3
Pgm
f(a) f(b) f(c) f(d) f(e) f(f) f(g)
= = = = = = =
0.088 f 0.213 k 0.273 + 0.136 f 0.150 + 0.087 f 0.053 *
0.017 0.024 0.026 0.020 0.021 0.017 0.012
0.033
0.798
25.4
21
Idh
f(a) = 0.122 f 0.019 f(b) = 0.836 ? 0.022 f(c) = 0.042 + 0.011
0.095
0.266
2.7
3
Mdh2
f(a) = 0.010 k 0.006 f(b) = 0.976 f 0.009 f(c) = 0.014 * 0.007
- 0.015
0.050
0.2
1
Adk2
f(a) = 0.087 f 0.017 f(b) = 0.895 f 0.018 f(c) = 0.018 f 0.008
0.197
0.151
3.1
1
Pgi2
f(a) f(b) f(c) f(d)
- 0.023
0.079
0.4
1
= = = =
0.010 f 0.962 + 0.014 * 0.014 f
0.006 0.011 0.007 0.007
’ Sample size is 143 clams for all loci, except 139 for Est. ’ Rare alleles have been combined with common alleles for G statistic. *** P < 0.0001.
HETEROZYGOSITY
AND CLEARANCE
127
RATE IN A CLAM
clearance rate. There was no association between heterozygosity and estimated flesh dry weight (P > 0.05). TABLEIII Numbers of individuals with different levels of multiple-locus heterozygosities and expected numbers based on single-locus heterozygosities in R. cunearu. No. of heterozygous loci
Observed no. of individuals
0
1
1 2 3 4 5 6 7 8 9
11 25 53 29 15 4 1 0 0
Expected no. of individuals 1.14 9.73 29.77 44.28 35.04 15.05 3.52 0.43 0.03 0.01
. 1.8.
5”/-
. .
1.4. C-R’
.
.
i.o:
0.6 0.2 -
... .
:
. . .
i.
7
:: :: .. :.
.
.
” .. :
. : .
l
. 1.0. C-R’
I5%,
0.2-
.
l
.
C-R’
.
0.6.
a.
:...
2
3
.
. 7
;;I 0
I
4
5
6
7
H Fig. 1. Linear regressions of individual weight-specific adjusted clearance rate (dR’) on number of heterozygous loci (H) for three salinity treatments (5, 15 and 25%,) in R.cuneatu.
128
M.E. HOLLEYAND D.W. FOLTZ DISCUSSION
Environmental biologists have long been concerned with the physiological effects of salinity on estuarine organisms. In the range of sublethal salinity conditions, the performance of estuarine organisms may be modified in various ways. Among these responses are modifications of rates and efficiencies of metabolism, activity, growth, and reproduction (Kinne, 1966). Changes in whole animal rate functions are well documented (Kinne, 1963; Bayne, 1976). These changes in the various rates may be used as indices of stress (Bayne, 1976). Mean weight-specific clearance rates of R. cuneata decreased significantly with increasing salinity (Table I). These results may be indicative of stress due to salinity, and are consistent with the finding by Theede (1963) that filtration rates of M. edulis decreased upon transfer to either higher or lower salinities. Another indication of stress might be the increased number of clams with an observed clearance rate of 0 in the higher salinity treatments (Table I). This pattern may be a result of valve closure by the animal as a behavioral response to stress (Burton, 1983). In a similar fashion, marine invertebrate carnivores show increased percentages of animals not feeding at extreme conditions along environmental stressor gradients (Stickle, 1985). Six of the nine loci studied appeared to be in Hardy-Weinberg equilibrium; however; Est, Mpi, and Lap were not (Table II). Each of these three loci exhibited deficiencies in numbers of heterozygotes. Departures from Hardy-Weinberg proportions due to deficiencies in the numbers of heterozygotes have been reported in many other marine bivalves (Berger, 1983; Singh & Green, 1984; Zouros & Foltz, 1984). Possible explanations of these deficiencies include nonrandom mating (e.g., inbreeding), selection in either the larval or adult stage of the life cycle, or the effects of population structure (Wahlund effect). Another possibility, though, could be the occurrence of nonreactive (null) alleles, which might result in a deficiency in the number of heterozygotes if the null heterozygote is wrongly scored as an active homozygote. In a recent study, Foltz (1986) reported the existence of null alleles at two loci (Mpi and Lap) in the oyster C. virginica. However, any of these theories, alone or in combination, could explain the deficiencies in the numbers of heterozygotes observed in R. cuneata. More information concerning the life history as well as the genetic history of the population sampled is needed to answer questions involving deviations from Hardy-Weinberg genotypic proportions in this species. Heterozygosity at nine allozyme loci was positively associated with weight-specific adjusted clearance rate. The percentage of the total variance in clearance rate that was explained by heterozygosity was small, and similar findings have been reported in other studies of heterozygosity, feeding rate and scope for growth (Garton et al., 1984). This result suggests that much of the variance in clearance rate in R. cuneata is of environmental origin, or is due to effects of loci other than the nine included in the study. There are no significant differences between the weight-specific adjusted clearance rates for heterozygotes and homozygotes at any single allozyme locus in R. cuneata (Holley,
HETEROZYGOSITYANDCLEARANCERATEINACLAM
129
1986). The effect of heterozygosity at a locus on clearance rate appears to be small but cumulative, resulting in an overall relationship between heterozygosity and clearance rate. Similar findings have been reported for other studies of heterozygosity and physiological energetics in marine molluscs (Garton, 1984; Garton et al., 1984). These results provide further evidence supporting the positive relationship between litnessrelated characters and allozyme heterozygosity in marine bivalves. The lack of an association between heterozygosity and estimated flesh dry weight appears to contradict other studies (e.g., Zouros et al., 1980; Koehn & GatIney, 1984) that have reported a significant positive correlation between allozyme heterozygosity and weight or size. However, the present study was not designed specifically to detect such a correlation. In particular, the sample of clams used for determination of heterozygosity, weight, and clearance rate was small in number and probably of mixed ages. These factors would tend to obscure any correlation between heterozygosity and weight that existed in the clam population that was sampled (Zouros & Foltz, 1987). The study was designed to detect an interaction between salinity and heterozygosity in determining clearance rate, but that objective was made more difficult by the failure of many clams to clear at elevated salinities, resulting in reduced sample sizes at 15 and 25s0 salinity. Therefore, the present data do not provide strong support either for or against the suggestion that the effect of heterozygosity on clearance rate depends on salinity. The superiority of heterozygotes over homozygotes in relation to growth, fecundity and viability has now been demonstrated in many species of marine invertebrates (Koehn & Gtiney, 1984; Zouros & Foltz, 1987). The actual genetic mechanism responsible for translating heterozygosity at loci coding for soluble proteins into greater metabolic efficiency and higher feeding rates are presently unknown, although several hypotheses have been proposed. It is possible that the growth advantage and metabolic efficiency of relatively heterozygous individuals results from a lower rate of enzyme biosynthesis in multiple-locus heterozygotes (Koehn, 1985). A second hypothesis is that the enzyme loci are acting as indicators of heterozygosity at linked loci affecting growth and metabolism, the “associative overdominance” explanation (Zouros, 1987; Zouros & Foltz, 1987). Further study is needed to determine more accurately the relationships among stress, metabolic efficiency, feeding rate, and multiple-locus heterozygosity in marine invertebrates.
ACKNOWLEDGMENTS
We thank Drs. M. Kapper, R. Roller and S. Wang for assistance with the clearance rate measurements and/or specimen collection; M. Rotenberry and M. N. Freeman for their assistance in specimen preparation; and Drs. E. Zouros, W. B. Stickle, Jr., J. W. Fleeger, and two anonymous reviewers for comments on an earlier draft of the manuscript. This research was supported in part by National Science Foundation Grant BSR-8407450 to D.W. Foltz.
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& R. H. GREEN, 1984. Excess of allozyme homozygosity in marine molluscs and its possible biological significance. Mulucol., Vol. 25, pp. 569-581. SINGH, S.M. & E. ZOUROS, 1978. Genetic variation associated with growth rate in the American oyster (Crassostrea virgbtica). Evolution, Vol. 32, pp. 342-353. STICKLE, W. B., 1985. Effects of environmental factor gradients on scope for growth in several species of carnivorous marine invertebrates. In, Marine biology of polar regions and effects of stress on marine organisms, edited by J. S. Gray & M.E. Christiansen, John Wiley & Sons, New York, pp. 601-616. STICKLE,W. B. & B. L. BAYNE, 1987. Energetics of the muricid gastropod Thais (Nucellu) lupillus (L.). J. Exp. Mar. Biol. Ecol., Vol. 107, pp. 263-278. THEEDE, H., 1963. Experimentelle Untersuchungen iiber die Filtrations-leistung der Miesmuschel Mytilus edulis L. Kiel. Meeresforsch., Vol. 19, pp. 20-41. WINBERG,G.C., 1956. Rate of metabolism and food requirement of fishes. Fish. Res. Board Can. Trunsl. Ser., Vol. 194, pp. l-253. ZOUROS,E., 1987. On the relation between heterozygosity and heterosis: an evaluation ofthe evidence from marine mollusks. In, Isozymes: current topics in biological and medical research, Vol. IS, edited by M.C. Rattazzi, J. G. Scandalios & G. S. Whitt, Alan R. Liss, New York, in press. ZOUROS, E. & D. W. FOLTZ, 1984. Possible explanations of heterozygote deficiency in bivalve molluscs. Malacol.. Vol. 25, pp. 583-591. ZOUROS, E. & D.W. FOLTZ, 1987. The use of allelic isozyme variation for the study of heterosis. In, Isozymes: current topics in biologicaland medical research, Vol. 13, edited by M. C. Rattazzi, J. G. Scandalios & G. S. Whitt, Alan R. Liss, New York, pp. l-59. ZOUROS, E., S.M. SINGH & H.E. MILES, 1980. Growth rate in oysters: an overdominant phenotype and its possible explanations. Evolution, Vol. 34, pp. 856-867.
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JEM 00926
Effects of multiple-locus heterozygosity and salinity on clearance rate in a brackish-water clam, Rangia cuneata (Sowerby) M. E. Holley and D. W. Foltz Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana, U.S.
(Received 11 August 1986; revision received 17 April 1987; accepted 30 April 1987) Abstract: Previous studies of several species of marine bivalves and gastropods have reported a positive
correlation between growth or size and level of multiple-locus heterozygosity. There is some evidence that the growth advantage of relatively heterozygous individuals is due to a lower rate of standard or routine metabolism, compared with more homozygous individuals, although heterozygosity-dependent differences in feeding rate may also be involved. The present study examined the relationship between clearance rate in three salinity treatments (5,15, and 25x,,) and multiple-locus heterozygosity at nine polymorphic allozyme loci in the clam Rangiu cuneata (Sowerby). Clearance rates were determined by disappearance of an algal suspension from a flowing-water system. Allozyme genotypes were determined using starch-gel electrophoresis. The polymorphic loci examined were those coding for a nonspecific esterase (Est), mannosephosphate isomerase (Mpi), leucine aminopeptidase (Lap), 6-phosphogluconate dehydrogenase (6-Pgd), phosphoglucose isomerase (Pgi), isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), adenylate kinase (Adk), and phosphoglucomutase (Pgm). Weight-corrected clearance rates increased significantly (P < 0.05) with increasing multiple-locus heterozygosity and decreased significantly (P < 0.05) with increasing salinity. These data support the idea that heterozygosity-growth correlations may be due in part to differences in clearance rate. However, further study is needed to understand the exact physiological processes which relate heterozygosity and growth. Key words: Clam; Clearance rate; Heterozygosity; Salinity; Rungib cuneatu
INTRODUCTION
There are now numerous reports of positive correlations between several titnessrelated traits, such as growth, viability and fecundity, and multiple-locus heterozygosity at electrophoretically detected allozyme loci (Mitton & Grant, 1984; Zouros & Foltz, 1987). Much of this evidence is based on research involving marine molluscs, especially members of the Gastropoda and Bivalvia. Recent investigations on bivalves, such as Mytilus edulis (Koehn & Gatfney, 1984), Mulinia lateralis (Garton et al., 1984), and Crassastrea virginica (Singh & Zouros, 1978; Zouros et al., 1980), have demonstrated a positive relationship between multiple-locus heterozygosity and growth rate.
Correspondence address: M.E. Holley, Department University, Baton Rouge, LA 70803, U.S.
of Zoology and Physiology, Louisiana State
0022-0981/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
122
M.E. HOLLEY AND D.W. FOLTZ
The growth advantage of heterozygotes could result from any of several physiological mechanisms. In the balanced energy equation (Winberg, 1956), net assimilated energy (or) equals amount of food consumed (C) minus energetic losses through respiration (R), feces (F), and excretory products (U), or Pr = C - (R + F t U). One way for heterozygotes to have a growth advantage is if their standard metabolic rate is lower than that of homozygotes. Koehn & Shumway (1982) found that oxygen consumption in starved oysters (C. virginica) was negatively correlated with heterozygosity, and a similar finding in M. edulis was reported by Diehl et al. (1985). Garton et al. (1984) found that differences in routine metabolic costs in the clam M. later& explained most of the variance in growth rate among heterozygosity classes, after removing the effect of feeding rate differences. However, Rodhouse & Gaffney (1984) found no detectable effect of m~tiple-locus heterozygosity on oxygen consumption in C. v~rgj~~ca, and Diehl et al. (1986) found a si~~c~t ~~~~~veassociation between heterozygosity at five allozyme loci and oxygen consumption in M. edulis starved for 18 days in one of two comparisons. Differences in experimental design among these studies make comparisons among them difficult. A second way for heterozygotes to have a growth advantage over homozygotes is if their feeding rate is higher than that of homozygotes. For example, Garton (1984) found that differences in feeding rate explained most of the difference in scope for growth between relatively homozygous and relatively heterozygous snails (Thais ~ae~ast~~a). Growth in molluscs is more likely to depend on rates of feeding and assimilation than on metabolic costs (Stickle, 1985; Diehl et al., 1986; Stickle & Bayne, 1987). However, except for the studies of Garton (1984) and Garton et al. (1984), there have been no attempts to test the hypothesis that the correlation between growth rate and multiple-locus heterozygosity commonly observed in molluscs is due in part to difference in feeding rate. In bivalves, the advantage of heterozygous individuals over more homozygous ones may be more apparent when the animals are stressed (Koehn & Shumway, 1982; Diehl et al., 1986). For estuarine organisms, one important source of environmental stress is the salinity gradient (Kinne, 1966; Bayne, 1976). The present research was undertaken to study the association of multiple-locus heterozygosity and clearance rate in a brackish-water clam, Rangia cuneata (Sowerby), at different salinities. The hypotheses tested were (1) whether there was a positive association between clearance rate and multiple-locus heterozygosity, and (2) if there is a positive association, is it dependent on salinity. R. cuneatu was chosen for this ~vestigation because (1) it is an estuarine species that is abundant in Louisiana (Hoese, 1973), (2) it is a sessile filter-feeder, and (3) it is extremely euryhaline (Deaton, 1981). To date, there has been no investigation in R. cuneatu of the amount of allozyme variation or of the relationships among clearance rate, salinity, and heterozygosity.
H~TEROZYGOSITYAND CLEARANCERATE IN A CLAM
123
MATERIALSAND METHODS Specimens (N = 210) were collected by dredge from Vermilion Bay, Louisiana, in July 1985 and brought to the laboratory. No data on field salinity at the time of collection were taken. The clams were divided equally among three 30-gal, aerated aquaria containing 5z0 water (Instant Ocean) at a constant temperature of 23 ‘C. The salinity was increased by 2x, per day in two of the aquaria to their final salinities of 15 and 25%,. The clams were then allowed 2 wk to adapt to the final salinities. Clams were fed Tha~assi~sirapseu~o~a~a, reared by the methods of Guillard (1975), at a ration of 0.5 1 of concentrated algae ~750,000 cells/ml, determined via hemac~ometer) per aquarium per day throughout the adaptation and treatment periods. After adaptation, 20 clams were randomly chosen from each salinity for the purpose of estimating flesh dry weight. These clams were oven dried at 60 *C for 72 h, then weighed. Flesh dry weights in grams were then estimated by linear regression for the remaining animals in each salinity treatment, using soft tissue wet weight (in grams) as the dependent variable. Individual clearance rates (CR) were determined using the following formula (Mohlenberg & Riisgard, 1979): CR = FL[ 1 - (C,jCi)], where FL (flow rate) is the volume of water passing through the clearance-rate chamber, C, (excurrent concentration) is the concentration of algae leaving an occupied chamber, and Ci (incurrent concentrations is the concen~ation of algae leaving the control chamber. To make clearance rates independent of flow rates, the following adjustment was used (Hildreth & Crisp, 1976, p. 118): CR’ = (CR x FL)I(FL - CR). Measurements of clearance rates were made over a &day period at the end of the 2-wk acclimation. Clearance rates were determined for each clam by measuring the rate of disappearance of an algal suspension in a flowing-water system using a model ZB Coulter Counter. Clams were placed in clearance rate chambers and allowed 1 h to adapt to the experimental conditions. Then, flow rate was measured and outflow samples were coilected at times 0, 30, and 60 min in the second hour. Algal concentration and flow rate (20,000 cells/ml and 30 ml/mm, respectively) were kept constant during both the adaptation and measurement periods. Clearance rates were homogeneous among days within salinity treatments (P > 0.05) as tested by analysis of variance. Weight-specific adjusted clearance rates (CR’) were calculated from the whole animal rate function using the formula: c;R’ = CR’lWb, where CR’ is the weight-speciIic rate variable in units per gram dry weight of a standard (i.e., constant weight) animal; CR’ is the whole animal rate variable; W is the flesh dry
I24
M.E.HOLLEYANDD.W.FOLTZ
weight of a standard animal; and b is the slope of the regression equation of adjusted clearance rate on estimated flesh dry weight. The slopes of the regression of logic-tram+ formed adjusted clearance rate on estimated flesh dry weight were homogeneous among the three salinity treatments (P > 0.05), so a single pooled slope was fit to the data (b = 0.263). This common-slope model explained 64.3% of the variance in adjusted clearance rates. For this study, the “standard-sized” animals were the mean flesh dry weights per salinity (0.89 g at S%,, 0.67 g at 15%,,,and 0.64 g at 25x,). Samples were prepared for electrophoresis by homogenizing the soft body parts in distilled water, followed by centrifugation at 20,000 rpm for 20 min. The supernatant was used as the enzyme source. Thirteen allozyme systems were examined: esterase (EST), mannosephosphate isomerase (MPI), leucine aminopeptidase (LAP), 6-phosphogluconate dehydrogenase (6-PGD), phosphoglucose isomerase (PGI), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), adenylate kinase (ADK), phosphoglucomutase (PGM), glutamate oxaloacetate transaminase (GOT), indophenol oxidase (IPO), xanthine dehy~ogenase (XDH), and peptidase (PEP). El~trophoresis for all of the allozymes was done in ho~zont~ starch gels, according to the general method of Selander et al. (197 1). EST, MPI, LAP, PGI, PEP, IPO, and GOT were demonstrate on a lithium hydroxide (pH 8.1) buffer system, 6-PGD, IDH, MDH, ADK, and PGM on a t&citrate EDTA (pH 7.0) system, and XDH on a &is-citrate (pH 8.0) system. Nine loci were both polymorphic and scoreable, and were used to determine the number of heterozygous loci per individual: Est, Mpi, Lap, ci-Pgd, Pgm, fdh, Mdh, Adk, and Pgi. Alleles (electromorphs) were designated by lower-case letters, with “a” denoting the fastest-migrating allele, and so on. Allele frequencies, fixation indices (F), and observed heterozygosities (Ho) were estimated by the methods of Hendrick (1983). The remaining statistical procedures were done using the General Linear Models (GLM) and Frequency (FREQ) procedures of the Statistical Analysis System (SAS Institute, 1985).
RESULTS
Of the 148 clams used to initiate the clearance rate experiment, 143 survived the 3 l-day experimental period and the manipulations necessary for determining clearance rates, a mortality of -C4%. Deaths occurred only in the 15 and 25x0 salinity treatments. Mean wet weights and mean estimated flesh dry weights for each treatment are presented in Table I. Wet weights and dry weights decreased si~~c~tly (P c 0.05) with increasing salinity. The number of clams not clearing (i.e., clearance rate of 0) increased si~~c~tly with increasing salinity (G = 36.3,2 df, P -=c0.001); such clams were excluded from further analysis. Excluding animals that did not clear introduced a conservative bias, in that it made the demonstration of a significant difference in clearance rate among treatments more difficult. The mean weight-specific clearance rates were significantly different among treatments by analysis of variance (P < 0.05),
HETEROZYGOSITY
125
AND CLEARANCE RATE IN A CLAM
with rates decreasing with increasing salinity. These differences in clearance rate probably accounted for the difference in wet weight and estimated flesh dry weight observed among the salinity treatments. TABLE I
Sample size (N), mean wet weights, mean estimated flesh dry weights, mean weight-specific clearance rates, and percent animals not clearing for three salinity treatments in R. cuneafa. Mean values are f 1 SE. Salinity (%0)
N
Wet weight (g)
Flesh dry weight (g)
Clearance rate’ (l/h/g)
Percent not clearing (%)
5 15 25
48 48 47
4.43 f 0.11 3.83 f 0.13 3.81 f 0.10
0.89 i: 0.03 0.67 + 0.03 0.64 f 0.02
0.766 f 0.059 0.216 f 0.034 0.084 + 0.018
0.0 16.7 44.7
’ Includes only those animals with clearance rates > 0.0 l/h.
The nine allozyme loci studied were selected from a total of 13 loci examined because they were polymorphic and readily scoreable (the other four loci were excluded from further analysis). The results of tests for fit to Hardy-Weinberg genotypic proportions and estimates of allele frequencies, fixation indices (F), and observed heterozygosities (H,) are presented in Table II. Six of the nine loci were in Hardy-Weinberg equilibrium; genotypic frequency distributions for three loci (Est, Mpi, and Lap) differed significantly from Hardy-Weinberg expectations, with fewer heterozygotes than expected. The number of clams examined for each locus was 143, except that only 139 animals were scored for Est. The four clams not examined for list all had clearance rates of 0, and were not included in the analysis of clearance rates in relation to multiple-locus heterozygosity. Because the 29 animals that did not clear were not significantly more or less heterozygous than the 114 that did clear (P > 0.05), excluding animals with zero clearance rate did not bias the genetic composition of the sample. Also, there were no differences in multiple-locus heterozygosity among the three salinity treatments. The distribution of the 139 individuals across the heterozygosity classes is given in Table III, along with the expected number of individuals in each class. The expected numbers were calculated from the Ho values in Table II, and hence did not assume Hardy-Weinberg equilibrium. The individual weight-specific adjusted clearance rates (CR’) were regressed on multiple-locus heterozygosity (number of heterozygous loci out of nine). Preliminary analysis indicated that the slopes of the regression of clearance rate on heterozygosity were homogeneous among salinity treatments (P > 0.05), although the intercepts were different. Therefore, a single pooled slope (b = 0.053) was tit to the data (Fig. 1). This value was significantly different from 0 (P c 0.05), indicating that heterozygosity at nine allozyme loci was positively associated with weight-specific adjusted clearance rate. However, heterozygosity explained relatively little (2.6%) of the total variance in
126
M. E. HOLLEY AND D. W. FOLTZ TABLEII
Allele frequency estimates ( + 1 SE),fixation index (F), observed heterozygosity (H,), G statistic and degrees of freedom (df) for test of fit to Hardy-Weinberg genotypic proportions for nine polymorphic allozyme loci in R. cuneata. Locus’
Allele frequency estimates
F
HO
G statistic
df
Est
f(a) f(b) f(c) f(d) f(e) f(f)
= = = = = =
0.050 f 0.255 f 0.115 f 0.306 k 0.230 f 0.044 f
0.013 0.026 0.019 0.028 0.025 0.012
0.274
0.561
48.8***
15
Mpi’
f(a) f(b) f(c) f(d) f(e) f(f)
= = = = = =
0.119 f 0.430 f 0.259 f 0.147 * 0.038 f 0.007 +
0.019 0.029 0.026 0.021 0.011 0.005
0.255
0.546
43.8***
10
Lap
f(a) = 0.098 + 0.018 f(b) = 0.832 f 0.022 f(c) = 0.070 * 0.015
0.310
0.194
1s.4***
6-Pgd*
f(a) f(b) f(c) f(d)
= = = =
0.007 f 0.503 f 0.476 f 0.014 k
0.005 0.030 0.030 0.007
- 0.045
0.532
1.3
Pgm
f(a) f(b) f(c) f(d) f(e) f(f) f(g)
= = = = = = =
0.088 f 0.213 k 0.273 + 0.136 f 0.150 + 0.087 f 0.053 *
0.017 0.024 0.026 0.020 0.021 0.017 0.012
0.033
0.798
25.4
21
Idh
f(a) = 0.122 f 0.019 f(b) = 0.836 ? 0.022 f(c) = 0.042 + 0.011
0.095
0.266
2.7
3
Mdh2
f(a) = 0.010 k 0.006 f(b) = 0.976 f 0.009 f(c) = 0.014 * 0.007
- 0.015
0.050
0.2
1
Adk2
f(a) = 0.087 f 0.017 f(b) = 0.895 f 0.018 f(c) = 0.018 f 0.008
0.197
0.151
3.1
1
Pgi2
f(a) f(b) f(c) f(d)
- 0.023
0.079
0.4
1
= = = =
0.010 f 0.962 + 0.014 * 0.014 f
0.006 0.011 0.007 0.007
’ Sample size is 143 clams for all loci, except 139 for Est. ’ Rare alleles have been combined with common alleles for G statistic. *** P < 0.0001.
HETEROZYGOSITY
AND CLEARANCE
127
RATE IN A CLAM
clearance rate. There was no association between heterozygosity and estimated flesh dry weight (P > 0.05). TABLEIII Numbers of individuals with different levels of multiple-locus heterozygosities and expected numbers based on single-locus heterozygosities in R. cunearu. No. of heterozygous loci
Observed no. of individuals
0
1
1 2 3 4 5 6 7 8 9
11 25 53 29 15 4 1 0 0
Expected no. of individuals 1.14 9.73 29.77 44.28 35.04 15.05 3.52 0.43 0.03 0.01
. 1.8.
5”/-
. .
1.4. C-R’
.
.
i.o:
0.6 0.2 -
... .
:
. . .
i.
7
:: :: .. :.
.
.
” .. :
. : .
l
. 1.0. C-R’
I5%,
0.2-
.
l
.
C-R’
.
0.6.
a.
:...
2
3
.
. 7
;;I 0
I
4
5
6
7
H Fig. 1. Linear regressions of individual weight-specific adjusted clearance rate (dR’) on number of heterozygous loci (H) for three salinity treatments (5, 15 and 25%,) in R.cuneatu.
128
M.E. HOLLEYAND D.W. FOLTZ DISCUSSION
Environmental biologists have long been concerned with the physiological effects of salinity on estuarine organisms. In the range of sublethal salinity conditions, the performance of estuarine organisms may be modified in various ways. Among these responses are modifications of rates and efficiencies of metabolism, activity, growth, and reproduction (Kinne, 1966). Changes in whole animal rate functions are well documented (Kinne, 1963; Bayne, 1976). These changes in the various rates may be used as indices of stress (Bayne, 1976). Mean weight-specific clearance rates of R. cuneata decreased significantly with increasing salinity (Table I). These results may be indicative of stress due to salinity, and are consistent with the finding by Theede (1963) that filtration rates of M. edulis decreased upon transfer to either higher or lower salinities. Another indication of stress might be the increased number of clams with an observed clearance rate of 0 in the higher salinity treatments (Table I). This pattern may be a result of valve closure by the animal as a behavioral response to stress (Burton, 1983). In a similar fashion, marine invertebrate carnivores show increased percentages of animals not feeding at extreme conditions along environmental stressor gradients (Stickle, 1985). Six of the nine loci studied appeared to be in Hardy-Weinberg equilibrium; however; Est, Mpi, and Lap were not (Table II). Each of these three loci exhibited deficiencies in numbers of heterozygotes. Departures from Hardy-Weinberg proportions due to deficiencies in the numbers of heterozygotes have been reported in many other marine bivalves (Berger, 1983; Singh & Green, 1984; Zouros & Foltz, 1984). Possible explanations of these deficiencies include nonrandom mating (e.g., inbreeding), selection in either the larval or adult stage of the life cycle, or the effects of population structure (Wahlund effect). Another possibility, though, could be the occurrence of nonreactive (null) alleles, which might result in a deficiency in the number of heterozygotes if the null heterozygote is wrongly scored as an active homozygote. In a recent study, Foltz (1986) reported the existence of null alleles at two loci (Mpi and Lap) in the oyster C. virginica. However, any of these theories, alone or in combination, could explain the deficiencies in the numbers of heterozygotes observed in R. cuneata. More information concerning the life history as well as the genetic history of the population sampled is needed to answer questions involving deviations from Hardy-Weinberg genotypic proportions in this species. Heterozygosity at nine allozyme loci was positively associated with weight-specific adjusted clearance rate. The percentage of the total variance in clearance rate that was explained by heterozygosity was small, and similar findings have been reported in other studies of heterozygosity, feeding rate and scope for growth (Garton et al., 1984). This result suggests that much of the variance in clearance rate in R. cuneata is of environmental origin, or is due to effects of loci other than the nine included in the study. There are no significant differences between the weight-specific adjusted clearance rates for heterozygotes and homozygotes at any single allozyme locus in R. cuneata (Holley,
HETEROZYGOSITYANDCLEARANCERATEINACLAM
129
1986). The effect of heterozygosity at a locus on clearance rate appears to be small but cumulative, resulting in an overall relationship between heterozygosity and clearance rate. Similar findings have been reported for other studies of heterozygosity and physiological energetics in marine molluscs (Garton, 1984; Garton et al., 1984). These results provide further evidence supporting the positive relationship between litnessrelated characters and allozyme heterozygosity in marine bivalves. The lack of an association between heterozygosity and estimated flesh dry weight appears to contradict other studies (e.g., Zouros et al., 1980; Koehn & GatIney, 1984) that have reported a significant positive correlation between allozyme heterozygosity and weight or size. However, the present study was not designed specifically to detect such a correlation. In particular, the sample of clams used for determination of heterozygosity, weight, and clearance rate was small in number and probably of mixed ages. These factors would tend to obscure any correlation between heterozygosity and weight that existed in the clam population that was sampled (Zouros & Foltz, 1987). The study was designed to detect an interaction between salinity and heterozygosity in determining clearance rate, but that objective was made more difficult by the failure of many clams to clear at elevated salinities, resulting in reduced sample sizes at 15 and 25s0 salinity. Therefore, the present data do not provide strong support either for or against the suggestion that the effect of heterozygosity on clearance rate depends on salinity. The superiority of heterozygotes over homozygotes in relation to growth, fecundity and viability has now been demonstrated in many species of marine invertebrates (Koehn & Gtiney, 1984; Zouros & Foltz, 1987). The actual genetic mechanism responsible for translating heterozygosity at loci coding for soluble proteins into greater metabolic efficiency and higher feeding rates are presently unknown, although several hypotheses have been proposed. It is possible that the growth advantage and metabolic efficiency of relatively heterozygous individuals results from a lower rate of enzyme biosynthesis in multiple-locus heterozygotes (Koehn, 1985). A second hypothesis is that the enzyme loci are acting as indicators of heterozygosity at linked loci affecting growth and metabolism, the “associative overdominance” explanation (Zouros, 1987; Zouros & Foltz, 1987). Further study is needed to determine more accurately the relationships among stress, metabolic efficiency, feeding rate, and multiple-locus heterozygosity in marine invertebrates.
ACKNOWLEDGMENTS
We thank Drs. M. Kapper, R. Roller and S. Wang for assistance with the clearance rate measurements and/or specimen collection; M. Rotenberry and M. N. Freeman for their assistance in specimen preparation; and Drs. E. Zouros, W. B. Stickle, Jr., J. W. Fleeger, and two anonymous reviewers for comments on an earlier draft of the manuscript. This research was supported in part by National Science Foundation Grant BSR-8407450 to D.W. Foltz.
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