Journal of Chemical Ecology, Vol. 22, No. 1. 1996
ANT REPELLENT EFFECT OF THE STERNAL GLAND SECRETION OF Polistes dominulus (Christ) AND P. sulcifer (Zimmermann). (Hymenoptera: Vespidae)
F R A N C E S C A R. D A N I , I ' * S A R A C A N N O N I , I S T E F A N O T U R I L L A Z Z I , I and E. D A V I D M O R G A N 2 ~Dipartimento di Biotogia Animale e Genetica Universit& di Firenze Via Romana 17. Firenze, 50125 Italy 'Department of Chemistry Keele Universio,. Staffordshire England. ST5 5BG. UK
(Received February 22, 1995; accepted August 23, 1995) Abstract--The long-chain carboxylic acids identified in the sternal gland secretion of Polistes dominulus and P. sulcifer females were tested individually on three species of ants. Crematogaster scutetlaris, Formica cunicularia, and Lasius sp., in order to verily if they have a repellent effect. The unsaturated acids (palmitoleic, linoleic, and oleic) act as repellents of all three ant species, while the saturated acids (lauric, myristic, palmitic, and stearic) have no effect. The mixture reproducing the secretion of the sternal glands of P. dominulus maintained its repellency for at least four days. Key Words--Vespidae, Polistinae, Polistes, sternal glands, nest defense, ant repellents, allomones, unsaturated carboxylic acids.
INTRODUCTION Several studies have s h o w n that ants, especially in tropical and subtropical areas, exert high predatory pressure on the nests o f social wasps, preying on their immature brood (Jeanne, 1975, 1979; Yamane, 1996). As active defense is less effective when only a few wasps constitute the colony, chemical defense against *To whom correspondence should be addressed.
37 IX)gB-O3M/9610100-O037509,50/O (~ 1996 Plenum Publishing Corporation
38
DANI, CANNONI, TURILLAZZt, AND MORGAN
ants is used by many species. In at least some species of Stenogastrinae, the Dufour's gland secretion, consisting of a gelatinous substance, is used as a storage substrate for the larval food as well as a barrier against ants (Turillazzi, 1991, 1994). In females of the independently nest-founding Polistinae, the gland cells of the last gastral sternites secrete ant repellent substances (see Downing, 1991; Jeanne, 1996). By rubbing the abdomen on it, females apply these substances to the pedicel, which suspends the nest from the substrate and constitutes the only access to the nest for pedestrian predators. Recently, the production of ant-repellent secretions by gastral sternal glands has also been reported for two species belonging to the subfamily Vespinae (Martin, 1992). Bioassays on several species of ants have been carried out using nest pedicels (Jeanne, 1970; Hermann and Dirks, 1974; Post and Jeanne, 1981; Turillazzi and Ugolini, 1979; Martin, 1992), glass capillaries or small wooden sticks stroked along the last abdominal sternites (Jeanne, 1970; Turillazzi and Ugolini, 1979; Post and Jeanne, 1981; Kojima, 1983; Martin, 1992), and solventextracted sternal glands (Keeping, 1990; Kojima, 1992). All the bioassays have shown that the sternal gland secretion and the substances coating the pedicel have a repellent effect on ants. Turillazzi and Ugolini (1979) and Kojima (1983) report that in Polistes and Ropalidia, respectively, the secretion loses activity a short time after the application, while Martin (1992) found that the repellent action lasts for several hours in Vespa. From the sixth sternite glands of Polistes fuscatus, Post et al. (1984) isolated two components active against ants. One of these was identified as methyl bexadecanoate (methyl palmitate). Bioassays using methyl palmitate and some related methyl esters (methyl myristate, methyl linoleate, methyl linolenate, and methyl stearate) on several ants species (Post et al., 1984; Henderson and Jeanne, 1989) and on flies (Henderson et al., 1991) showed that only methyl myristate and methyl palmitate had a repellent effect, the former being more active than the latter. Also in these trials, the repellent effect lasted only for some hours. More recently, hexadecanoic (palmitic) and octadecenoic (oteic) acid have been identified in the sixth sternite glands of P. annularis workers (Espelie and Hermann, 1990). The same compounds, together with other carboxylic acids (dodecanoic or lauric, tetradecanoic or myristic, hexadecenoic or palmitoleic, octadecadienoic or linoleic, and octadecanoic or stearic acid), have been found by Dani et al. (1995) in the fifth and sixth sternite glands of P. dominulus. P. sulcifer, a social parasite of P. dominulus (see Turillazzi, 1992; Cervo and Dani, 1996) has been shown to have most of the same fatty acids (palmitoleic, palmitic, oleic, and stearic) in its sternal glands also (Dani et al., 1995). In the present research, bioassays have been performed in order to test if the acids found in the P. dorninulus and P. sulcifer gastral stemite glands have an effect in repelling ants.
ANT REPELLENTS BY
Polistes WASPS
39
METHODS AND MATERIALS
Ant Species The experiments were conducted in the laboratory at the Dipartimento di Biologia Animale e Genetica of Florence University. Most of the bioassays were carried out on Crematogaster scutellaris, but tests were also done on Formica cunicularia and a Lasius species. Turillazzi and Ugolini (1979) observed Lasius sp. and Crematogaster sp. invading nests of European Polistes, while Dani and Cervo (personal observation) have found nests invaded by Formica sp. Crematogaster scutellaris foragers were captured with an aspirator in the garden of the department and quickly used for the experiments. Formica cunicularia and Lasius sp. workers were collected in the Appennines near Pesaro (together with some immature brood coming from their nests) and used for the experiments the day after collection.
Experimental Apparatus In all the experiments we used an experimental apparatus of the type employed by Turillazzi and Ugolini (1979). In each trial, about 40 ants were placed on an inverted Petri dish (9.5 cm in diameter), placed in the center of a bigger Petri dish (15 cm in diameter) containing water. A bridge was constructed with glass rods to permit the ants to escape from their water-surrounded dish. It consisted of a large glass rod (30 mm long and 2 mm in diameter), connected via a Y junction to two narrow glass rods (80 mm long and 1 mm in diameter). The end of the larger rod was put on the smaller Petri dish while the other two rods were placed on the border of the bigger Petri dish. Ants arriving at the Y junction had to make a decision to continue on one or the other branch to escape confinement. In each trial, before placing the bridge, one of the two smaller rods (experimental branch), near its junction with the main stem, was treated with a hexane solution containing synthetic carboxylic acids (the solvent was allowed to evaporate) while the other tube (control branch) was treated only with the same quantity of hexane. If the solution had a repellent effect, at the branching point, the ants tended to follow the control branch. Each trial lasted 20 min, unless all the ants crossed the branches in a shorter time. During the trial, the position of the experimental and control rods was alternated about every 5 min. If ants were walking on the rods, we waited until they had reached the end of the rods before alternating them. Four trials were made for each kind of experiment. The results (i.e., the number of ants crossing the experimental and the control rods) of the four trials of each experiment were tested with the X 2 homogeneity test. When the probability given by the test was not significant,
40
DANI, CANNONI, TURILLAZZI, AND MORGAN
the data were cumulated and compared, using the G test, with a two-class uniform distribution (50%-50%), to find if a significantly different number of ants chose one of the two routes. When the probability given by the homogeneity test was significant, the G test was done for each single trial. For graphic representation, we report the index of repellency of each acid as the percentage of ants that crossed the control route with respect to the total number of ants crossing both routes.
Experiments Control Experiments. Prior to the experiments, nine control trials in which neither branch was treated were carded out on Crematogaster scutellaris to verify that the ants walked indiscriminantly on the two branches of the experimental device. Effect of Each Acid. Oleic acid is the main component found in the sternal gland secretion of P. dominulus; the sixth stemite glands of one worker contained about 2.5 /,g of this compound (Dani et al., 1995). In experiments on Crematogaster scutellaris, we tested separately lauric (dodecanoic), myristic (tetradecanoic), palmitoleic [(Z)-9-hexadecenoic], palmitic (hexadecanoic), linoleic [(Z,Z)-9,12-octadecadienoic], oteic [(Z)-9-octadecenoic], and stearic (octadecanoic) acid, by applying to the experimental branch 2.5, 25, or 250 tzg of one of these acids diluted in 2/,1 of hexane (from solutions previously prepared), except that stearic and palmitic acid at 2 5 0 / , g were each dissolved in 4/,1 of hexane because of the limited solubility of these acids in hexane. Only the 250-p.g quantity was tested on Formica cunicularia and on Lasius sp. Duration of Repellent Effect. The sixth sternal glands of one P. dominulus worker contained, on average, the acids in the following percentages (Dani et al., 1995): lauric acid, 2.9% (195 rig); myristic acid, 5.9% (393 rig); palmitoleic acid, 4.1% (271 ng); palmitic acid, 21.4% (1410 ng) linoleic acid, 21.3% (1405 ng); oleic acid, 38.3% (2523 ng); and stearic acid, 5.9% (390 ng). A solution of pure acids, mixed in these proportions was tested on Crematogaster scutellaris by applying 2/,1 of hexane containing the equivalent of the sixth sternite glands of 10 workers. The effect of this solution was tested at 0, 24, 48, and 96 hr after application to the rods. RESULTS
Control Experiments. The control experiments showed that the device was balanced. In fact, although it was not possible to cumulate the data of the nine trials (homogeneity test; X z = 6.58, P < 0.05), in none of them did we find that significantly more ants preferred one of the two branches.
ANT REPELLENTS BY
Pol&teswAsPS
41
Effect of Each Acid. We report the average percentage (of the four trials) of Crematogaster scutellaris foragers that chose the control branch at the Y junction, using 2.5, 25, or 250/~g of each acid in Figure 1. At 2.5 ~g, palmitoleic, linoleic, and oleic acids had a highly repellent effect. It was not possible to cumulate the results of the four trials for patmitoleic acid (homogeneity test; X2 = 8.76, P < 0.01), but in all of them significantly more than 50% of the ants crossed the control branch (Trial 1, G = 21.26, P < 0.001; Trial 2, G = 33.00, P < 0.001; Trial 3, G = 39.57, P < 0.001; Trial 4, G = 6.70, P < 0.01). The data of the other two unsaturated acids were cumulated (X 2 = 3.12, NS for linoleic acid; X2 = 1.65, NS for oleic acid); in both the experiments significantly more ants followed the control branch (G = 20.90, P < 0.001 for linoleic acid; G = 18.60, P < 0.001 for oteic acid). No repellent effect was found for lauric, myristic, palmitic, and stearic acids. At the 25-/zg quantity, no ants at all crossed the rods to which linoleic acid (X 2 = 0.01, NS; G = 250.93, P < 0.001) or oleic acid (x 2 = 0.21, NS; G = 141.42, P < 0.001) had been applied, and only very few crossed the rods treated with palmitoleic acid (X 2 = 0.31, NS; G = 215.I9, P < 0.001). No repellent effect was shown by lauric, myristic, palmitic, and stearic acids. Because at 25/zg linoleic and oleic acids repelled all the ants (100%), we did not test these substances in the 250-/~g experiments. In the trials with 250 /zg, only very few ants chose the path with palmitoleic acid (X 2 = 0.09, NS; G = 200.30, P < 0.001), and no repellent effect was found for lauric, myristic, palmitic, and stearic acids. Similar results were obtained in the experiments with Formica cunicularia (Figure 2) and with Lasius sp. (Figure 3) using 250 /zg of acid. No Formica cunicularia ants used the branch treated with palmitoleic acid (X 2 = 0.042, NS; G = 94.17, P < 0.001). Only very few used that treated with oleic (X 2 = 0.25, NS; G = 148.47, P < 0.001) or linoleic acids (X 2 = 0.12, NS; G = 158.20, P < 0.001), and no repellent effect was shown by lauric, myristic, palmitic, and stearic acids. Only a few Lasius sp. crossed the path treated with palmitoleic, linoleic, and oleic acids (respectively: X2 = 0.12, NS; G = 136.55, P < 0.001; X2 = 0.08, NS; G = 148.02, P < 0.001; X2 = 0.72, NS; G = 121.37, P < 0.001), while lauric, myristic, palmitic, and stearic acids had no repellent effect. In the case of Crematogaster scutellaris, we compared the results obtained in the experiments with the three different amounts of each acid. No differences in the repellent effect of the saturated acids were found between the 2.5- and 25-t~g and between the 25-/~g and 250-#g experiments. For linoleic and oleic acids, it was found that 25 p.g of each substance had a greater repellent effect than 2.5/zg (respectively: G = 87.92, P < 0.001; G = 65.11, P < 0.001).
42
DAN], CANNONI, TURILLAZZI, AND MORGAN %
A
100 80 60 40
20
P
ANT REPELLENT EFFECT OF THE STERNAL GLAND SECRETION OF Polistes dominulus (Christ) AND P. sulcifer (Zimmermann). (Hymenoptera: Vespidae)
F R A N C E S C A R. D A N I , I ' * S A R A C A N N O N I , I S T E F A N O T U R I L L A Z Z I , I and E. D A V I D M O R G A N 2 ~Dipartimento di Biotogia Animale e Genetica Universit& di Firenze Via Romana 17. Firenze, 50125 Italy 'Department of Chemistry Keele Universio,. Staffordshire England. ST5 5BG. UK
(Received February 22, 1995; accepted August 23, 1995) Abstract--The long-chain carboxylic acids identified in the sternal gland secretion of Polistes dominulus and P. sulcifer females were tested individually on three species of ants. Crematogaster scutetlaris, Formica cunicularia, and Lasius sp., in order to verily if they have a repellent effect. The unsaturated acids (palmitoleic, linoleic, and oleic) act as repellents of all three ant species, while the saturated acids (lauric, myristic, palmitic, and stearic) have no effect. The mixture reproducing the secretion of the sternal glands of P. dominulus maintained its repellency for at least four days. Key Words--Vespidae, Polistinae, Polistes, sternal glands, nest defense, ant repellents, allomones, unsaturated carboxylic acids.
INTRODUCTION Several studies have s h o w n that ants, especially in tropical and subtropical areas, exert high predatory pressure on the nests o f social wasps, preying on their immature brood (Jeanne, 1975, 1979; Yamane, 1996). As active defense is less effective when only a few wasps constitute the colony, chemical defense against *To whom correspondence should be addressed.
37 IX)gB-O3M/9610100-O037509,50/O (~ 1996 Plenum Publishing Corporation
38
DANI, CANNONI, TURILLAZZt, AND MORGAN
ants is used by many species. In at least some species of Stenogastrinae, the Dufour's gland secretion, consisting of a gelatinous substance, is used as a storage substrate for the larval food as well as a barrier against ants (Turillazzi, 1991, 1994). In females of the independently nest-founding Polistinae, the gland cells of the last gastral sternites secrete ant repellent substances (see Downing, 1991; Jeanne, 1996). By rubbing the abdomen on it, females apply these substances to the pedicel, which suspends the nest from the substrate and constitutes the only access to the nest for pedestrian predators. Recently, the production of ant-repellent secretions by gastral sternal glands has also been reported for two species belonging to the subfamily Vespinae (Martin, 1992). Bioassays on several species of ants have been carried out using nest pedicels (Jeanne, 1970; Hermann and Dirks, 1974; Post and Jeanne, 1981; Turillazzi and Ugolini, 1979; Martin, 1992), glass capillaries or small wooden sticks stroked along the last abdominal sternites (Jeanne, 1970; Turillazzi and Ugolini, 1979; Post and Jeanne, 1981; Kojima, 1983; Martin, 1992), and solventextracted sternal glands (Keeping, 1990; Kojima, 1992). All the bioassays have shown that the sternal gland secretion and the substances coating the pedicel have a repellent effect on ants. Turillazzi and Ugolini (1979) and Kojima (1983) report that in Polistes and Ropalidia, respectively, the secretion loses activity a short time after the application, while Martin (1992) found that the repellent action lasts for several hours in Vespa. From the sixth sternite glands of Polistes fuscatus, Post et al. (1984) isolated two components active against ants. One of these was identified as methyl bexadecanoate (methyl palmitate). Bioassays using methyl palmitate and some related methyl esters (methyl myristate, methyl linoleate, methyl linolenate, and methyl stearate) on several ants species (Post et al., 1984; Henderson and Jeanne, 1989) and on flies (Henderson et al., 1991) showed that only methyl myristate and methyl palmitate had a repellent effect, the former being more active than the latter. Also in these trials, the repellent effect lasted only for some hours. More recently, hexadecanoic (palmitic) and octadecenoic (oteic) acid have been identified in the sixth sternite glands of P. annularis workers (Espelie and Hermann, 1990). The same compounds, together with other carboxylic acids (dodecanoic or lauric, tetradecanoic or myristic, hexadecenoic or palmitoleic, octadecadienoic or linoleic, and octadecanoic or stearic acid), have been found by Dani et al. (1995) in the fifth and sixth sternite glands of P. dominulus. P. sulcifer, a social parasite of P. dominulus (see Turillazzi, 1992; Cervo and Dani, 1996) has been shown to have most of the same fatty acids (palmitoleic, palmitic, oleic, and stearic) in its sternal glands also (Dani et al., 1995). In the present research, bioassays have been performed in order to test if the acids found in the P. dorninulus and P. sulcifer gastral stemite glands have an effect in repelling ants.
ANT REPELLENTS BY
Polistes WASPS
39
METHODS AND MATERIALS
Ant Species The experiments were conducted in the laboratory at the Dipartimento di Biologia Animale e Genetica of Florence University. Most of the bioassays were carried out on Crematogaster scutellaris, but tests were also done on Formica cunicularia and a Lasius species. Turillazzi and Ugolini (1979) observed Lasius sp. and Crematogaster sp. invading nests of European Polistes, while Dani and Cervo (personal observation) have found nests invaded by Formica sp. Crematogaster scutellaris foragers were captured with an aspirator in the garden of the department and quickly used for the experiments. Formica cunicularia and Lasius sp. workers were collected in the Appennines near Pesaro (together with some immature brood coming from their nests) and used for the experiments the day after collection.
Experimental Apparatus In all the experiments we used an experimental apparatus of the type employed by Turillazzi and Ugolini (1979). In each trial, about 40 ants were placed on an inverted Petri dish (9.5 cm in diameter), placed in the center of a bigger Petri dish (15 cm in diameter) containing water. A bridge was constructed with glass rods to permit the ants to escape from their water-surrounded dish. It consisted of a large glass rod (30 mm long and 2 mm in diameter), connected via a Y junction to two narrow glass rods (80 mm long and 1 mm in diameter). The end of the larger rod was put on the smaller Petri dish while the other two rods were placed on the border of the bigger Petri dish. Ants arriving at the Y junction had to make a decision to continue on one or the other branch to escape confinement. In each trial, before placing the bridge, one of the two smaller rods (experimental branch), near its junction with the main stem, was treated with a hexane solution containing synthetic carboxylic acids (the solvent was allowed to evaporate) while the other tube (control branch) was treated only with the same quantity of hexane. If the solution had a repellent effect, at the branching point, the ants tended to follow the control branch. Each trial lasted 20 min, unless all the ants crossed the branches in a shorter time. During the trial, the position of the experimental and control rods was alternated about every 5 min. If ants were walking on the rods, we waited until they had reached the end of the rods before alternating them. Four trials were made for each kind of experiment. The results (i.e., the number of ants crossing the experimental and the control rods) of the four trials of each experiment were tested with the X 2 homogeneity test. When the probability given by the test was not significant,
40
DANI, CANNONI, TURILLAZZI, AND MORGAN
the data were cumulated and compared, using the G test, with a two-class uniform distribution (50%-50%), to find if a significantly different number of ants chose one of the two routes. When the probability given by the homogeneity test was significant, the G test was done for each single trial. For graphic representation, we report the index of repellency of each acid as the percentage of ants that crossed the control route with respect to the total number of ants crossing both routes.
Experiments Control Experiments. Prior to the experiments, nine control trials in which neither branch was treated were carded out on Crematogaster scutellaris to verify that the ants walked indiscriminantly on the two branches of the experimental device. Effect of Each Acid. Oleic acid is the main component found in the sternal gland secretion of P. dominulus; the sixth stemite glands of one worker contained about 2.5 /,g of this compound (Dani et al., 1995). In experiments on Crematogaster scutellaris, we tested separately lauric (dodecanoic), myristic (tetradecanoic), palmitoleic [(Z)-9-hexadecenoic], palmitic (hexadecanoic), linoleic [(Z,Z)-9,12-octadecadienoic], oteic [(Z)-9-octadecenoic], and stearic (octadecanoic) acid, by applying to the experimental branch 2.5, 25, or 250 tzg of one of these acids diluted in 2/,1 of hexane (from solutions previously prepared), except that stearic and palmitic acid at 2 5 0 / , g were each dissolved in 4/,1 of hexane because of the limited solubility of these acids in hexane. Only the 250-p.g quantity was tested on Formica cunicularia and on Lasius sp. Duration of Repellent Effect. The sixth sternal glands of one P. dominulus worker contained, on average, the acids in the following percentages (Dani et al., 1995): lauric acid, 2.9% (195 rig); myristic acid, 5.9% (393 rig); palmitoleic acid, 4.1% (271 ng); palmitic acid, 21.4% (1410 ng) linoleic acid, 21.3% (1405 ng); oleic acid, 38.3% (2523 ng); and stearic acid, 5.9% (390 ng). A solution of pure acids, mixed in these proportions was tested on Crematogaster scutellaris by applying 2/,1 of hexane containing the equivalent of the sixth sternite glands of 10 workers. The effect of this solution was tested at 0, 24, 48, and 96 hr after application to the rods. RESULTS
Control Experiments. The control experiments showed that the device was balanced. In fact, although it was not possible to cumulate the data of the nine trials (homogeneity test; X z = 6.58, P < 0.05), in none of them did we find that significantly more ants preferred one of the two branches.
ANT REPELLENTS BY
Pol&teswAsPS
41
Effect of Each Acid. We report the average percentage (of the four trials) of Crematogaster scutellaris foragers that chose the control branch at the Y junction, using 2.5, 25, or 250/~g of each acid in Figure 1. At 2.5 ~g, palmitoleic, linoleic, and oleic acids had a highly repellent effect. It was not possible to cumulate the results of the four trials for patmitoleic acid (homogeneity test; X2 = 8.76, P < 0.01), but in all of them significantly more than 50% of the ants crossed the control branch (Trial 1, G = 21.26, P < 0.001; Trial 2, G = 33.00, P < 0.001; Trial 3, G = 39.57, P < 0.001; Trial 4, G = 6.70, P < 0.01). The data of the other two unsaturated acids were cumulated (X 2 = 3.12, NS for linoleic acid; X2 = 1.65, NS for oleic acid); in both the experiments significantly more ants followed the control branch (G = 20.90, P < 0.001 for linoleic acid; G = 18.60, P < 0.001 for oteic acid). No repellent effect was found for lauric, myristic, palmitic, and stearic acids. At the 25-/zg quantity, no ants at all crossed the rods to which linoleic acid (X 2 = 0.01, NS; G = 250.93, P < 0.001) or oleic acid (x 2 = 0.21, NS; G = 141.42, P < 0.001) had been applied, and only very few crossed the rods treated with palmitoleic acid (X 2 = 0.31, NS; G = 215.I9, P < 0.001). No repellent effect was shown by lauric, myristic, palmitic, and stearic acids. Because at 25/zg linoleic and oleic acids repelled all the ants (100%), we did not test these substances in the 250-/~g experiments. In the trials with 250 /zg, only very few ants chose the path with palmitoleic acid (X 2 = 0.09, NS; G = 200.30, P < 0.001), and no repellent effect was found for lauric, myristic, palmitic, and stearic acids. Similar results were obtained in the experiments with Formica cunicularia (Figure 2) and with Lasius sp. (Figure 3) using 250 /zg of acid. No Formica cunicularia ants used the branch treated with palmitoleic acid (X 2 = 0.042, NS; G = 94.17, P < 0.001). Only very few used that treated with oleic (X 2 = 0.25, NS; G = 148.47, P < 0.001) or linoleic acids (X 2 = 0.12, NS; G = 158.20, P < 0.001), and no repellent effect was shown by lauric, myristic, palmitic, and stearic acids. Only a few Lasius sp. crossed the path treated with palmitoleic, linoleic, and oleic acids (respectively: X2 = 0.12, NS; G = 136.55, P < 0.001; X2 = 0.08, NS; G = 148.02, P < 0.001; X2 = 0.72, NS; G = 121.37, P < 0.001), while lauric, myristic, palmitic, and stearic acids had no repellent effect. In the case of Crematogaster scutellaris, we compared the results obtained in the experiments with the three different amounts of each acid. No differences in the repellent effect of the saturated acids were found between the 2.5- and 25-t~g and between the 25-/~g and 250-#g experiments. For linoleic and oleic acids, it was found that 25 p.g of each substance had a greater repellent effect than 2.5/zg (respectively: G = 87.92, P < 0.001; G = 65.11, P < 0.001).
42
DAN], CANNONI, TURILLAZZI, AND MORGAN %
A
100 80 60 40
20
P
