Fish Physiology and Biochemistry vol. 12 no. 2 pp 131-141 (1993) Kugler Publications, Amsterdam/New York
Protein metabolism during sexual maturation in female Atlantic salmon (Salmo salar L) N.B. Martin,' D.F. Houlihan,l C. Talbot 2 and R.M. Palmer 3 'Departmentof Zoology, University of Aberdeen, U.K. AB9 2TN; 2FreshwaterFisheriesLaboratory, Pitlochry, PertshireU.K. PH16 5LB; 3 Rowett Research Institute, Bucksburn, Aberdeen, U.K. AB9 2SB
Accepted: April 8, 1993 Keywords: protein synthesis, sexual maturation, Salmo salar
Abstract Body composition and fractional rates of protein synthesis (percentage of the protein mass synthesized per day) were determined in female Atlantic salmon returning to the River Tay, Scotland in July and in October after a 95 day period without food, during which time the animals became sexually mature. During the 95 day period of starvation/sexual maturation the ventricle and red muscle remained as a constant proportion of fresh weight whereas the liver, gill and ovary increased and the stomach and white muscle decreased. Fractional rates of protein synthesis increased markedly in the liver, stomach and ovary during the period of starvation/sexual maturation. In the gill, ventricle and white muscle fractional protein synthesis rates increased slightly or remained constant. From the estimated rates of protein loss or gain in the various tissues it is concluded that there is considerable protein turnover and repartitioning of amino acids during the period of starvation and sexual maturation. The absolute rate of protein synthesis rates in the ovary indicates that this tissue made the largest contribution to the energy and amino acid demands of the fish, whilst most of the amino acids required for maturation of the ovary were derived from white muscle, principally as the result of increased muscle protein degradation.
Introduction Changes in the chemical composition of tissues and in physiology of sexually maturing fish have been well documented, particularly in salmonid species (Greene 1926; Idler and Clemens 1959; Love 1980; Ando et al. 1986; Talbot et al. 1986). Adult Atlantic salmon enter freshwater up to 12 months before they spawn between October and December. Adult salmon do not feed in freshwater and during this period of starvation protein of some organs acts as an important source of energy and tissue building
material, especially during the later stages of the migration, when lipid stores become depleted (Love 1980; Mommsen et al. 1980). Tissue protein breakdown and subsequent amino acid metabolism are therefore important metabolic events which occur during sexual migration of salmon (Mommsen et al. 1980). Certain tissues undergo an increase in their protein degradation rate and it is now accepted that the major source of protein in migrating fish comes from the breakdown of white muscle (Love 1980; Ando et al. 1986. From studies on mammals, it is known that pro-
Correspondenceto: D.F. Houlihan, Department of Zoology, University of Aberdeen, U.K. AB9 2TN.
132 teins are subject to the continuous processes of synthesis and degradation (Schoenheimer 1942). Fish similarly exhibit control over the mass of different tissue by their ability to modify the rate of protein synthesis and protein degradation in the tissues (Fauconneau 1985; Houlihan 1991). However, little is known about the changes in protein metabolism of individual fish tissues during periods of sexual maturation when nutritional input is zero. This paper reports on the modification of protein metabolism in the tissues of sexually maturing Atlantic Salmon (Salmo salar L). Body composition was determined in sexually immature fish and three months later when the fish were sexually mature. Rates of protein synthesis were measured in 7 tissues by the method of Garlick et al. (1980).
Materials and methods Fish A group of 12 sexually immature Atlantic salmon (weight range 1.3-1.6 kg) was used in preliminary studies to examine the equilibrium and incorporation of injected [3 H]-phenylalanine into the intracellular and extracellular free pool and protein of various tissues. These sea water fish were obtained from the Highland Fish Farm Ltd., Loch Carron, Scotland. After transportation of the fish to the Zoology Department, University of Aberdeen, they were maintained for three days in aerated sea water at 9 + 0.5C without food. A second group of Atlantic salmon was captured by Seine net from the river Tay, Scotland early in July. The fish were transported to covered fresh water holding tanks at the Freshwater Fisheries Laboratories, Pitlochry, Scotland, and kept at ambient water temperature (15 + 2°C in July to 8 + 2°C in October). On arrival at the holding tanks and prior to being treated for skin infections all the fish were weighed (initial body weights 1.8-3.0 kg) and individually marked. Five days later 8 males and 10 females were killed for the determination of the initial (July) body compositions. Rates of protein synthesis, RNA and protein contents were measured in various tissues of a further 8 female
fish. The remaining fish were kept without food until late October, when all were killed for body composition analysis. Seven sexually mature female fish were used at this time to measure tissue protein synthesis rates and RNA and protein contents.
Body composition of sexually maturingfish The fish used for the analysis of body composition were killed by a sharp blow to the back of the head followed by severance of the spinal cord. Fish were blot dried and weighed. The liver, gill, heart ventricle, red muscle, white muscle, gonad, stomach and caecum were removed and weighed. Gill filaments were removed from the branchial arches, blotted dry and weighed. Red muscle mass was determined by doubling the value obtaining after carefully dissecting out the red fibres along one side of the body. White muscle was determined similarly. The condition factor was calculated from the equation: condition factor = (gutted body weight (g)/fork length 3 (cm)) x 100 (Aksens et al. 1986), where the fork length is a measurement taken from the snout point to the central fork in the tail.
Measurement of protein synthesis As the fish were larger than any used previously (e.g., Houlihan et al. 1988) minor modifications of the method to measure protein synthesis were adopted. Individual fish were weighed, anaesthetized with benzocaine (0.3 g/l) (Laird and Oswald 1975) and injected into the caudal vein with 150 mM phenylalanine containing 20 /Ci (7.4 x 106 Bq/ml) of L-[2,6,- 3 H] phenylalanine/ml (specific radioactivity = 300 d.p.m./nmole phenylalanine) at a dose of 1 ml/100 g body weight. The fish were returned to aerated water and left for various times (20, 40 and 60 min for the initial time course, 45 to 50 min in the main experiment). In all cases, samples of liver, gill, ventricle, gonad, stomach, red muscle and white muscle were rapidly dissected on ice, immediately frozen in liquid nitrogen and stored at -20 0C.
Tissue protein and RNA concentrations and
133 phenylalanine specific radioactivity of individual fish were determined with a modification of the method previously described by Houlihan et al. (1986) and McMillan and Houlihan (1989). Frozen tissue samples of approximately 1 g were homogenized in 8 ml of 0.5M ice cold perchloric acid (PCA). The homogenate was centrifuged for 20 min and the supernatant (free pool) decanted into stoppered tubes and stored at 40 C. This supernatant was used to measure the specific radioactivity of the homogenate L-phenylalanine (free pool, Sa). The protein pellet was washed in a further 5 ml of 0.5M PCA, centrifuged and the supernatant discarded. The subsequent pellet was re-suspended in 10.8 ml of distilled water and 1.2 ml of 3M NaOH added (final concentration of 0.3M NaOH) and incubated at 37°C for lh. Then, 200 /A1 of the solution was used to determine the protein concentration, 2 ml of 20% PCA was added to the remaining solution to precipitate the protein. After centrifugation the resulting supernatant was decanted and RNA levels measured. Total tissue RNA was determined by the dual wavelength method (Ashford and Pain 1986). The results were validated using the Orcinol method (Mejbaum 1939); since RNA was measured by solubilization in warm NaOH, possible denaturation by RNases was not considered significant. Tissue protein concentrations were determined by the method of Lowry et al. (1951). Methods for the analysis of the specific radioactivity of free and protein-bound phenylalanine have previously been described (Garlick et al. 1980; Houlihan et al. 1986).
for each animal was estimated from its live weight, the tissue composition of the animals dissected in July and their protein concentrations. The fractional rate of protein synthesis was calculated from the specific radioactivities (d.p.m./ nmole) of the free (Sa) and protein-bound phenylalanine (Sb) using the equation of Garlick et al. (1980): k (%/day) = (Sb/Sa) x (1440/t) x 100,
where 1440 is the number of minutes in a day and t is the time after injection. The rate of protein degradation was calculated in tissues of the October salmon (kd, %/day) as the difference between the mean July and October rates of protein synthesis and the growth or weight loss rates (kg) during the July to October period: kd = [(ksJuly) + (ksoctober)/2] - kg. The mean ks value was used because
growth rate is a change over several months while k s is measured over minutes and the mean k s value may therefore be more representative of ks changes over the growth period. Absolute rates of protein synthesis (As: g protein synthesized/tissue/day) were calculated by multiplying the fractional rates of protein synthesis (ks, %/day) by the total protein content (g) of the tissue.
Statistics The data were analyzed using Student's unpaired ttest for equal variance if the F value between the data were not significant. However, if the F value was significantly different in variance between the two sets of data, then a unequal variance t-test was used to determine significant differences.
Calculations Results Rates of protein loss or gain (kg, %/day) were calculated using changes in protein contents between
Time-course of free pool and protein labelling
July to October: kg = (In W 2 - In Wl/t) x 100,
where W 1 = the initial total protein content of the tissues in July and W2 = the final total protein content of the tissues in October and t = the time in days (95 days). Total protein contents of the tissues in October were determined from their measured fresh weight and protein concentration. W1
Houlihan et al. (1986) reported that the flooding dose technique could be successfully applied to measure fractional rates of protein synthesis of tissues, such as ventricle, gill, red and white muscle in small rainbow trout (approximately 80 g body weight). As the present work entailed the use of
134 300 -
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--
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red musde
-
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--
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0 0
40
20
60
j
20
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Time, min
Time, min
Fig. 1. Mean free phenylalanine specific radioactivity (d.p.m/ nmole) ( SEM) of 3 day fasted female Atlantic salmon in sea water. The animals (n=9) were injected at time zero with a flooding dose of phenylalanine containing [3H] phenylalanine with a specific radioactivity of 300 dpm/nmole. Three animals were sampled at each time point.
Fig. 2. Mean free phenylalanine concentration (± SEM) (nmole phenylalanine/g fresh weight of tissue) of tissues of Atlantic salmon at various times after injection of a flooding dose. Other details as in Figure 1. 20-
2.515c
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.
Ce oC,
i---p--,
.9 10-
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5-
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0.0
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20
60
I=---·a
20
40
Time, min
60
Time, min
Fig. 4. Mean fractional rates of protein synthesis (ks, %/day) (+ SEM) of various tissues of Atlantic salmon calculated at var-
Fig. 3. Mean protein-bound phenylalanine specific radioactivity
ious times after an injection of a flooding dose of phenylalanine.
(d.p.m./nmole) (± SEM) of fasted Atlantic salmon in sea water
Each fractional rate was calculated from the protein-bound and
following a flooding dose of phenylalanine. Other details as in Figure 1.
Other details as in Figure 1.
salmon of over 1000 g it was important to determine if flooding of the tissues occurred in order to validate the techniques. The specific radioactivity of the free phenylalanine (Sa) 20, 40 and 60 min after injection is shown in Figure 1. Ideally, the free pool specific radioactivity of the tissues should rapidly reach that of the
plasma and remain constant over the time studied. In all tissues except white muscle, the levels were close to that of the injection solution by 20 min and remained at these levels throughout 60 min. Figure 2 shows the recovery of free L-phenylalanine at 20, 40 and 60 min after injection. The free phenylalanine content of the gill, stomach, red
free phenylalanine specific radioactivity at each time point.
135 Table 1. Mean body weight ( SEM) and percentage weight change of Atlantic salmon after a short period without food soon after capture in July and after 95 days without food in October. Mean condition factors (+ SEM) are also indicated Initial body weight (g)
Males (n=8) Females (n= 18) Males (n = 8) Females (n=7)
July 2816 ±184 2573 150 July 3000 + 153 2314 + 84
Final weight (g)
Days
July 2785 ± 171 2571 ± 146 October 2700 + 194 1965 + 76
5 5
95 95
°/o change
Condition factor
1
1.2 +0.04 1.1 + 0.03
< I1
10
0.90 + 0.02** 0.85 0.03** ±+
15
**p
Protein metabolism during sexual maturation in female Atlantic salmon (Salmo salar L) N.B. Martin,' D.F. Houlihan,l C. Talbot 2 and R.M. Palmer 3 'Departmentof Zoology, University of Aberdeen, U.K. AB9 2TN; 2FreshwaterFisheriesLaboratory, Pitlochry, PertshireU.K. PH16 5LB; 3 Rowett Research Institute, Bucksburn, Aberdeen, U.K. AB9 2SB
Accepted: April 8, 1993 Keywords: protein synthesis, sexual maturation, Salmo salar
Abstract Body composition and fractional rates of protein synthesis (percentage of the protein mass synthesized per day) were determined in female Atlantic salmon returning to the River Tay, Scotland in July and in October after a 95 day period without food, during which time the animals became sexually mature. During the 95 day period of starvation/sexual maturation the ventricle and red muscle remained as a constant proportion of fresh weight whereas the liver, gill and ovary increased and the stomach and white muscle decreased. Fractional rates of protein synthesis increased markedly in the liver, stomach and ovary during the period of starvation/sexual maturation. In the gill, ventricle and white muscle fractional protein synthesis rates increased slightly or remained constant. From the estimated rates of protein loss or gain in the various tissues it is concluded that there is considerable protein turnover and repartitioning of amino acids during the period of starvation and sexual maturation. The absolute rate of protein synthesis rates in the ovary indicates that this tissue made the largest contribution to the energy and amino acid demands of the fish, whilst most of the amino acids required for maturation of the ovary were derived from white muscle, principally as the result of increased muscle protein degradation.
Introduction Changes in the chemical composition of tissues and in physiology of sexually maturing fish have been well documented, particularly in salmonid species (Greene 1926; Idler and Clemens 1959; Love 1980; Ando et al. 1986; Talbot et al. 1986). Adult Atlantic salmon enter freshwater up to 12 months before they spawn between October and December. Adult salmon do not feed in freshwater and during this period of starvation protein of some organs acts as an important source of energy and tissue building
material, especially during the later stages of the migration, when lipid stores become depleted (Love 1980; Mommsen et al. 1980). Tissue protein breakdown and subsequent amino acid metabolism are therefore important metabolic events which occur during sexual migration of salmon (Mommsen et al. 1980). Certain tissues undergo an increase in their protein degradation rate and it is now accepted that the major source of protein in migrating fish comes from the breakdown of white muscle (Love 1980; Ando et al. 1986. From studies on mammals, it is known that pro-
Correspondenceto: D.F. Houlihan, Department of Zoology, University of Aberdeen, U.K. AB9 2TN.
132 teins are subject to the continuous processes of synthesis and degradation (Schoenheimer 1942). Fish similarly exhibit control over the mass of different tissue by their ability to modify the rate of protein synthesis and protein degradation in the tissues (Fauconneau 1985; Houlihan 1991). However, little is known about the changes in protein metabolism of individual fish tissues during periods of sexual maturation when nutritional input is zero. This paper reports on the modification of protein metabolism in the tissues of sexually maturing Atlantic Salmon (Salmo salar L). Body composition was determined in sexually immature fish and three months later when the fish were sexually mature. Rates of protein synthesis were measured in 7 tissues by the method of Garlick et al. (1980).
Materials and methods Fish A group of 12 sexually immature Atlantic salmon (weight range 1.3-1.6 kg) was used in preliminary studies to examine the equilibrium and incorporation of injected [3 H]-phenylalanine into the intracellular and extracellular free pool and protein of various tissues. These sea water fish were obtained from the Highland Fish Farm Ltd., Loch Carron, Scotland. After transportation of the fish to the Zoology Department, University of Aberdeen, they were maintained for three days in aerated sea water at 9 + 0.5C without food. A second group of Atlantic salmon was captured by Seine net from the river Tay, Scotland early in July. The fish were transported to covered fresh water holding tanks at the Freshwater Fisheries Laboratories, Pitlochry, Scotland, and kept at ambient water temperature (15 + 2°C in July to 8 + 2°C in October). On arrival at the holding tanks and prior to being treated for skin infections all the fish were weighed (initial body weights 1.8-3.0 kg) and individually marked. Five days later 8 males and 10 females were killed for the determination of the initial (July) body compositions. Rates of protein synthesis, RNA and protein contents were measured in various tissues of a further 8 female
fish. The remaining fish were kept without food until late October, when all were killed for body composition analysis. Seven sexually mature female fish were used at this time to measure tissue protein synthesis rates and RNA and protein contents.
Body composition of sexually maturingfish The fish used for the analysis of body composition were killed by a sharp blow to the back of the head followed by severance of the spinal cord. Fish were blot dried and weighed. The liver, gill, heart ventricle, red muscle, white muscle, gonad, stomach and caecum were removed and weighed. Gill filaments were removed from the branchial arches, blotted dry and weighed. Red muscle mass was determined by doubling the value obtaining after carefully dissecting out the red fibres along one side of the body. White muscle was determined similarly. The condition factor was calculated from the equation: condition factor = (gutted body weight (g)/fork length 3 (cm)) x 100 (Aksens et al. 1986), where the fork length is a measurement taken from the snout point to the central fork in the tail.
Measurement of protein synthesis As the fish were larger than any used previously (e.g., Houlihan et al. 1988) minor modifications of the method to measure protein synthesis were adopted. Individual fish were weighed, anaesthetized with benzocaine (0.3 g/l) (Laird and Oswald 1975) and injected into the caudal vein with 150 mM phenylalanine containing 20 /Ci (7.4 x 106 Bq/ml) of L-[2,6,- 3 H] phenylalanine/ml (specific radioactivity = 300 d.p.m./nmole phenylalanine) at a dose of 1 ml/100 g body weight. The fish were returned to aerated water and left for various times (20, 40 and 60 min for the initial time course, 45 to 50 min in the main experiment). In all cases, samples of liver, gill, ventricle, gonad, stomach, red muscle and white muscle were rapidly dissected on ice, immediately frozen in liquid nitrogen and stored at -20 0C.
Tissue protein and RNA concentrations and
133 phenylalanine specific radioactivity of individual fish were determined with a modification of the method previously described by Houlihan et al. (1986) and McMillan and Houlihan (1989). Frozen tissue samples of approximately 1 g were homogenized in 8 ml of 0.5M ice cold perchloric acid (PCA). The homogenate was centrifuged for 20 min and the supernatant (free pool) decanted into stoppered tubes and stored at 40 C. This supernatant was used to measure the specific radioactivity of the homogenate L-phenylalanine (free pool, Sa). The protein pellet was washed in a further 5 ml of 0.5M PCA, centrifuged and the supernatant discarded. The subsequent pellet was re-suspended in 10.8 ml of distilled water and 1.2 ml of 3M NaOH added (final concentration of 0.3M NaOH) and incubated at 37°C for lh. Then, 200 /A1 of the solution was used to determine the protein concentration, 2 ml of 20% PCA was added to the remaining solution to precipitate the protein. After centrifugation the resulting supernatant was decanted and RNA levels measured. Total tissue RNA was determined by the dual wavelength method (Ashford and Pain 1986). The results were validated using the Orcinol method (Mejbaum 1939); since RNA was measured by solubilization in warm NaOH, possible denaturation by RNases was not considered significant. Tissue protein concentrations were determined by the method of Lowry et al. (1951). Methods for the analysis of the specific radioactivity of free and protein-bound phenylalanine have previously been described (Garlick et al. 1980; Houlihan et al. 1986).
for each animal was estimated from its live weight, the tissue composition of the animals dissected in July and their protein concentrations. The fractional rate of protein synthesis was calculated from the specific radioactivities (d.p.m./ nmole) of the free (Sa) and protein-bound phenylalanine (Sb) using the equation of Garlick et al. (1980): k (%/day) = (Sb/Sa) x (1440/t) x 100,
where 1440 is the number of minutes in a day and t is the time after injection. The rate of protein degradation was calculated in tissues of the October salmon (kd, %/day) as the difference between the mean July and October rates of protein synthesis and the growth or weight loss rates (kg) during the July to October period: kd = [(ksJuly) + (ksoctober)/2] - kg. The mean ks value was used because
growth rate is a change over several months while k s is measured over minutes and the mean k s value may therefore be more representative of ks changes over the growth period. Absolute rates of protein synthesis (As: g protein synthesized/tissue/day) were calculated by multiplying the fractional rates of protein synthesis (ks, %/day) by the total protein content (g) of the tissue.
Statistics The data were analyzed using Student's unpaired ttest for equal variance if the F value between the data were not significant. However, if the F value was significantly different in variance between the two sets of data, then a unequal variance t-test was used to determine significant differences.
Calculations Results Rates of protein loss or gain (kg, %/day) were calculated using changes in protein contents between
Time-course of free pool and protein labelling
July to October: kg = (In W 2 - In Wl/t) x 100,
where W 1 = the initial total protein content of the tissues in July and W2 = the final total protein content of the tissues in October and t = the time in days (95 days). Total protein contents of the tissues in October were determined from their measured fresh weight and protein concentration. W1
Houlihan et al. (1986) reported that the flooding dose technique could be successfully applied to measure fractional rates of protein synthesis of tissues, such as ventricle, gill, red and white muscle in small rainbow trout (approximately 80 g body weight). As the present work entailed the use of
134 300 -
2500'
C ._
o
_e 0)
250 -
>, E
2000-
E
C
1500 .C co
._ `3 .0 2
200 -
-a
.o .o
150 -
-0-
gill
--
vende v-
-D-
stmach
0 CL U_
LL.
1000-
500-
red musde
-
u.
--
whitemuscl
inn
0 0
40
20
60
j
20
0
40
60
Time, min
Time, min
Fig. 1. Mean free phenylalanine specific radioactivity (d.p.m/ nmole) ( SEM) of 3 day fasted female Atlantic salmon in sea water. The animals (n=9) were injected at time zero with a flooding dose of phenylalanine containing [3H] phenylalanine with a specific radioactivity of 300 dpm/nmole. Three animals were sampled at each time point.
Fig. 2. Mean free phenylalanine concentration (± SEM) (nmole phenylalanine/g fresh weight of tissue) of tissues of Atlantic salmon at various times after injection of a flooding dose. Other details as in Figure 1. 20-
2.515c
2.0-
.
Ce oC,
i---p--,
.9 10-
a
1.5-
a 0
u_
1.0-
5-
._ 9 1
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o0 0
0.0
40
20
60
I=---·a
20
40
Time, min
60
Time, min
Fig. 4. Mean fractional rates of protein synthesis (ks, %/day) (+ SEM) of various tissues of Atlantic salmon calculated at var-
Fig. 3. Mean protein-bound phenylalanine specific radioactivity
ious times after an injection of a flooding dose of phenylalanine.
(d.p.m./nmole) (± SEM) of fasted Atlantic salmon in sea water
Each fractional rate was calculated from the protein-bound and
following a flooding dose of phenylalanine. Other details as in Figure 1.
Other details as in Figure 1.
salmon of over 1000 g it was important to determine if flooding of the tissues occurred in order to validate the techniques. The specific radioactivity of the free phenylalanine (Sa) 20, 40 and 60 min after injection is shown in Figure 1. Ideally, the free pool specific radioactivity of the tissues should rapidly reach that of the
plasma and remain constant over the time studied. In all tissues except white muscle, the levels were close to that of the injection solution by 20 min and remained at these levels throughout 60 min. Figure 2 shows the recovery of free L-phenylalanine at 20, 40 and 60 min after injection. The free phenylalanine content of the gill, stomach, red
free phenylalanine specific radioactivity at each time point.
135 Table 1. Mean body weight ( SEM) and percentage weight change of Atlantic salmon after a short period without food soon after capture in July and after 95 days without food in October. Mean condition factors (+ SEM) are also indicated Initial body weight (g)
Males (n=8) Females (n= 18) Males (n = 8) Females (n=7)
July 2816 ±184 2573 150 July 3000 + 153 2314 + 84
Final weight (g)
Days
July 2785 ± 171 2571 ± 146 October 2700 + 194 1965 + 76
5 5
95 95
°/o change
Condition factor
1
1.2 +0.04 1.1 + 0.03
< I1
10
0.90 + 0.02** 0.85 0.03** ±+
15
**p
