NATURAL TOXINS I:96-99 (1992)
Inhibition of Insect Acetylcholinesterase by the Potato Glycoalkaloid a-Chaconine Joel M. Wierenga and Robert M. Hollingworth Pesticide Research Center and Department of Entomology, Michigan State University, East Lansing, Michigan
ABSTRACT Homogenates from several insect species were assayed for inhibition of acetylcholinesterase by the potato gtycoalkaloid a-chaconine. Colorado potato beetle acetylcholinesterase was up to 150-fold less sensitive than other species tested. Acetylcholinesterasefrom an insecticide-resistantstrain of Colorado potato beetles was more sensitive to inhibition than the susceptible strain. Most insect species tested had inhibitory concentrations causing a 50% reduction in activity in the 5 to 40 pM range. Sensitive insect acetylcholinesterases were similar to mammalian cholinesterases in their response to u-chaconine. The results indicate that pesticides and host plant resistance factors may interact at the same target. Changes in the target due to selection pressure from either pesticides or host plant resistance factors could affect the efficacy of both 1999 WiIey-Liss, Inc. control strategies. @
Key Words: Leptinotana decemlineata, Musca domestica, Aedes aegypti, Blattella germanica, Chrysomela scr1pta
INTRODUCTION
The major glycoalkaloid components of potatoes are a-solanine and a-chaconine, alkaloids differing only in their sugar moieties (Fig. 1). These molecules have been implicated in instances of food poisoning [e.g., MacMilIan and Thompson, 19791, and several toxic effects in mammals have been identified. Among the effects described are membrane disruption [Roddick et al., 19881, teratogenicity, and acetylcholinesterase (AChE) inhibition [see review by Jadhav et al., 19811. Entomologists and potato breeders have investigated the role of glycoalkaloids in host plant resistance to insect pests, especially the Colorado potato beetle (Leptinotarsa decemlineata (Say)). These studies have examined the effects on insect behavior [Harrison and Mitchell, 19881, growth and de-
R-0
w
Chaconlne
, ‘
,
Glucose
RIGahdDse,
Ahamnose
Fig. 1. The structures of chaconine and solanine 0
1992 Wiley-Liss, Inc.
MATERIALS AND METHODS
All insects were maintained at 28°C and 16:8 L:D photoperiod with relative humidity uncontrolled. Colorado potato beetles were fed fresh potato foliage. In addition to a susceptible strain, two insecticide-resistant strains were tested. The multiresistant strain (Long Island strain [Ioannidis et al., 19921) was highly resistant to carbofuran (225-fold) and azinphosmethyl (445-fold), with low resistance (ninefold) to permethrin. The carbofuran-resistant strain (Montcalm-C strain [Ioannidis et al., 19921) was highly resistant to carbofuran (576-fold) with low resistance to azinphosmethyl (ninefold) and permethrin (eightfold). Susceptible German cockroaches, Blattella germanica L., were fed dog chow and water ad lib. Adult mosquitoes, Aedes aegypti L., from
Sofanine
Rhafi’IMSe
R-GluCOS
velopment [Sinden et al.. 19861, and crop damage [Sanford et al., 19901. Potato breeders have successfully exploited high alkaloid levels to confer host plant resistance to the Colorado potato beetle, although one variety (Lenape) was withdrawn from the market due to high levels of glycoalkaloids in the tuber [Nishie et al., 19711. In the present study we evaluate the ability of a-chaconine to inhibit acetylcholinesterase from several insect species, including Colorado potato beetles susceptible and resistant to conventional insecticides.
Rhamnose
Received February 26, 1992; accepted for publication August 10, 1992. Address reprint requests to Dr. Joel M. Wierenga, Pesticide Research Center and Department of Entomology, Michigan State University, East Lansing, MI 48824.
97
a-CHACONINE INHIBITS INSECT AChE
the laboratory of Dr. A. Raikhel (Michigan State University), and houseflies, Muscn domesticn L., purchased from Carolina Biological Supply (Burlington, NC) were fed sugar water ad lib. Cottonwood leaf beetles, Chrysomelu scripra F., were from the laboratory of Dr. L. Bauer (USDA, E. Lansing, MI) and raised on cottonwood foliage. All chemicals used, including a-chaconine, were purchased from Sigma Chemical Co. (St. Louis, MO). Adult insects 2 to 10 days old were used in all of the in vitro assays for AChE activity. Heads and thoraces (Colorado potato beetle and German cockroach) or whole insects were used to produce tissue homogenates. Insects were homogenized with a Vertishear (Gardiner, NY) microfine homogenizer (2 x 10 sec setting 80) in 50 mM phosphate buffer (pH 7.4). The homogenate was centrifuged at 5,OOOg for 20 min (4"C), and the supernatant centrifuged at 100,OOOg (4°C) for 1 h. The resulting pellet was resuspended to give 0.1 to 5 mg/ml protein with approximately 7.3 units of activity per ml, and assayed for AChE activity [Ellman et al., 19611. Briefly, phosphate buffer (including 0.0625 N HCI) and a-chaconine (2.5 mM in 0.0625 N HCI) totaling 200 p1 were added to 300 p1 homogenate (20 to 500 pg protein). Following preincubation for 10 min at 30"C, 100 pI 5,5-dithio-bis(2-nitrobenzoate) (3 mM) and 400 pl acetylthiocholine iodide (10 mM) were added for a total volume of 1 ml. The reaction rate at 3 0 ° C was measured using a Shimadzu UV265 spectrophotometer (Kyoto, Japan). Absorbance readings (at 412 nm) were taken every 30 sec for 10 min. Corrections for the spontaneous hydrolysis of acetylthiocholine and microsomal interaction with 53,dit hio- bis-(2-nitrobenzoate) were made. The reaction was linear for over 10 min. Data points between 2 and 8 min were used to calculate the reaction rates and correlation coefficients. The reaction rates at various chaconine concentrations were used to calculate the mean percent inhibition and construct concentration-inhibition curves. The Lowry method as modified by Guegenrich [1984] was used for protein determination. For housefly and cottonwood leaf beetle homogenates the 100,OOOg supernatant (100 to 2,000 pg protein) was assayed for AChE activity using the procedure above. In vivo toxicity studies were carried out with Colorado potato beetles. For feeding studies, 25 mg of a-chaconine was applied to potato leaves (petioles with 5 leaflets each, ca. 225 pg/cm2). Young second instars (12 per petri dish) were placed on the leaves (two petioles per dish), and mortality was assessed daily for 5 days. For injection studies, adult beetles 2 to 10 days old were injected in the ventral abdomen with 40 pg a-chaconine in 2 pl 0.25N HCI. Controls were injected with 0.25N HCI only. In some cases adults were pretreated with piperonyl butoxide solution 2 hr before injection with chaconine. The piperonyl butoxide was applied topically in 2 p1 (12.5
mg/ml acetone) to the ventral abdomen. Each treatment group consisted of either 10 or 15 beetles (n = 2). Mortality was assessed daily for 3 days. Calculated IC,, (concentration causing 50% inhibition) values were tested for significance by analysis of variance, and paired t-tests. RESULTS AND DISCUSSION
On a milligram protein basis, the beetle species had generally low AChE activity (ca. 7 nmole/mg protein/ min) (Table I), while the housefly had high specific activity, similar to that seen by others [e.g., Lenoir-Rousseaux, 19851. Comparison of 100,OOOg supernatants and microsomal fractions indicated that most AChE activity was membrane-bound. For the inhibition studies, homogenates with high activity were diluted to give approximate 7.3 units per ml. Initial AChE assays with the susceptible strain of Colorado potato beetles showed that at 200 pM,a-chaconine was slightly more potent than solanine (25% and 20%)inhibition, respectively). This is consistent with the findings of others in numerous species [see Roddick et al., 19881. For most of the insects assayed, the concentration causing 50% inhibition (IC,,) was in the 5 to 40 pM range (Fig. 2). This is similar to the sensitivity of mammalian AChE to a-chaconine. Alozie et al. [I9781 reported ICso values of 4 and 30 pM for bovine erythrocyte AChE and horse serum butyrylcholinesterase, respectively. Analysis of variance showed that there were significant differences between species ( F = 152, d.J = 31, P < 0.005). Colorado potato beetle AChE was not sensitive to a-chaconine (1C5, ca. 300 to 900 pM) compared with the other species tested (Table I). Although the cottonwood leaf beetle is a closely related herbivorous species,
TABLE 1. Inhibition of Insect Acetylcholinesterases by a-Chaconine Insect German cockroach (M)C Mosquito (M) Housefly (M) Housefly (S) Cottonwood leaf beetle (M) Cottonwood leaf beetle (S) Colorado potato beetle (M)d Susceptible Carbofuran-resistant Multiresistant
Specific activitya (nrnole/mg prot./min) 56.4 32.7 155.5 8.4 7.5 1.4
f 4.1 f 2.9 f 2.1 f 1.8 0.4 f 0.1
+
7.0 f 0.64 17.1 f 1.74 11.3 f 0.84
8.7 f 0.4' 9.4 f 0.3' 35.2 f 4.6' 36.1 f 5.g2 6.7 f 1.4' > >40 863 f 163 913 f 93 223 f 44
aData are the mean f SEM, n = 4-6. bData computed from dose response curves, n = 4-6. Values followed by different numbersaresignificantlydiRerentfStudent'sttest, P
Inhibition of Insect Acetylcholinesterase by the Potato Glycoalkaloid a-Chaconine Joel M. Wierenga and Robert M. Hollingworth Pesticide Research Center and Department of Entomology, Michigan State University, East Lansing, Michigan
ABSTRACT Homogenates from several insect species were assayed for inhibition of acetylcholinesterase by the potato gtycoalkaloid a-chaconine. Colorado potato beetle acetylcholinesterase was up to 150-fold less sensitive than other species tested. Acetylcholinesterasefrom an insecticide-resistantstrain of Colorado potato beetles was more sensitive to inhibition than the susceptible strain. Most insect species tested had inhibitory concentrations causing a 50% reduction in activity in the 5 to 40 pM range. Sensitive insect acetylcholinesterases were similar to mammalian cholinesterases in their response to u-chaconine. The results indicate that pesticides and host plant resistance factors may interact at the same target. Changes in the target due to selection pressure from either pesticides or host plant resistance factors could affect the efficacy of both 1999 WiIey-Liss, Inc. control strategies. @
Key Words: Leptinotana decemlineata, Musca domestica, Aedes aegypti, Blattella germanica, Chrysomela scr1pta
INTRODUCTION
The major glycoalkaloid components of potatoes are a-solanine and a-chaconine, alkaloids differing only in their sugar moieties (Fig. 1). These molecules have been implicated in instances of food poisoning [e.g., MacMilIan and Thompson, 19791, and several toxic effects in mammals have been identified. Among the effects described are membrane disruption [Roddick et al., 19881, teratogenicity, and acetylcholinesterase (AChE) inhibition [see review by Jadhav et al., 19811. Entomologists and potato breeders have investigated the role of glycoalkaloids in host plant resistance to insect pests, especially the Colorado potato beetle (Leptinotarsa decemlineata (Say)). These studies have examined the effects on insect behavior [Harrison and Mitchell, 19881, growth and de-
R-0
w
Chaconlne
, ‘
,
Glucose
RIGahdDse,
Ahamnose
Fig. 1. The structures of chaconine and solanine 0
1992 Wiley-Liss, Inc.
MATERIALS AND METHODS
All insects were maintained at 28°C and 16:8 L:D photoperiod with relative humidity uncontrolled. Colorado potato beetles were fed fresh potato foliage. In addition to a susceptible strain, two insecticide-resistant strains were tested. The multiresistant strain (Long Island strain [Ioannidis et al., 19921) was highly resistant to carbofuran (225-fold) and azinphosmethyl (445-fold), with low resistance (ninefold) to permethrin. The carbofuran-resistant strain (Montcalm-C strain [Ioannidis et al., 19921) was highly resistant to carbofuran (576-fold) with low resistance to azinphosmethyl (ninefold) and permethrin (eightfold). Susceptible German cockroaches, Blattella germanica L., were fed dog chow and water ad lib. Adult mosquitoes, Aedes aegypti L., from
Sofanine
Rhafi’IMSe
R-GluCOS
velopment [Sinden et al.. 19861, and crop damage [Sanford et al., 19901. Potato breeders have successfully exploited high alkaloid levels to confer host plant resistance to the Colorado potato beetle, although one variety (Lenape) was withdrawn from the market due to high levels of glycoalkaloids in the tuber [Nishie et al., 19711. In the present study we evaluate the ability of a-chaconine to inhibit acetylcholinesterase from several insect species, including Colorado potato beetles susceptible and resistant to conventional insecticides.
Rhamnose
Received February 26, 1992; accepted for publication August 10, 1992. Address reprint requests to Dr. Joel M. Wierenga, Pesticide Research Center and Department of Entomology, Michigan State University, East Lansing, MI 48824.
97
a-CHACONINE INHIBITS INSECT AChE
the laboratory of Dr. A. Raikhel (Michigan State University), and houseflies, Muscn domesticn L., purchased from Carolina Biological Supply (Burlington, NC) were fed sugar water ad lib. Cottonwood leaf beetles, Chrysomelu scripra F., were from the laboratory of Dr. L. Bauer (USDA, E. Lansing, MI) and raised on cottonwood foliage. All chemicals used, including a-chaconine, were purchased from Sigma Chemical Co. (St. Louis, MO). Adult insects 2 to 10 days old were used in all of the in vitro assays for AChE activity. Heads and thoraces (Colorado potato beetle and German cockroach) or whole insects were used to produce tissue homogenates. Insects were homogenized with a Vertishear (Gardiner, NY) microfine homogenizer (2 x 10 sec setting 80) in 50 mM phosphate buffer (pH 7.4). The homogenate was centrifuged at 5,OOOg for 20 min (4"C), and the supernatant centrifuged at 100,OOOg (4°C) for 1 h. The resulting pellet was resuspended to give 0.1 to 5 mg/ml protein with approximately 7.3 units of activity per ml, and assayed for AChE activity [Ellman et al., 19611. Briefly, phosphate buffer (including 0.0625 N HCI) and a-chaconine (2.5 mM in 0.0625 N HCI) totaling 200 p1 were added to 300 p1 homogenate (20 to 500 pg protein). Following preincubation for 10 min at 30"C, 100 pI 5,5-dithio-bis(2-nitrobenzoate) (3 mM) and 400 pl acetylthiocholine iodide (10 mM) were added for a total volume of 1 ml. The reaction rate at 3 0 ° C was measured using a Shimadzu UV265 spectrophotometer (Kyoto, Japan). Absorbance readings (at 412 nm) were taken every 30 sec for 10 min. Corrections for the spontaneous hydrolysis of acetylthiocholine and microsomal interaction with 53,dit hio- bis-(2-nitrobenzoate) were made. The reaction was linear for over 10 min. Data points between 2 and 8 min were used to calculate the reaction rates and correlation coefficients. The reaction rates at various chaconine concentrations were used to calculate the mean percent inhibition and construct concentration-inhibition curves. The Lowry method as modified by Guegenrich [1984] was used for protein determination. For housefly and cottonwood leaf beetle homogenates the 100,OOOg supernatant (100 to 2,000 pg protein) was assayed for AChE activity using the procedure above. In vivo toxicity studies were carried out with Colorado potato beetles. For feeding studies, 25 mg of a-chaconine was applied to potato leaves (petioles with 5 leaflets each, ca. 225 pg/cm2). Young second instars (12 per petri dish) were placed on the leaves (two petioles per dish), and mortality was assessed daily for 5 days. For injection studies, adult beetles 2 to 10 days old were injected in the ventral abdomen with 40 pg a-chaconine in 2 pl 0.25N HCI. Controls were injected with 0.25N HCI only. In some cases adults were pretreated with piperonyl butoxide solution 2 hr before injection with chaconine. The piperonyl butoxide was applied topically in 2 p1 (12.5
mg/ml acetone) to the ventral abdomen. Each treatment group consisted of either 10 or 15 beetles (n = 2). Mortality was assessed daily for 3 days. Calculated IC,, (concentration causing 50% inhibition) values were tested for significance by analysis of variance, and paired t-tests. RESULTS AND DISCUSSION
On a milligram protein basis, the beetle species had generally low AChE activity (ca. 7 nmole/mg protein/ min) (Table I), while the housefly had high specific activity, similar to that seen by others [e.g., Lenoir-Rousseaux, 19851. Comparison of 100,OOOg supernatants and microsomal fractions indicated that most AChE activity was membrane-bound. For the inhibition studies, homogenates with high activity were diluted to give approximate 7.3 units per ml. Initial AChE assays with the susceptible strain of Colorado potato beetles showed that at 200 pM,a-chaconine was slightly more potent than solanine (25% and 20%)inhibition, respectively). This is consistent with the findings of others in numerous species [see Roddick et al., 19881. For most of the insects assayed, the concentration causing 50% inhibition (IC,,) was in the 5 to 40 pM range (Fig. 2). This is similar to the sensitivity of mammalian AChE to a-chaconine. Alozie et al. [I9781 reported ICso values of 4 and 30 pM for bovine erythrocyte AChE and horse serum butyrylcholinesterase, respectively. Analysis of variance showed that there were significant differences between species ( F = 152, d.J = 31, P < 0.005). Colorado potato beetle AChE was not sensitive to a-chaconine (1C5, ca. 300 to 900 pM) compared with the other species tested (Table I). Although the cottonwood leaf beetle is a closely related herbivorous species,
TABLE 1. Inhibition of Insect Acetylcholinesterases by a-Chaconine Insect German cockroach (M)C Mosquito (M) Housefly (M) Housefly (S) Cottonwood leaf beetle (M) Cottonwood leaf beetle (S) Colorado potato beetle (M)d Susceptible Carbofuran-resistant Multiresistant
Specific activitya (nrnole/mg prot./min) 56.4 32.7 155.5 8.4 7.5 1.4
f 4.1 f 2.9 f 2.1 f 1.8 0.4 f 0.1
+
7.0 f 0.64 17.1 f 1.74 11.3 f 0.84
8.7 f 0.4' 9.4 f 0.3' 35.2 f 4.6' 36.1 f 5.g2 6.7 f 1.4' > >40 863 f 163 913 f 93 223 f 44
aData are the mean f SEM, n = 4-6. bData computed from dose response curves, n = 4-6. Values followed by different numbersaresignificantlydiRerentfStudent'sttest, P
