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Physiological variation among clonal genotypes in the sea anemone Haliplanella lineata: growth and biochemical content.


Many species of sea anemones reproduce asexually, resulting in the production of genetically identical individuals in natural populations. Local populations may consist of a single clone (e.g., the actinian Haliplanella lineata; Shick, 1976; Shick and Lamb, 1977), or they may be composed of many clones and approach the genetic diversity expected for a sexually outcrossing population (see Shick, 1991, pp. 270-277). Such differences in population genetic structure may have multiple causes. For example, monoclonal populations may result from genetic founder effects (i.e., settlement of a single adult or planula larva, followed by asexual proliferation), followed by competitive exclusion of other clonal genotypes (Ayre, 1982, 1983, 1995; Hoffmann, 1986). Alternatively, monoclonal populations could result from the settlement of multiple genotypes, followed by differential selection leading to the elimination of all but one highly locally adapted genotype (see Ayre, 1985, 1995). Multiclonal populations could result from the asexual proliferation of multiple genotypes that have the same relative fitness, although formal tests of fitness differences among clonal genotypes are rare (Ayre, 1995).

On theoretical grounds, asexual reproduction may be viewed as a means of preserving and increasing the size of locally adapted multiple-locus (i.e., clonal) genotypes (Williams, 1975), and a number of researchers have provided evidence consistent with this theoretical prediction of local adaptation (see especially, Ayre, 1985, 1995). However, the functional basis for such local adaptation has not been explored. Indeed, Shick (1991) points out that studies yielding evidence of local adaptation have not tested whether locally adapted clones exist in any other localities, i.e., the extent to which any single clone is a broadly adapted, "general purpose genotype" (Shick, 1976; Shick and Lamb, 1977).

Although the ecological physiology of sea anemones is rather well-studied (cf. Shick, 1991), there have been surprisingly few investigations of physiological variation among clonal genotypes present in individual populations or individual habitats within populations of asexually reproducing anemones (e.g., Shick and Dowse, 1985). Such information could provide the initial basis for a mechanistic understanding of localized adaptation, and for determining whether natural selection or genetic founder effect is more important to the genetic structure of these populations. Of particular note in this context is a study by Shick and Dowse (1985), who concluded, on the basis of an examination of literature data on sea anemones, that intraclonal variance in a variety of physiological measurements is smaller than interclonal variance in these traits, and that the variance for some traits could be explained largely by clonal identity. Thus they provided strong evidence of genetically correlated performance variability. However, much of their work focused on comparisons of variances between geographically separated monoclonal and multiclonal populations of the same species, or between genetically diverse populations of one species and monoclonal populations of another species. Although many studies have persuasively shown that localized adaptation of clones occurs (e.g., Ayre 1985, 1995; Sebens, 1981; Zamer and Shick, 1989), such comparisons reflect the fact that few studies have directly examined physiological variation among clonal genotypes collected from a single population or habitat, and maintained under the same environmental conditions so as to remove acclimation effects on performance.

Some field-based studies have provided limited evidence for physiological variation among clonal genotypes within and between populations. For example, Jennison (1979) interpreted differences in tissue lipid among clones of the anemone Anthopleura elegantissima to be the result of either genetically encoded differences in lipid metabolism or micro-environmental differences in food availability between clones at a single field site. Differences in reproductive characteristics between high- and low-intertidal clones of A. elegantissima were attributed to higher temperatures in the upper intertidal (Sebens, 1981), the likeliest explanation, but the possibility remains that these clones were genetically adapted to the different habitats in this single population.

More persuasive evidence of localized genetic adaptation among anemone clones comes from controlled experimental studies. When acclimated to common conditions, high-intertidal clones of A. elegantissima exhibit different physiological energetic characteristics (e.g., absorption efficiency, net growth efficiency) than low-inter-tidal clones of this anemone; these results were interpreted to mean that adaptive, genetic divergence of the clones had occurred in response to low food availability in the upper intertidal zone (Zamer, 1986; Zamer and Shick, 1987, 1989). Ayre (1985) showed that local clones of the anemone Actinia tenebrosa had greater capacities for asexual reproduction than did transplanted foreign clones; he concluded that highly localized adaptation of the clones had occurred in colonies of the anemone.that were only 2-4 km apart. And Shick et al. (1979) transplanted clones of the anemone H. lineata from a Rhode Island population to a Maine population site and observed 100% mortality of the transplants. These investigators also interpreted their results to mean that the separate populations were genetically distinct and contained locally adapted clones. However, little information about functional diversity among clones within any single habitat or population is available from the aforementioned studies. More recently, variation in performance traits among clones has been explored in polyps of the jellyfish Aurelia aurita (Keen and Gong, 1989), in the corallimorpharian Corynactis californica (Chadwick and Adams, 1991), and in the anemone A. elegantissima (Tsuchida and Potts, 1994).

Genetic variation in growth among individual organisms has been examined generally by two approaches. The first, quantitative genetics, statistically partitions phenotypic variance among relatives (e.g., parents, offspring, half-siblings) into genetic and environmental components (Falconer, 1989). This approach has successfully documented significant genetic variation and genotype-environment interactions in growth in a number of shellfish species (Jones et al., 1996; Rawson and Hilbish, 1991). Such studies cannot, however, address the physiological mechanisms underlying genetic variation in growth (cf Clark, 1990; Koehn, 1991), and therefore cannot provide relevant information about the mechanisms of localized adaptation among clones of sea anemones.

The second approach is physiological energetics, which focuses on performance traits that constitute the energy and materials budgets of an organism (e.g., Hilbish and Koehn, 1985; Hawkins et al., 1986; Zamer and Shick, 1987; 1989; Koehn and Bayne, 1989; Present and Conover, 1992). We have taken this approach to examine physiological variation among clonal genotypes in the eurytolerant sea anemone H. lineata collected from a single population. The present paper focuses on growth and on the biochemical content of the tissue. The balanced energy equation (Winberg, 1956) has been used as a conceptual framework in examining variation in organismal performance. Bayne and Newell (1983) expressed the equation as

Pg + Pr = C [multiplied by] AE - ([R.sub.m] + [R.sub.r])

where Pg is somatic production (growth), Pr is reproductive production (gametes), C is energy consumed, AE is the efficiency with which consumed energy is absorbed, [R.sub.m] is the metabolic cost of body maintenance, and [R.sub.r] includes all other metabolic costs (Koehn and Bayne, 1989). Variation in growth among individuals can originate from differences in the components of the balanced energy equation, such as metabolic costs, consumption, and absorption efficiency (Koehn and Bayne, 1989; Present and Conover, 1992).

Haliplanella lineata is a widely distributed, colonizing species, which in natural populations rapidly increases in numbers by asexual reproduction and may disappear from an area suddenly (Shick, 1976). Although this species is extremely euryhaline and eurythermal (Shick, 1976), sudden disappearances have been attributed to the existence of one or only a few clonal genotypes in populations and to environmental factors that exceed genetically based tolerance limits (Shick, 1976; Shick et al., 1979). Although sexually reproducing populations occur in Japan (Fukui, 1991), North American and laboratory populations of H. lineata reproduce only asexually, mainly by longitudinal fission (Johnson and Shick, 1977; Minasian, 1979), and the species can be classified as an agametic, cloning anemone (Hughes, 1989; Carvalho, 1994).

A physiological advantage of studying anemones that reproduce exclusively by asexual fission is that sexual reproduction (Pr) is eliminated from consideration of energy balance, simplifying the analysis. The animal is easily cultured in the laboratory, and the rate of longitudinal fission may be increased by maintaining the anemones at relatively high temperatures (Minasian, 1979; Minasian and Mariscal, 1979; Zamer and Mangum, 1979), so that large numbers of genetically identical clonemates are obtained easily. It is this latter feature, and the eurytolerant nature of the animal, that affords an ecophysiological and genetic advantage, in that clonemates may be studied at a variety of relevant environmental conditions. Using this approach we can replicate our physiological measurements on genetically identical individuals. Eventually, to better understand localized adaptation in this species, we can study the physiological characteristics of clonal genotypes from separate populations. We will also be able to partition physiological variation in H. lineata into genetic (comparisons among anemones from different clones) and nongenetic (comparisons within each clone) components (Shick and Dowse, 1985; Vrijenhoek, 1994).

In this study genetic variation in growth was examined in anemones from different clones, all of which were fed similar, measured food rations, thereby eliminating consumption differences in the energy balance equation as a source of variation in growth. In terms of physiological energetics we asked: Do growth, absorption efficiency, and growth efficiency differ among anemones from different clones? Because genetic variation in physiological energetics has been associated with differences in lipid and protein metabolism in some organisms (Medrano and Gall, 1976a, b; Hawkins et al., 1986), we examined the biochemical composition of the ration-fed anemones from the different clones. Finally, we measured the biochemical composition of tissues from anemones that were fed Artemia nauplii to test whether consumption differences associated with the capture of suspended prey could affect any of the biochemical patterns.

Materials and Methods

Collection and maintenance

In October 1990, individuals of H. lineata were collected from Indian Field Creek, a tributary of the York River in Virginia (37 [degrees] 16 [minutes] N, 76 [degrees] 33 [minutes] W), and shipped by air to our laboratory at Lake Forest College. Anemones were collected from tens of square meters at the site (C. P. Mangum, pers. comm.), so that this original sample was likely to have included a representative sampling of clones in this population. At the time of collection, water temperature was about 20 [degrees] C (C.P. Mangum, pers. comm.). Salinity ranges from 15-18 parts per thousand (ppt) at this site (W.E.Z., pers. obs., and C.P. Mangum, pers. comm.), and surface water temperature ranges annually from about 5 [degrees] C to 27 [degrees] C (Coast and Geodetic Survey, 1960, as cited in Sassaman and Mangum, 1970). Air temperatures to which the anemones are exposed at low tide may reach 30 [degrees]-32 [degrees] C during the summer, and may be near freezing during the winter (C. P. Mangum, pers. comm.).

Each anemone was placed in its own beaker (30 ml) containing 16 ppt seawater (Instant Ocean), held at room temperature (15 [degrees]-25 [degrees] C), and fed Anemia nauplii (San Francisco Bay brand) ad libitum every other day. Under these conditions, most anemones underwent repeated longitudinal fission. Fission products within individual beakers constitute a separate genetic lineage, and were eventually transferred to individual "stock" aquaria (9.5 1), where the separate lineages are currently maintained under continuous immersion in recirculating filtered seawater. Other maintenance conditions are the same as stated above.

Anemones from each of the aquaria were genotyped by using starch gel electrophoresis at five polymorphic loci [superoxide dismutase (SOD), isocitrate dehydrogenase 1 (IDH 1), glucose-6-phosphate isomerase (GPI), octopine dehydrogenase (ODH), and 6-phosphogluconate dehydrogenase (6Pgdh)] with standard gel electrophoresis techniques that will be described elsewhere (Zamer, unpubl. data). Based on the five polymorphic loci, five unique, multiple-locus genotypes, designated as A, B, C, D, E, and referred to hereafter as clones, were detected among the different lineages of anemones. For most of the work described below, three clones (A-C) were used.

Anemones from these different clones were acclimated in monoclonal aquaria (9.5 1) in an incubator at 15 [degrees] C for at least 5 weeks prior to the experiments. At this temperature these anemones do not readily undergo longitudinal fission, but they do grow. All acclimation and experimental conditions described in this paper included continuous immersion of the anemones.

Growth experiment

In 1994 we initiated a controlled growth experiment using anemones from our stock cultures. Sixty-five anemones from clones A, B, and C were selected from the stock cultures so as to minimize any size differences among the clones (Table I). Sample sizes were n = 13, 26, and 26, in clones A, B, and C, respectively (unequal sample size was due to the slow rate of fission by clone A anemones in the stock aquaria). Before weighing each anemone, adhering debris was removed, and the gastrovascular water was removed to absorbent paper by applying [TABULAR DATA FOR TABLE 1 OMITTED] gentle pressure to the body wall with a small spatula. Anemones were allowed to attach to individual 15-ml plastic beakers, which were then floated on the surface of monoclonal aquaria at 15 [degrees] C. Anemones were fed Artemia nauplii in the aquaria by submerging the beakers.

From the 65 anemones, an initial group of 25 was chosen for calculation of dry-to-wet-mass regressions. Each anemone in this initial group (n = 5, 10, and 10 respectively for clones A, B, and C) was removed from its beaker, sliced longitudinally to release all gastrovascular water, rinsed briefly with deionized water to remove salts, and blotted. The anemones were dried at 50 [degrees] C for 24 h, cooled to room temperature in a desiccator, and weighed to the nearest 0.01 mg. For each of the clones, the dry mass of the anemones in the initial group was regressed on their wet mass (previous paragraph). Clonal regression equations were used to estimate the starting dry mass from the wet mass of the remaining individual anemones in each clone. These dry mass estimates were used in the growth experiment, and the size range of initial anemones was selected to ensure that the estimates were interpolations and not extrapolations of the regressions (Weisburg, 1985).

The 40 remaining anemones used in the growth experiment (hereafter called the experimental anemones) were attached to individual glass beakers that were randomly assigned to one of two 38-1 aquaria maintained at 15 [degrees] C and filled with 24 l of 16-ppt salt water. Anemones were randomly assigned to a position on the floor of each aquarium. The box filter in each aquarium was moved once a day to a different corner of the aquarium to prevent any bias in airflow and filtration. These aquaria served as experimental blocks, which minimized any effects of temperature heterogeneity within the incubator.

Each experimental anemone received a daily ration of frozen adult Artemia (San Francisco Bay brand) for the next 10 days. For the first 2 days, the size of the ration was 6% of the estimated starting dry mass of each anemone. To make the weighing of the Artemia easier, the ration size was increased to 8.5% for the remaining 8 days. Each ration was placed on the oral disk of each anemone to ensure that it was ingested. On the following day, egesta produced from each anemone was removed a few hours prior to that day's feeding. The daily egesta for each anemone was rinsed with deionized water to remove salts and stored on a weigh boat in a desiccator. Egesta obtained from individual anemones was pooled over the 10-day experiment, dried at 50 [degrees] C, and then weighed. The pooled egesta mass was used in the calculation of gravimetric absorption efficiency for each anemone (see below). On the day following collection of the last egesta, "blotted" wet mass (described below) and dry mass (described earlier) were determined for each of the 40 experimental anemones.

Physiological energetics

The relative growth (RGR) of the experimental anemones was calculated as: RGR = [(final dry mass - estimated starting dry mass) [multiplied by] [(estimated starting dry mass).sup.-1]] x 100%. Gravimetric absorption efficiency [[A.sub.g], (g ingested - g egested) [multiplied by] [(g ingested).sup.-1] x 100%] and net growth efficiency [[K.sub.2], g growth [multiplied by] [(g absorbed ration).sup.-1] x 100%] were calculated as in Zamer (1986).

Biochemical analyses and energetic content

Tissue hydration, and protein, carbohydrate, and lipid content of tissues were measured. Tissue hydration was calculated for the initial and experimental groups of anemones as: (blotted wet mass - dry mass) [multiplied by] [(blotted wet mass).sup.-1] x 100%. Blotted wet mass (mean [+ or -] SE = 83.1 [+ or -] 4.55 mg) was obtained on anemones after cutting their body walls to express gastrovascular cavity water, rinsing to remove salts, and blotting as described previously for the initial group of anemones.

After the dry mass of an anemone was measured, the material was placed in a shell vial, ground with a glass rod, and stored at -70 [degrees] C. Total protein and carbohydrate was measured in samples (3.1-8.4 mg) of oven-dried tissue sonicated in 500 [[micro]liter] of deionized water. An 800-[[micro]liter] volume of cold 10% trichloroacetic acid (TCA) was added to a 200-[[micro]liter] aliquot of the homogenate, the sample was kept on ice for 10 min and swirled every 5 min, then centrifuged at 4500 x g at 2 [degrees] C for 30 min. The supernatant was discarded, and the tube containing the precipitated protein was inverted and drained for 1 h at room temperature. The protein was then dissolved in 1.0 ml of 10% sodium hydroxide (NaOH). Two 200-[[micro]liter] aliquots of this protein solution were removed and each was mixed with 400 [[micro]liter] of 10% NaOH and 1.4 ml of de-ionized water. Microbiuret (Itzhaki and Gill, 1964) determinations of protein were made on this solution. Bovine serum albumin is an inappropriate standard for estimating protein content in sea anemones (Zamer et al., 1989). Protein was isolated from anemones (kept in the stock aquaria) representing all five clones, using the procedures of Zamer et al. (1989). This Haliplanella protein was dissolved in 10% NaOH and used as the standard.

Total carbohydrate was isolated from the remaining 300 [[micro]liter] of homogenate as described in Zamer et al. (1989) and quantified spectrophotometrically using the method of Dubois et al. (1956). The relatively mild wet biochemical methods that we used to isolate carbohydrate do not cleave the carbohydrate residues from glycoproteins associated with collagen in anemones (Zamer et al., 1989). Consequently, this source of structural carbohydrate was not included in our measurements of carbohydrate content of the tissues.

Total lipid was extracted from 1.4-6.0 mg of oven-dried tissue by using a modified Bligh and Dyer (1959) technique with methylene chloride and methanol as the solvents (Carlson, 1985). Lipid class composition was measured by thin-layer chromatography/flame ionization detection (TLC/FID) on an Iatroscan TH-10 TLC/FID Analyzer (Iatron Laboratories, Tokyo, Japan). About 20 [[micro]gram] of lipid from each extracted lipid sample was spotted onto activated S-III chromarods in duplicate in 1-2 [[micro]liter] of methylene chloride:methanol (1:1) for TLC/FID lipid class analysis. As controls for both lipid class retention time and FID efficiency, we included with each sample analysis two chromarods, which were spotted with a standard mixture of phosphatidyl choline, cholesterol, triolein, 1-O-hexadecyl-2-3-dipalmitoyl-rac-glycerol (glycerol ethers) and cholesteryl oleate in proportions similar to those found in anemone samples, as determined by a preliminary analysis of the samples. Rods spotted with lipids were prefocused twice in chloroform:methanol (1:1), then developed in hexane:diethyl ether:formic acid (85:15:0.1) for 45 min. Racks containing the chromarods were then dried at 100 [degrees] C for 5 min before being scanned. Developed chromarods were scanned at 30 cm [multiplied by] [min.sup.-1]. Gas flow rates for hydrogen and air were 190 ml [multiplied by] [min.sup.-1] and 20 1 [multiplied by] [min.sup.-1], respectively. Peak areas for each lipid component were quantified by using a Hewlett Packard 3390A integrator.

Standard curves from 0 to 20 [[micro]gram] were created for phospholipids, sterols, fatty acids, triacylglycerols, glycerol ethers, sterol esters, and wax esters. The specific lipid used to represent each lipid class and the [r.sup.2] value for each standard curve are as follows: phospholipids (phosphatidyl choline), [r.sup.2] = 0.992; sterols (cholesterol), [r.sup.2] = 0.996; fatty alcohols (hexadecanol), [r.sup.2] = 0.998; fatty acids (oleic acid), [r.sup.2] = 0.985; triacylglycerols (triolein), [r.sup.2] = 0.984; glycerol ethers. (1-O-hexadecyl-2-3-dipalmitoyl-rac-glycerol), [r.sup.2] = 0.992; sterol esters (cholesteryl oleate), [r.sup.2] = 0.987 and wax esters (cetyl oleate), [r.sup.2] = 0.914. Sterol and wax esters are not resolved in the solvent system used. Periodic checks of area responses for standards indicated that standard errors of replicates were less than 3% of mean values in all cases. Total lipid was estimated by summing peak areas for each of the lipid classes.

Anemones in the experimental group were of sufficient size so that enough tissue was available for protein, carbohydrate, and lipid measurements, including the six lipid classes. The masses (in milligrams) of protein, lipid, and carbohydrate in each experimental anemone were multiplied by their corresponding specific enthalpy of combustion ([[Delta].sub.c]h, kJ [multiplied by] [g.sup.-1]; Gnaiger, 1983), and the products were summed to estimate tissue energetic content (in kilojoules) of each anemone.

Anemones in the initial group, which were fed nauplii and used for estimating starting dry mass, had only enough tissue for determination of lipid class content. Therefore, in two other experimental groups (X and Y), we measured biochemical composition of tissues of anemones. These two groups of anemones were maintained simultaneously (in this case for over 5 weeks) in a single set of five monoclonal aquaria under the same conditions of feeding, temperature, and salinity as the initial group of anemones. Anemones in groups X and Y consisted of individuals from all five clones (A, B, C, D, and E), and were attached to the surfaces of the aquaria; unlike the anemones in the initial and experimental groups, they were not confined to beakers. The initial group of anemones and anemones in groups X and Y allowed us to test for the effects of clonal identity on biochemical composition when H. lineata consumed suspended nauplii as opposed to rations of frozen adult Artemia. Protein, carbohydrate, and tissue hydration were measured on the group X anemones; ash content and tissue hydration were measured on group Y anemones. Ash content was determined after the dried anemone tissue was combusted at 500 [degrees] C for 6 h in a muffle furnace.

Statistical analysis

Relative growth, gravimetric absorption efficiency, and gravimetric net growth efficiency of the experimental anemones were analyzed with a randomized block analysis of variance (ANOVA) (Steel and Torrie, 1980). The categorical factors were block, represented by the two aquaria, and clone. Relative growth was arcsine transformed, whereas gravimetric absorption efficiency required a logit transformation (Cox and Snell, 1992) to meet the assumptions of the analysis. Gravimetric net growth efficiency did not require any transformation. Based on regression analysis, these three physiological energetic traits did not vary with body size.

Tissue hydration (percent) was measured on anemones from all four data sets: initial, experimental, and groups X and Y. The analysis of tissue hydration for the experimental anemones followed the randomized block design. Tissue hydration was examined by using an analysis of covariance (ANCOVA) for anemones in the other three data sets because significant regression slopes showed size-dependence, which was not the case for the experimental group. Tissue hydration in all cases was arcsine transformed, and the covariate, dry mass of the anemones, was transformed using natural logarithms.

All the biochemical variables (protein, carbohydrate, total lipid, and the six lipid classes) were initially expressed as proportions of the dry mass of tissue samples. These biochemical variables were "scaled up" (and expressed in milligrams) by multiplying the proportions by the dry mass of each anemone. The estimate of ash was made from an entire anemone, not from a tissue sample, so "scaling up" of that variable (milligrams of ash) was not needed. All the biochemical response variables, ash and anemone dry mass were transformed using natural logs.

Next, to acknowledge the metabolic relationships and potential covariance among similar dependent variables (e.g., protein, carbohydrate, and lipid in tissues of experimental anemones), all of which had dry mass as a covariate, we grouped such variables and initially analyzed them with a multivariate analysis of covariance (MANCOVA; Huitema, 1980). This set of analyses also guards against over-interpretation of only a series of univariate analyses for these same variables. We used MANCOVA to analyze the effect of clone on protein, carbohydrate, and total lipid in experimental anemone tissue; the effect of clone on the six lipid classes in tissues of initial and experimental anemones; and the effect of clone on protein and carbohydrate content in tissues of group X anemones. When a MANCOVA resulted in a significant effect of clone on the group of dependent variables being analyzed, we proceeded with a series of ANCOVA, in which the effect of clone was examined for individual dependent variables having dry mass as covariate. Block and clone were the categorical variables for the experimental group of anemones for all these analyses. Clone was the only categorical variable for the other data sets (initial group, and groups X and Y) in all of these analyses.

The energetic contents of the experimental anemones were size-dependent, so they were analyzed with an ANCOVA. Block and clone were the categorical variables, and the natural log of dry mass was the covariate. No transformation was necessary for energetic content.

When ANOVA or ANCOVA resulted in a significant effect of clone on a dependent variable, we employed unplanned multiple comparison procedures (Day and Quinn, 1989). For the ANOVAs, all pairwise comparisons of means were made with the Tukey-Kramer procedure, which adjusts for unequal sample sizes (Day and Quinn, 1989). In the case of significant clonal effects from ANCOVAs, the Bryant-Paulson-Tukey (BPT) procedure was used to compare the size-adjusted means (Huitema, 1980). The BPT procedure takes into account that these size-adjusted means are not statistically independent due to the use of a pooled regression slope (Day and Quinn, 1989).

The relationship between the amount of water in the anemones (measured as the difference between blotted wet mass and dry mass; in milligrams) and the amount of total lipid (in milligrams) in the experimental and initial anemones was examined by partial correlation analysis, which measures the correlation between this pair of variables, keeping anemone body size constant (Sokal and Rohlf, 1981).

In the tables, the clonal means are presented with their 95% confidence limits (CL), and in the graphs, the clonal means are presented with their 95% confidence intervals (CI). For the size-independent variables (physiological energetic traits and tissue hydration of experimental anemones), their transformed means and standard errors were used to calculate the 95% CL, the means and 95% CL were back-transformed, and these values are reported here (Sokal and Rohlf, 1981, p. 419). The untransformed means for gravimetric absorption efficiency are presented. For the size-dependent variables (tissue hydration for the initial, X, and Y groups of anemones, and all the biochemical data) transformed, size-adjusted means, and their size-adjusted standard errors (Sokal and Rohlf, 1981, p. 525) were used to calculate 95% CL, and both means and confidence, limits were then back-transformed. We present these back-transformed values of means and confidence intervals. The significant differences from the unplanned multiple comparison procedures, Tukey-Kramer or BPT, are indicated on the graphs, where different lowercase letters indicate significantly different clonal means.


Physiological energetics

Our comparison of the physiological energetics of the anemones from the different clones revealed substantial intraclonal variation. Average relative growth for the ration-fed anemones was 35% [ILLUSTRATION FOR FIGURE 1 OMITTED], and the effect of clone was not significant ([F.sub.2,36] = 1.3, P = 0.29). The initial dry mass and final dry mass averages did not differ significantly among the clones ([F.sub.2,37] = 0.60, P = 0.56, [F.sub.2,36] = 0.80, P = 0.46, respectively; Table I). Nor was there a significant effect of clone on gravimetric absorption efficiency ([F.sub.2,26] = 0.36, P = 0.70), for which clonal averages ranged between 92.7% and 93.5%. The clonal pattern in net growth efficiency ([K.sub.2]) was similar to that for relative growth. Clonal averages for [K.sub.2] were 40.4%, 43.8%, and 51.3% for clones A, B, and C, respectively. There was no significant effect of clone on [K.sub.2] ([F.sub.2,26] = 1.9, P = 0.16).

Tissue hydration and lipid content

At the biochemical level of organization, interclonal variation was frequently greater than intraclonal variation. Clonal genotype significantly affected biochemical content in the experimental anemones, as revealed by a MANCOVA in which carbohydrate, protein, and lipid values were analyzed collectively (Wilks' [Lambda] = 0.33, [F.sub.6,32] = 7.6, P [less than] 0.001).

Tissue hydration differed significantly among clones of the experimental anemones ([F.sub.2,36] = 15.2, P [less than] 0.001), with those from clone B having the highest average [ILLUSTRATION FOR FIGURE 2A OMITTED]. The effect of clone accounted for 43% of the variation in tissue hydration. For the initial group, anemones from the three clones also differed significantly in tissue hydration ([F.sub.2,21] = 14.8, P [less than] 0.001); anemones from clone B had the highest average at 84.5% [ILLUSTRATION FOR FIGURE 2B OMITTED]. Tissue hydration was not significantly different among the anemones from the five clones of group X ([F.sub.4,34] = 2.4, P = 0.07; Table II), but it was in group Y anemones ([F.sub.4,24] = 2.8, P = 0.048; Table II). However, in group Y none of the pairwise comparisons among clonal means were significant.
Table II

Tissue hydration for group X and Y anemones

                            Group X                  Group Y
Clone                 Tissue Hydration (%)     Tissue Hydration (%)

A                      82.3 (81.6, 83.0)        81.8 (81.0, 82.6)
B                      82.3 (81.6, 83.0)        82.2 (81.4, 83.0)
C                      81.0 (80.3, 81.8)        81.6 (80.8, 82.5)
D                      81.6 (80.8, 82.4)        81.5 (80.4, 82.6)
E                      82.1 (81.3, 82.8)        83.1 (82.2, 84.1)

For each of the five clones, the sample sizes are n = 8 in group X
and n = 6 in group Y. All values are the size-adjusted,
back-transformed means and their 95% confidence limits. Tissue
hydration was calculated for an anemone with an average dry mass of
8.73 mg for group X and 17.80 mg for group Y.

The amount of water and lipid (both in milligrams) in tissues of the experimental anemones showed an inverse relationship that was significant according to the partial correlation analysis (r = -0.34; [r.sub.0.05, 35df] = -0.32). The effect of clone was significant on the amount of total lipid ([F.sub.2,35] = 3.3, P = 0.05; [ILLUSTRATION FOR FIGURE 3A OMITTED]), which composed 15.5% of the dry mass, on average, of the experimental anemones. On the basis of the Tukey-Kramer procedure, none of the pairwise comparisons were significant. For the initial group of anemones, amounts of tissue water and total lipid were not significantly correlated (partial correlation coefficient = -0.30, [r.sub.0.05, 22df] = -0.40). Also, initial anemones from the three clones were not significantly different in the amount of total lipid ([F.sub.2,21] = 2.8, P = 0.08; [ILLUSTRATION FOR FIGURE 3B OMITTED]). Total lipid constituted 20.4% of the dry mass, on average, of the initial anemones.

The analysis of the individual lipid classes proved more informative than the examination of total lipid. Overall, lipid class content differed significantly among clones of both the experimental anemones (Wilks' [Lambda] = 0.245, [F.sub.12,60] = 5.10, P [less than] 0.001) and the initial group of anemones (Wilks' [Lambda] = 0.107, [F.sub.12,32] = 5.48, P [less than] 0.001). In general, anemones from clone B had less lipid than anemones from clones A and C. The three clones of experimental anemones differed significantly in the amounts of triacylglycerols ([F.sub.2,35] = 4.3, P = 0.02; [ILLUSTRATION FOR FIGURE 4A OMITTED]), sterol esters and wax esters ([F.sub.2,35] = 7.3, P = 0.002; [ILLUSTRATION FOR FIGURE 5A OMITTED]), glycerol ethers ([F.sub.2,35] = 15.1, P [less than] 0.001; [ILLUSTRATION FOR FIGURE 6A OMITTED]), and free fatty acids ([F.sub.2,35] = 5.5, P = 0.009; [ILLUSTRATION FOR FIGURE 7A OMITTED]). In the initial anemones, clonal identity significantly affected levels of triacylglycerols ([F.sub.2,21] = 3.9, P = 0.04; [ILLUSTRATION FOR FIGURE 4B OMITTED]), glycerol ethers ([F.sub.2,21] = 8.3, P = 0.002; [ILLUSTRATION FOR FIGURE 6B OMITTED]), and free fatty acids ([F.sub.2,21] = 4.3, P = 0.03; [ILLUSTRATION FOR FIGURE 7B OMITTED]).

On average, experimental anemones from clone B had 35.6% less triacylglycerol than anemones in clone C [ILLUSTRATION FOR FIGURE 4A OMITTED], and clone B anemones in the initial group had the lowest levels of triacylglycerol [ILLUSTRATION FOR FIGURE 4B OMITTED]. However, comparison of the average amount of triacylglycerol between clones B and C in the initial group yielded a generalized Studentized range statistic ([Q.sub.P(0.05,1,2,21)] = 3.47) from the BPT procedure (Huitema, 1980) that was not significant ([Q.sub.P(crit)] = 3.67).

Among the experimental anemones, those in clone B had the lowest amount of sterol esters and wax esters, averaging 36.4% less than anemones in clone A and 22.5% less than anemones in clone C [ILLUSTRATION FOR FIGURE 5A OMITTED]). Although the effect of clone was significant on the amount of sterol esters and wax esters in the initial group of anemones ([F.sub.2,21] = 3.8, P = 0.04; [ILLUSTRATION FOR FIGURE 5B OMITTED]), an outlier, the maximum value for sterol esters and wax esters, was identified, and dropping this value resulted in no detectable effect of clone ([F.sub.2,20] = 1.5, P = 0.25).

Similar patterns were found for glycerol ethers and free fatty acids in both experimental and initial anemones. In the experimental group of anemones, clone B anemones had at least 41% less glycerol ethers than anemones in clones A and C [ILLUSTRATION FOR FIGURE 6A OMITTED], and they had 36% less free fatty acids than anemones in clone C [ILLUSTRATION FOR FIGURE 7A OMITTED]. Among initial anemones, clone B anemones had 64% less glycerol ethers than anemones in clone A, and 45% less than anemones in clone C [ILLUSTRATION FOR FIGURE 6B OMITTED]. And free fatty acids in initial clone B anemones were 62% less than the value in anemones in clone C [ILLUSTRATION FOR FIGURE 7B OMITTED].

Sterol content did not vary among the clones of experimental anemones ([F.sub.2,35] = 0.20, P = 0.82; [ILLUSTRATION FOR FIGURE 8A OMITTED]), or among clones of the initial anemones ([F.sub.2,21] = 2.6, P = 0.10; [ILLUSTRATION FOR FIGURE 8B OMITTED]). Similarly, there was no effect of clone on the amount of phospholipids for the experimental anemones ([F.sub.2,35] = 0.45, P = 0.64; [ILLUSTRATION FOR FIGURE 9A OMITTED]) or the initial anemones ([F.sub.2,21] = 0.92, P = 0.41; [ILLUSTRATION FOR FIGURE 9B OMITTED]).

Carbohydrate, protein, and ash contents

Clones of experimental anemones differed significantly in tissue carbohydrate content ([F.sub.2,33] = 27.0, P [less than] 0.001), which constituted 6.7% of anemone dry mass, on average [ILLUSTRATION FOR FIGURE 10A OMITTED]. Carbohydrate, like lipid, was lowest in anemones from. clone B - on average, 24% less than that of anemones in clones A and C [ILLUSTRATION FOR FIGURE 10A OMITTED]. In group X anemones, a MANCOVA revealed significant clonal effects on protein and carbohydrate contents (Wilks' [Lambda] = 0.43, [F.sub.8,52] = 3.4, P [less than] 0.001), and univariate analysis of carbohydrate content showed that clonal identity affected levels of this biochemical class ([F.sub.4,27] = 5.0, P = 0.004; [ILLUSTRATION FOR FIGURE 10B OMITTED]. Anemones in clones A and C had higher amounts of carbohydrate than anemones in clones B, D, and E, and the latter clones had similar levels [ILLUSTRATION FOR FIGURE 10B OMITTED]. Carbohydrate constituted, on average, 8.3% of the dry mass among group X anemones.

The clonal pattern for the amount of protein in the experimental anemones resembled that seen for relative growth, with anemones in clone A having the lowest amount and those in clone C the highest amount [ILLUSTRATION FOR FIGURE 11A OMITTED]. Like relative growth, the amount of protein in the experimental anemones showed no significant effect of clone ([F.sub.2,34] = 1.7, P = 0.19). Protein constituted the bulk of the anemone's dry mass, averaging 69.2%. For group X anemones, the ranking of lowest to highest amount of protein was the same as for the experimental anemones. The effect of clone was not significant on amount of protein in group X anemones ([F.sub.4,28] = 1.2, P = 0.34; [ILLUSTRATION FOR FIGURE 11B OMITTED]), and protein composed, on average, 77.8% of the anemone dry mass.

On average, ash constituted 8.6% of the dry mass of an anemone (group Y; Table III). Clonal genotype did not affect ash content ([F.sub.4,23] = 1.5, P = 0.24; Table III). The amount of ash in H. lineata is comparable to the 9.1% ash content in the sea anemone A. elegantissima fed Artemia nauplii in the laboratory (Zamer, 1986).
Table III

Ash content of group Y anemones

Clone                      Ash (mg)

A                      1.44 (1.33, 1.55)
B                      1.51 (1.41, 1.61)
C                      1.46 (1.36, 1.57)
D                      1.57 (1.44, 1.72)
E                      1.61 (1.49, 1.75)

The sample size is n = 6 for all of the clones except clone A, where
[n.sub.A] = 5. All values are the size-adjusted, back-transformed
means and their 95% confidence limits. Ash content was calculated
for an anemone with an average dry mass of 17.83 mg.

Energetic content

Tissue energetic content (in kilojoules), as calculated from biochemical composition, differed significantly among the three clones of experimental anemones ([F.sub.2,33] = 5.6, P = 0.008). The average energetic content of anemones from clone B was 17% less than that of anemones from clone C [ILLUSTRATION FOR FIGURE 12 OMITTED].


We observed a consistent, significant pattern of genetic variation in tissue hydration, carbohydrate content, and the content of several lipid classes among clones of H. lineata. The same clonal differences occurred in anemones from the two feeding regimes. Compared to anemones from clones A and C, clone B anemones consistently had lower averages for the lipid and carbohydrate contents of their tissues, and higher values for tissue hydration. Similar to clone B, anemones from clones D and E had less carbohydrate in their tissues than anemones from clone A [ILLUSTRATION FOR FIGURE 10B OMITTED]. The net result of these differences in tissue constituents was lower energetic content in tissues of clone B anemones.

Although the similarity in clonal pattern for the tissue constituents between nauplii-fed and ration-fed anemones may reflect the relatively short duration of the growth experiment (10 days of ration feedings) compared with the extended acclimation of the nauplii-fed anemones (groups X and Y; more than 5 weeks), the experimental anemones (ration fed) tended to contain less protein (69.2%) and carbohydrate (6.7%) than the anemones in group X (average protein and carbohydrate contents: 77.8% and 8.3%, respectively), which were fed ad libitum on suspensions of Anemia nauplii. Thus biochemical composition responded to the change in feeding regime during the 10-day experimental period. Changes in biochemical constituents in tissues of A. elegantissima also were evident after just 6 days of feeding on even smaller rations (1% of dry body mass) of adult Artemia (Zamer, 1986; Zamer and Shick, 1989), and the same trend in protein and carbohydrate content was found between groups of A. elegantissima that were fed adults and nauplii of Artemia (Zamer, 1986; Zamer and Shick, 1989). Clearly, cnidarian biochemical composition can be altered by differences in diet composition and feeding frequency (Szmant-Froelich and Pilson, 1980; Fitt and Pardy, 1981; Zamer and Shick, 1989).

Yet despite this trend in biochemical content associated with the different feeding regimes in our study, we observed overriding and repeatable clonal patterns in biochemical composition of the tissues of il. lineata. We cannot convincingly argue that the 10-day period of feeding on adult Artemia was sufficient time to remove a clonal pattern in biochemical content differences that could have been established by clonal differences in tentacular capture of suspended prey. However, the similar and rapid changes in protein and carbohydrate that occur when both H. lineata and A. elegantissima are switched from nauplii to ration feeding suggest that the persistent pattern of clonal differences observed under both feeding regimes is not the result of prey capture differences, but rather the result of differences in metabolic rates or food conversion efficiencies among the separate clones of il. lineata (see below).

The high amounts of storage lipids and carbohydrate in anemones in clones A and C compared to anemones in clone B is the same pattern of genetic covariation in these biochemical classes that has been found in different genetic lines of Drosophila melanogaster (Clark and Keith, 1988; Clark, 1990) and in different species of Drosophila (Clark and Wang, 1994). Mechanisms that may produce this covariation include genetic modulation of the activities of enzymes associated with lipid and carbohydrate pathways of metabolism (Clark and Keith, 1988), and such genetic modulation of metabolic pathway performance has been associated with genotypes at the glucose-phosphate isomerase locus in the anemone Metridium senile (Zamer and Hoffmann, 1989). Alternatively, clone B anemones may have been unable to absorb energy from the digested rations as well as anemones from the other two clones. Differential substrate-specific absorption has been demonstrated in anemones (Zamer and Shick, 1989), and could produce differences in the biochemical and energetic content of their tissues. Low amounts of carbohydrate and lipid in tissues of clone B anemones resulted in low values for the calculated energetic content of these tissues, even though anemones in clone B received the same rations as anemones in clones A and C.

The lower energetic content of clone B anemones derives primarily from the lower levels of what may be considered energy-storage forms of lipid: fatty acids, triacylglycerols, and sterol esters and wax esters. All of these lipid classes have been associated with energy storage in anemones (e.g., Pollero, 1983; Hill-Manning and Blanquet, 1979). We know of no data concerning the turnover of glycerol ethers in tissues of sea anemones, but given the low levels of this class of lipids in clone B anemones, we infer that it may also be a storage form.

In contrast, sterol (with cholesterol being the principal sterol in the anemone Actinostola callosa; Bergmann et al., 1956) and phospholipids (often considered to be important as structural or membrane classes) are not variable among the three anemone clones examined here. These results indicate that storage of energy in the form of lipid is somehow impaired in clone B anemones.

In this context, we also note the inverse relationship between tissue water and total lipid in the experimental anemones. Small differences in tissue hydration have been reported between high- and low-intertidal individuals of A. elegantissima and between continuously immersed individuals of H. lineata and those maintained under fluctuating immersion conditions (Shick, 1991; Johnson and Shick, 1977). In both cases, anemones periodically exposed to air have slightly higher tissue hydration. In A. elegantissima freshly collected from upper and lower intertidal areas, a significant difference in lipid content was not observed, although high-shore specimens tended to have lower lipid than low-shore ones, consistent with the inverse relationship between tissue hydration and lipid content found in this study for H. lineata. Shick (1991) also points out that tissue hydration may be genetically correlated, given that variance in tissue hydration is significantly greater in multiclonal populations of H. lineata than in monoclonal ones (Shick and Dowse, 1985). Our findings of significant differences in tissue hydration among clones of H. lineata are consistent with this observation.

No genetic variation in tissue protein was detected among the anemones. Likewise, none was found for the flour beetle Tribolium castaneum, either in genetic lines selected for 21-day pupa weight or in control, unselected, genetic lines (Medrano and Gall, 1976a). Protein content in H. lineata is less variable than carbohydrate or lipid. For the experimental anemones (n = 38), the coefficient of variation of protein was 8.3%, whereas it was 15.4% for carbohydrate and 19.1% for lipid. Although our static measurements of protein were not different among our anemone clones, genetic variation has been associated with nitrogen metabolism, protein turnover rate, and physiological energetics of the blue mussel Mytilus edulis (Hilbish and Koehn, 1985; Hawkins et al., 1986).

The physiological energetic values reported here for H. lineata are within typical ranges for sea anemones (Shick, 1991), and the experimental anemones from clones A, B, and C did not differ in the traits of relative growth, absorption efficiency, and net growth efficiency, which are all expressed gravimetrically. Recently, Tsuchida and Potts (1994) also reported that, in two separate feeding experiments, clonal identity had no effect on weight change in A. elegantissima. Differences in absorption efficiencies and net growth efficiencies have been detected among clones of A. elegantissima from different shore levels (Zamer, 1986), and those differences could not be erased by acclimation to common conditions. However, variation among clones within each tidal regime was not examined in that study. A. elegantissima had an average gravimetric absorption efficiency of 69.6% (Zamer, 1986), compared to 93% measured for H. lineata in this study. The net gravimetric growth efficiencies for these two species are about 45% (cf Zamer, 1986). The relative growth reported here for H. lineata is about 15% greater than that measured in A. elegantissima (Zamer, 1986), but the difference is probably due to the larger rations received by H. lineata in this study (6%-8.5% of dry body mass compared to 4%-5.6% for A. elegantissima).

The lack of clonal differences in these components of the energy budget does not necessarily mean that the genotypes of H. lineata are equivalent in their physiological energetics. First, an examination of energetically expressed absorption and net growth efficiencies, in which the energy content (rather than mass) of tissue growth, rations, and egesta are used in the calculations of these quantities, may reveal features of the physiological energetics of these clones that are not apparent from the present analysis of gravimetric values (cf Zamer, 1986). The similar average body masses of anemones in all three clones examined (Table I), in combination with both the low energetic content of the tissues of clone B anemones and the similar growth rates in anemones among these clones, is circumstantial support for the hypothesis that energetic values for net growth efficiency may be more informative than the present gravimetric ones.

Moreover, estimates of the biochemical content of food and egesta, which are values needed for determining energetic contents of these substances, may also be used to test one hypothesis concerning the differences in biochemical and energetic content of the tissues from anemones in the three clones. If we find the biochemical composition of the egesta to be the same, regardless of clonal genotype, then we can eliminate the differential-substrate-absorption hypothesis as an explanation for differences in the biochemical content of tissue.

Second, metabolic rate was not measured in the present study. If clone B anemones have a higher metabolic rate, on average, than anemones in clones A and C, then a greater proportion of the absorbed ration would be catabolized for maintenance, and consequently less energy would be stored in the tissues. In comparing clone B anemones acclimated to 25 [degrees] C with anemones in clone C at the same temperature, we have observed consistently higher rates of longitudinal fission and smaller average body mass in clone B animals (Zamer, unpubl. data). The metabolic rate in clone B anemones may be elevated owing to greater costs associated with higher fission rate at 25 [degrees] C. But we do not know whether elevation of metabolic rate underlies the low energetic content of clone B anemone tissues at 15 [degrees] C. Genetic variation in metabolic rate, measured as oxygen consumption, has been detected among selected and control lines of T. castaneum (Medrano and Gall, 1976b), among D. melanogaster lines selected for desiccation resistance and control lines (Hoffmann and Parsons, 1989a; 1989b), in bivalves differing in multiple-locus heterozygosity (Bayne, 1987), in common garter snake (Thamnophis sirtalis) offspring from different families (Garland, 1994), and among strains and populations of the deer mouse Peromyscus maniculatus that differed in a-chain hemoglobin genotypes (Chappell and Snyder, 1984). Such variation in metabolic rate can be manifested as variation in maintenance efficiency, specifically in the rate of protein turnover (Hawkins et al., 1986). Our ongoing experiments are aimed at determining energetically expressed values for absorption efficiency and growth and measuring oxygen uptake rates among anemones from our different clones. These studies will yield information about the mechanisms underlying the present clonal differences in physiological energetic traits as well as additional data on genetically correlated physiological variation.

In addition to providing these two mechanistic hypotheses, our study of the physiological variation that is associated with clonal genotype potentially has implications for the relative fitness of anemones from the different clones. At 10 [degrees] C in the laboratory, individuals of H. lineata commonly encyst in mucous secretions, and in Indian Field Creek encysted and nonencysted individuals occur in the winter when the surface water temperature is below 100 [degrees] C (Sassaman and Mangum, 1970). Although metabolic rate is correspondingly low at this temperature, and is likely to be even less in encysted compared to nonencysted individuals, survivorship of encysted anemones probably depends on reserves of storage lipid and carbohydrate. During extended encystment, anemones from clone B, which have less of these reserves at 15 [degrees] C, may have lower survivorship than anemones in clones A and C, which have more of these reserves. The relationship between individual variation in the physiological characteristics of organisms and the variation among organisms in fitness is an essential component of organismal performance (Pough, 1989).


Thanks to Mike Lynch for assistance in the lipid analysis, to M. Amsler for doing the electrophoresis, and to C.P. Mangum for collecting the anemones at Indian Field Creek. This research was supported by NSF grant DCB-9057315 to W. E. Z. This is contribution No 294 from the Center of Marine Biotechnology, University of Maryland Biotechnology Institute. Versions of this manuscript benefitted from comments by C. O. Deetz, A. T. Weglinski, and two anonymous reviewers.

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Author:McManus, Michael G.; Place, Allen R.; Zamer, William E.
Publication:The Biological Bulletin
Date:Jun 1, 1997
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