(Opsanus Beta) Sonic Muscle Enzyme Activities

BULLETIN OF MARINE SCIENCE, 45(1): 68-75,1989 SCALING AND SEX-RELATED DIFFERENCES IN TOADFISH (OPSANUS BETA) SONIC MUSCLE ENZYME ACTIVITIES Patrick J. Walsh, Cindy Bedolla and Thomas P. Mommsen ABSTRACT <ul style="display: flex;"><li style="flex:1">Male toadfishes (genus Opsanus) use a sonic muscle on the swim bladder to produce </li><li style="flex:1">a</li></ul><ul style="display: flex;"><li style="flex:1">mating call-the boatwhistle-for </li><li style="flex:1">extended periods during the mating season. Previously, we </li></ul>noted significant differences in sonic muscle mass and activities of metabolic enzymes of the sonic muscles between male and female gulf toadfish, Opsanus beta, of a limited size range (12-130 grams). Notably, males had larger sonic muscles and elevated aerobic capacity, as indicated by higher mass-specific activities of citrate synthase (CS) and malate dehydrogenase (MDH). The present study examined the patterns of sonic muscle mass and activities of these enzymes (and lactate dehydrogenase) in sonic muscle and skeletal muscle as a function of a larger range of body size (7-400 grams) in an effort to determine the point of growth and development that these mate-female differences occur, and to shed light on the possible functional significances of these differences. Sonic muscle mass differences were apparent in the smallest toadfish, and identical rates of increase in sonic muscle mass in males and females maintained these differences throughout the size range examined. In contrast, mass-specific CS and MDH activities were similar in smaller toadfish and began to diverge when fish were about 2S g, While mass-specific MDH activity increased at different rates in males and females, mass-specific CS activity increased in males and decreased in females. The results are discussed in the context of possible control by steroid sex hormones, size at sexual maturity, and success in mate attraction. The toadfish (genus Opsa nus, family Batrachoididae) swimbladder has a sonic muscle which contracts to produce sound. The sonic muscle is used by both sexes to generate grunts when disturbed, but additionally, males are known to sound a <ul style="display: flex;"><li style="flex:1">mating call-the "boatwhistle" -intermittantly </li><li style="flex:1">for many hours during the mating </li></ul>season (Gray and Winn, 1961; Fine et al., 1977). The sonic muscle is characterized by one of the fastest contraction cycles in the animal kingdom (Skoglund, 1959), due in part to several specializations in innervation (Gainer and Klancher, 1965), ultrastructure (Franzini-Armstrong and Nunzi, 1983), and biochemistry of the contractile apparatus (i.e., parvalbumin isozyme proportions and total quantity differ from white skeletal muscle, Hamoir et a1., 1980). In characterizing the metabolic poise of this unique muscle, we discovered that biochemical specializations are apparent at the level of energy metabolism as well (Walsh et al., 1987). The capacity for anaerobic glycolysis in sonic muscle is similar to white skeletal muscle in Opsanus beta, as judged by high activities of lactate dehydrogenase (LOH) and other enzymes (Walsh et al., 1987). However, sonic muscle possesses a higher potential for aerobic metabolism than toadfish white skeletal muscle as indicated by activities of citrate synthase (CS) and malate dehydrogenase (MOH) (Walsh et al., 1987). Some details of the mechanisms of the sex-related differences in use ofthe sonic muscle in toadfish species are known. Males have larger sonic muscles than females (Fine, 1975; Walsh et al., 1987). There is a polymorphism in the degree of development of the sonic motor nucleus (SMN) in the posterior medulla and anterior spinal cord; some males have much larger SMN's than other males and females (Fine et al., 1984). At the biochemical level, significant differences in enzyme activities per gram sonic muscle exist for CS, LOH, and MDH between male and females ofa limited body mass range (12-130 grams, Walsh et al., 1987). However, 68 WALSH ET AL.: TOADFISH PHYSIOLOGY 69 in spite of these differences, both males and females can be made to produce the boatwhistle by electrically stimulating the SMN in the laboratory (Demski and Gerald, 1974; Fine, 1979). The current working hypothesis (see Pennypacker et aI., 1985) is that the production of the boatwhistle is produced by differential environmental/hormonal stimulation of similar (but not identical) nerve-muscle complexes in males and females. This hypothesis is supported by several lines of evidence: females do not produce the boatwhistle in nature (Gray and Winn, 1961; Fine et aI., 1977), and males produce it only during the summer months with some apparent dependence on water temperature (Fine et aI., 1977; Fine, 1978); testosterone is taken up by specific areas of the toadfish brain believed to be linked to sound production (Fine et aI., 1982). Finally, testosterone and dihydrotestosterone administration to ovariectomized females appears to increase the levels of some sonic muscle enzyme activities (Pennypacker et aI., 1985); however, Walsh et al. (1987) have raised methodological concerns about this study. With this background in mind, we decided to examine the manner in which the activities ofLDH, CS, and MDH scale versus body mass in order to determine if the sex-related differences (Walsh et aI., 1987) are apparent at all sizes/ages, or if some critical point in development/maturation exists at which these differences appear. This information would supply clues to the mechanisms maintaining these sexual differences, and would help in evaluating the hypothesis (Walsh et aI., 1987) that elevated CS and MDH activities lead to an enhanced capacity for sustained, aerobic sound production. Furthermore, the scaling of enzyme activities in a non-locomotory muscle may add interesting counterpoint to the discovery of size-scaling in enzyme activities in locomotory muscles (for review see Somero and Childress, 1985). We report significant differences between male and female toadfish in the manner in which CS and MDH scale with body mass. These differences are partly due to differences in enzyme activity established at an early developmental stage, as well as through differential accumulation of enzyme activity during later growth. MATERIALS AND METHODS Specimens of the gulf toadfish, Opsanus beta. were captured by local shrimp trawlers in south Biscayne Bay, Florida between June 1986 and July 1987, and were held without feeding in running saltwater aquaria at ambient temperature for no longer than 1 week prior to sacrifice. Fish were anesthetized with 0.5 g per liter tricaine methanesulfonate, weighed, the sonic muscle was dissected from the swimbladder and weighed, and the sex was recorded. Sex is relatively easy to determine in this species in the size range used by simple visual inspection. Ovaries are distinguished by a large ovarian artery and large (0.5 to 4 mm) yellow to orange oocytes. Testis are filled with a clear to offwhite material and no cells are visible to the naked eye. Additionally, a sample of white skeletal muscle was taken from the lateral area just behind the anus from a smaller subset of fish. In some cases the intact swimbladder or skeletal muscle sample was frozen at -80·C for I to 2 months before further processing. Control measurements of enzyme activities before and after freezing demonstrated no significant effect of freezing. Muscles were homogenized in 5 volumes of 50 mM NaHEPES, pH 7.5 (adjusted at room temperature) with glass Duall homogenizers on ice. Following sonication with a Heat Systems Ultrasonics Model WI85 for 15 sec at 50 watts, homogenates were centrifuged at 1,600 x g for 5 min. The resulting crude supernatants were used directly (CS) or diluted I: 10 with HEPES buffer (LDH, MDH) immediately prior to the assays. <ul style="display: flex;"><li style="flex:1">Enzymes were assayed spectrophotometrically </li><li style="flex:1">with an LKB Ultrospec 4050 by the methods of </li></ul>Walsh et al. (I 987). Assay temperature was 22·C, final reaction volume was 2.0 ml, and assays were buffered with 50 mM HEPES. Oxidation ofNADH was monitored at 340 nm (micromolar extinction coefficient E = 6.22) for LDH and MDH, and change in absorbance of 5,5' dithiobis-(2-nitrobenzoic acid) (DTNB) was monitored at 412 nm (E = 13.6) for CS. Specific assay conditions were as follows (final concentrations): Citrate synthase (E.C. 4.1.3.7)-0.1 mM DTNB, 0.3 mM Acetyl CoA, 20 Itl supernatant, 0.5 mM oxaloacetate, pH 8.0. Lactate dehydrogenase (E.C. 1.1.1.27) (LDH)-O.15 mM NADH, 20 Itll: 10 diluted supernatant, 0.5 mM pyrvate, pH 7.5. Malate dehydrogenase (E.C. 1.1.1.37) BULLETIN OF MARINE SCIENCE, VOL. 45, NO.1, 1989 70 R"VI "DE'" a: e4'" " •1: •~'" ... 0 ...• •••ua: 3<ul style="display: flex;"><li style="flex:1">"</li><li style="flex:1">D</li></ul>'" •••1: '" •=<ul style="display: flex;"><li style="flex:1">EI </li><li style="flex:1">13 EC </li><li style="flex:1">C</li></ul>"... ...• U1: ~~ 'I.••• "• Z·1 m<ul style="display: flex;"><li style="flex:1">I ! I . </li><li style="flex:1">•</li><li style="flex:1">• • </li></ul>'" =0'" 1: ~•0'" ...• zo'" oo<ul style="display: flex;"><li style="flex:1">100 </li><li style="flex:1">200 </li><li style="flex:1">300 </li><li style="flex:1">400 </li><li style="flex:1">1</li><li style="flex:1">2</li></ul><ul style="display: flex;"><li style="flex:1">BODY MRSS (g rllm s) </li><li style="flex:1">lOG BODY MRSS </li></ul>Figure I. A. Sonic muscle mass vs. body mass for males (open squares) and females (closed diamonds) of the gulf toad fish, Opsanus beta. Equations for linear regressions are y = 0.137 + O.Ollx, r = 0.95 (males) and y = 0.116 + 0.008x, r = 0.90 (females). B. Log sonic muscle mass vs. log body mass for male (y = -1. 732 + 0.912x, r = 0.99) and female (y = -1.882 + 0.9l3x, r = 0.97) toadfish. Symbols as in Figure IA. (MDH}-O.15 mM NADH, 20111 1:10 diluted supernatant, 0.5 mM oxaloacetate, pH 7.5. The last item for each assay was omitted as a control. In all cases this control activity, which was less than 5%, was subtracted from the activity with substrate. Reactions were initiated with 50 to 100 III of the substrate. Biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and all other chemicals were reagent grade. One unit is defined as I I1mol product min-I. Results were curve fitted with Cricket Graph ® (Cricket Software, Inc., Philadelphia, PA), and slopes and y-intercepts of linear regressions were compared by two-tailed Student's t-test (Zar, 1974), and P :5 0.05 is considered to indicate significant differences between groups. RESULTS Sonic Muscle Mass. -Sonic muscle mass increases regularly with body mass in Opsanus beta, but the mass of the sonic muscle is higher in males than in females (Fig. lA). Although the mean body weights (±SE, N) for the two groups are not significantly different (males = 143.2 grams ± 14.2, N = 50; females = 164.6 ± 17.2, N = 47; t = 0.96), the Sonic Muscle Somatic Index (SMSI = percentage of body weight that is sonic muscle) is significantly different in the two groups (male SMSI = 1.230 ± 0.035; female SMSI = 0.891 ± 0.030; t = 7.39, P ::5 0.001). The increase in sonic muscle mass is best described by a double logarithmic relationship in both sexes (Fig. 1B). The slopes of the linear regressions of log sonic muscle mass vs. log body mass are not significantly different (t = 0.25), but the y-intercepts are (t = 13.22, P ::5 0.00 I). These data indicate that the relative difference in sonic muscle mass between male and female toad fish is determined at a point in development earlier than the smallest (=youngest?) fish we sampled (7.3 grams), and that thereafter sonic muscle mass increases similarly in both sexes. Sonic Muscle CS Activity. -Mass-specific aerobic capacity, as indicated by citrate synthase activity per gram of sonic muscle, also scales differently for males and females (Fig. 2A). CS activity per gram of sonic muscle increases in males, whereas it decreases in females (Fig. 2A; slopes significantly different, t = 4.21, P::5 0.001). However, CS activities per gram in smaller fish are similar (y-intercepts are not significantly different, t = 1.30) indicating that the differentiation of CS activities occurs during later stages in development and maturation. When the total CS activity per sonic muscle is calculated highly significant logarithmic relationships WALSH ET AL.: TOADFISH PHYSIOLOGY 71 A B". !:: "D: . ". !:: ;:: : . u";" E;:: 4321o0: 1U0: I I ... D0: .. '" " "D"<ul style="display: flex;"><li style="flex:1">'</li><li style="flex:1">"</li></ul>•... ='" DZ.. 0-': •... Q•". -•D'c ••0'" •... ... •... D.=- .D0: DQ•D0: '" •... D'b •• •-' ~"..,.~ ••U D•• •<ul style="display: flex;"><li style="flex:1">•</li><li style="flex:1">•</li></ul>400 •~o<ul style="display: flex;"><li style="flex:1">100 </li><li style="flex:1">200 </li><li style="flex:1">300 </li><li style="flex:1">0</li><li style="flex:1">1</li><li style="flex:1">2</li></ul>BODY MASS (grams) lOG BODY MASS Figure 2. A. Mass-specific citrate synthase activity (units gram sonic muscle mass-I) vs. body mass (grams) for male (y = 1.159 + 0.006x, r = 0.51) and female (y = 1.477 - O.OOlx, r = 0.26) toadfish. B. Log total sonic muscle citrate synthase activity vs. log body mass for male (y = -2.074 + 1.206x, r = 0.91) and female (y = -1.358 + 0.678x, r = 0.78) toadfish. Total sonic muscle enzyme activity is calculated by multiplying mass-specific activity (Fig. 2A) by sonic muscle mass (Fig. IA) for each individual. Symbols as in Figure IA. with body weight are evident. The regression lines for male and female toadfish cross at a body size of about 25 grams (Fig. 2B). Sonic Muscle MDH Activity. - The mass-specific activity of MDH also increases as a function of body weight, and the differing rates of increase in males and females are best described by logarithmic and linear functions, respectively (Fig. 3A). We again compared total activity in the sonic muscle with body mass for MDH and found that these data were best fit by a log-log relationship (Fig. 3B). The slopes of these lines are not significantly different (t = 1.465, P ::: :0;.10), but the y-intercepts are (t = 2.418, P :S 0.02). Apparently, MDH activity is set at a higher titre in males at an earlier stage of development, but males continue to accrete M D H activity at a slightly higher rate than females. Sonic Muscle LDH Activity. - In contrast to CS and MDH, the mass-specific activity ofLDH decreases as a function of body mass (t = 3.01, P ::::; 0.005, males; t = 2.58, P ::::; 0.01, females) but the rates of decrease are not significantly different for the two sexes (t = 1.08) (Fig. 4A). We previously reported significantly higher mass-specific LDH activity in females vs. males of a limited size-range (female LDH = 481.9 ± 126.4 units g-I(7); female body mass = 72.5 ± 27.4 g; male LDH = 333.4 ± 89.0 (6); male body mass = 96.8 ± 21.3; x ± SD (N), Walsh et al., 1987). However, the increased variability in the present data set obscures these differences (y-intercepts not significantly different, Fig. 4A). When the total anaerobic capacities (as indicated by total LDH activity) of the sonic muscles are compared as a function of body mass, highly significant linear log-log relationships are obtained (Fig. 4B), but neither the slopes nor the y-intercepts of the relationship for males and females are significantly different. Thus, the lower LDH activity per gram in males (Walsh et al., 1987; Fig. 4A) is offset by a larger sonic muscle mass resulting in nearly identical total anaerobic capacities in the sonic muscles of the two sexes (Fig. 4B). Skeletal Muscle Enzyme Activities. -In the limited group of toadfish tested, no apparent differences existed between the sexes in skeletal muscle MDH, LDH, or CS activities (Table I). Correlations with body mass were weak for MDH and BULLETIN OF MARINE SCIENCE, VOL. 45, NO. I, 1989 72 600 500 DR"" !:: ::0 Di= u"" !:: a: ::0 3 D•D"i= '"'- <ul style="display: flex;"><li style="flex:1">.." </li><li style="flex:1">I</li></ul>400 300 ua: <ul style="display: flex;"><li style="flex:1">~</li><li style="flex:1">E</li></ul>.. ~<ul style="display: flex;"><li style="flex:1">'"' </li><li style="flex:1">=</li></ul>:; i: 2 ..... ~ .: a: •... "" .- <ul style="display: flex;"><li style="flex:1">ffi </li><li style="flex:1">;</li></ul>200 e:;1 e - •... '"' a: e100 ..... ..... a: 1: <ul style="display: flex;"><li style="flex:1">o</li><li style="flex:1">o</li></ul><ul style="display: flex;"><li style="flex:1">100 </li><li style="flex:1">200 </li><li style="flex:1">300 </li><li style="flex:1">400 </li><li style="flex:1">0</li><li style="flex:1">1</li><li style="flex:1">2</li></ul>°<ul style="display: flex;"><li style="flex:1">BOOY MflSS (g ro ffis) </li><li style="flex:1">LOG BODY MRSS </li></ul>Figure 3, A, Mass-specific malate dehydrogenase activity vs, body mass for male (y = 43,8 I2XO.369 ,r = 0.54) and female (y = 116.418 + 0,339x, r = 0.50) toadfish. B. Log total sonic muscle malate dehydrogenase activity vs. log body mass for male (y = -0.111 + 1.293x, r = 0.91) and female (y = -0.054 + 1.092x, r = 0.90) toadfish. Symbols and calculations as in Figures 1 and 2. LDH; however, a significant negative slope was noted for skeletal CS activity/ gram versus body mass with no difference between males and females (Table 1). DISCUSSION The results of this study are consistent with the hypothesis advanced by Walsh et al. (1987), that the ability to sustain the mating call in male toadfish (Opsanus beta) may be linked to aerobic metabolic capacity in the sonic muscle. Male toadfish possess significantly larger sonic muscles than females (Fig. lA). This difference already is apparent early in development, and sonic muscle mass is added equally and incrementally with body mass in both sexes; the exponents (b) in the allometric growth equation, Sonic Muscle Mass = a(body mass)b are equivalent for males and females and close to 1.0 (Fig. IB). Aerobic metabolic capacity, as indicated by CS activity, is similar in sonic muscles of smaller toadfish (~50 g), but increases in the two sexes at different rates (Fig. 2A, B) CS activity per gram increases in males and decreases in females (Fig. 2A), and, taking into consideration the increased sonic muscle mass with body mass in both sexes, total tissue CS increases more rapidly in males than in females (Fig. 2B). The exponent in the allometric equation total CS activity = a(body mass)b is greater than 1.0 in males and less than 1.0 in females, and the plots of log total CS activity vs. log body weight for the two sexes cross at a body weight of about 25 grams (Fig.

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(<I>Opsanus Beta</I>) Sonic Muscle Enzyme Activities