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Toxicity and metabolism of carbaryl in the European corn borer
PESTICIDE
BIOCHEMISTRY
Toxicity
AND
PHYSIOLOQY
and Metabolism
5,
of Entomology, Received
(1975)
of Carbaryl R. J.
Department
330-337
New
KUHR
York
August
in the European
AND
A. C.
State Agriculturul 27, 1974;
accepted
Corn
Borer’
DAVIS
Experiment January
Station,
Geneva,
New
York
17, 1975
Larvae from two strains of the European corn borer, Ostriniu nubilalis (Hiibner), were compared for differences in their tolerance and metabolism of carbaryl (I-naphthyl N-methylcarbamate). The Geneva strain was about twice as susceptible to carbaryl, but both Valley and Geneva borers converted carbaryl to oxidative metabolites at similar rates in viva and in vi&o. Maximum carbaryl-metabolizing activity WB present in last-instar larvae, particularly in the fat body and gut tissues. However, the specific activity of gut homogenates was highest in the Geneva strain and the specific activity of fat body was highest in the Valley strain. Other differences in the mixed-function oxidase systems of gut and fat body were also found. The major metabolite in vivo and in vitro was hydroxymethyl carbaril. INTRODUCTION
The European corn borer, Ostrinia nubilalis (Hiibner), is a pest of fresh and processing sweet corn as well as many other crops. In most parts of New York, this insect has been effectively controlled for many years with DDT, and more recently with carbaryl without showing any indications of resistance. However, borer control with carbaryl has not always been optimum. In the Hudson Valley region, where both broods of the borer must be controlled on fresh-market sweet corn, it is not unusual to use S-10 applications of carbaryl during a season at 3-5 day intervals for suppression of corn borer and corn earworm. Near Geneva, later plantings of processing corn require control measures for only the second brood of borers with three applications of carbaryl recommended but seldom used. Thus, because of greater insecticide pressure through continuous use, development of carbaryl resistance by 1 Supported search Project
in part 53.
by
Northeast
Regional
Re-
330 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in my form reserved.
borers in the Valley would appear to be more likely than by Geneva borers. Therefore, a thorough study of carbaryl toxicity and metabolism was conducted with laboratory strains of borers collected from the two areas of New York. Such instances, where insects have been treated year after year with the same insecticide with little or no resistance buildup, have not been given a great deal of attention with respect to toxicity and metabolism. MATERIALS
AND
METHODS
Chemicals. Technical samples of carbaryl and its metabolites were supplied by Union Carbide Corp., New York, N. Y. The C’4C]carbonyl insecticide (26.4 mCi/mmol) was purchased from Amersham/Searle Corp., Des Plaines, IL. Acetone solutions were prepared from stock by diluting with unlabeled carbaryl to give 55,000 cpm/5 pg of carbaryl. Ring-labeled insecticide was synthesized as before (1) and diluted to give 30,000 cpm/pg of carbaryl. The sources for all other chemicals and bio-
METABOLISM
OF
CARBARYL
IN
TABLE Toxicity
Instar
Average weight (mg/larvae)
Third Fourth Fifth
6.0 f 0.3 20 fl 64 f3
a Values
include
standard
of Carbaryl Ingested
(ppm
Geneva 66f 3 179 f 12 524 xk 50
331
BORER
1
to Di$erent LCsa
CORN
Instars
of Corn
Borersa
in diet)
Topical
Valley 90f 4 364 f 84 832 zk 96
L&o
(Mg/larvae)
Geneva 3.4 f 10 * 198 f
Valley 0.5 0.9 125
3.6 f 33 f 323 f
0.5 5 62
error.
chemicals used in these studies have been published (1, 2). Insects. Two laboratory strains of borers were started in 1971 from field collections taken from Geneva and from a concentrated sweet corn area in the Hudson Valley. Larvae were reared on a diet proposed by Chippendale and Beck (3) with modifications described by Miles (4). Both strains completed about 12 generations a year in the laboratory with a relative humidity of 70$& at 26°C and 16 hr of light. Toxicity tests. The toxicity of ingested carbaryl was determined by placing larvae on diet medium which had been prepared with water containing various concentrations of 80% WP carbaryl. After 48 hr, mortality counts were taken. Using 6 replicates of 15 insects at 5 dosage levels, LCSO values were calculated via probit analysis. Tests were conducted on third, fourth and fifth instars from each strain. Evaluation of contact toxicity was accomplished by topically treating third and fourth instars with 0.5 ~1 of acetone containing various amounts of technical carbaryl. Last-instar larvae were treated with 1 ~1 of acetone-insecticide solutions. Probit analysis of 4%hr mortalities from 6 replicates of 10 insects at each dose gave the reported LDso values. Metabolism studies. The rate of carbaryl metabolism in vivo was determined by injecting 1 Mg of ring-labeled carbaryl dissolved in 1 ~1 acetone into individual fifth-instar larvae and tracing the disappearance of parent compound by extrac-
tion, chromatography and radioassay as previous1.y described (1). The method also allowed for the tentative identification by cochromatography of ether-soluble metabolites with authentic standards. Watersoluble products were partially characterized after acidic and enzymic hydrolysis (1). The distribution of metabolic act’ivity was measured by incubating carbaryl with isolated whole tissues or tissue homogenates. Borers were dissected in ice-cold 1.15% KC1 and the isolated tissues were homogenized as described for cabbage loopers (2). Whole tissues or whole homogenates were placed in IO-ml beakers together with 5 fig of carbonyl-labeled carbaryl, phosphate buffer (5.0 X 1OF M), glucose-6-phosphate (2.4 X 10v3 M), glucose-6-phosphate dehydrogenase (1 unit), NADP (1.18 X 1O-4 M), KC1 (3.86 X 1O-3 M) and water in a final volume of 2 ml. Unless stated otherwise, the buffer pH was 7.4, fifth-instar larvae were used, and incubations took place with shaking at 25°C for 1 hr. Extraction of the reaction mixture with ether, chromatography and radioautography of the extracts, protein determinations, and other methods were identical to those already reported (2). RESULTS
AND
DISCUSSION
Toxicity. There was little difference in toxicity between the strains, although Valley borers generally tolerated about twice as much insecticide in each instar
332
KUHR
AND
DAVIS
lepidoptera such as the cabbage looper (6, 7) and various cutworms (S-10). In viva metabolism. The half-lives for injected carbaryl were 86 min and 96 min for the Valley and Geneva strains, respectively. This indicates very little difference in the rate at which borers from the two strains metabolized carbaryl in vivo. Not only were the rates similar, but the metabolic pathways were almost identical with each strain producing about 45y0 of the metabolites as ether-soluble materials (mainly hydroxymethyl carbaryl with some 5,6-dihydrodihydroxy carbaryl). The watersoluble products were cleaved by 55y0 with p-glucosidase or glusulase, 41% with Bglucuronidase-sulfatase, and 757, with heat carcms silk glandr fat body molp. tubules plus acid. Enzymic hydrolysis released gut primarily hydroxymethyl, 4-hydroxy and FIG. 1. Metabolism of mrbaryl by tissue homog5-hydroxy carbaryl, together with a mixture enates from jifth-instar European corn borer larvae. of dihydrodihydroxy derivatives and some Values are averages of three experiments with three unknown materials. Because of instability, cliffrent tissue preparations. the dihydroxy compounds were found in only minor quantities after acid treatment. (Table 1). When carbaryl was incorporated In all instances, from lo-25ye of t#he into the diet, younger larvae died at lower ether-extractable hydrolysatcs consisted of concentrations as expected. But when the 1-naphthol. body weight of each instar was considered, The rate of metabolism was considerably ingested carbaryl was almost equitoxie to slower than that reported for cabbage all three larval stages. On the other hand, loopers under identical conditions (1). carbaryl was considerably less toxic to Depending on the looper strain, half-lives fifth instars than to third or fourth instars for 1 pg of injected carbaryl ranged from 3 following topical application, even after to 13 min. This very active oxidative allowing for weight differences. metabolism of carbaryl in the loopers is A recent study comparing carbaryl partially responsible for carbaryl’s ineffectoxicity among 54 species of insects showed tiveness against this insect. Conversely, the that the European corn borer was one of slower metabolic rate in the borer may allow the insects most susceptible to carbaryl for greater toxicity. poisoning (5). However, the reported In vitro nletabolim. The in vitro distributopical LDS, for the borer larvae (12.3 tion of carbaryl metabolic activity among pg/g) was ca. 40X lower than that found different tissues from 5th-instar borers is in our studies with New York borers of shown in Fig. 1. When compared on a similar weight (fourth instars). With larger per-tissue basis, the fat body had the fifth-instar borers, the LD,o value fell in greatest capacity for carbaryl metabolism the range of the insects most tolerant to and, together with the gut, was probably carbaryl. Thus, the field-success of carbaryl responsible for over 95yo of the in vivo metabolism of carbaryl by borers from both for corn borer control must lie in its ability strains. Similar results were obtained when to kill early instars. This change in toxicity with ane with other~~ the tissues were homogenized or incubated -0 has been demonstrated
METABOLIRhl
Geneva
INSTAR
OF’
CARBARYL
Strain
PREPUPA
FIG. 2. Metabolism of curbaryl by gut and fat body homogenates from various developmental stages of European corn borers. Values are average of six experiments with six different tissue preparations.
as whole tissues. However, homogenization, which resulted in some loss of activity, allowed for quantitation on a protein basis and greater consistency from experiment to experiment. Thus, if specific activities are compared, the fat body of the Valley borers had the greatest metabolic capacity while the gut tissue from Geneva borers was most active (Fig. 1). The distribution of carbaryl metabolic activity in the Valley borer tissues was much like that found for cabbage loopers (2). On the other hand, high oxidase activity in the gut of the Geneva borers was like that reported for most other lepidoptera (11, 12). These differences between borer tissues emphasize the great variation that can exist among insects, not only between orders and species, but even between strains of the same species. With respect to age, fifth-instar gut and fat body from both strains were most active, with decreased metabolism evident in earlier instars and prepupae (Fig. 2). This bell-shaped curve of activity through the different developmental stages is wellestablished, particularly for holomctabolous species (11). On a per-insect basis the fat body was the most active tissue in all stages of both strains. The increase in specific activity with larval age, however, indicated
IN
CORN
333
BORER
that changes in metabolic activity were not only due to changes in size of tissues. Further comparative studies between 5th-instar larvae from the two corn borer strains yielded average specific activities for gut, and fat body homogenates as shown in Table 2. Maximum metabolism occurred at pH 7.2-7.4 after tissue dissection and homogenization in 1.15% KCl. Changing to a Tris buffer for incubation or a phosphate buffer for tissue dissection and homogenization did not alter activity significantly. All tissues gave linear reaction rates for at least 1 hr with 0.5 ml or less of homogenate. Boiled preparations were inactive and storage at 0°C also resulted in decreased act’ivity. The temperature optimum was bet’ween 20 and 3°C for all tissues. A similar range has been reported for cabbage looper tissues (2) and caddisfl larval tissues (13). Interestingly, the specific activities of the gut and fat body were almost exactly reversed in t’he two strains (Table 2). Considering that these tissues together probably comprised the principal sites of carbaryl metabolism in the insects (Fig. l), and using the average protein content found for each tissue, it is possible to multiply specific activity times weight expressed as mg protein to get an idea of the maximum possible metabolism in an individual borer. Thus, for a Geneva larva this amounts to 1.15 pg (fat) + 0.52 pg TABLE Speci’c
Actiwity enates from ~__-__
2
of Gut and Fat Hody HomogFifth-Instar Corn Borers PR carbaryl metzabolizedjmg protein/hr f SEa
Strain
Fat
Geneva Valley
0.44 f 0.77 f
Gut,
body 0.04 0.05
0.74 0.36
f 0.08 It 0.04
(2 Incubation medium contained phosphate buffer, KCl, NADPH generator, tissue homogenate, carbaryl, and water to tot,al2 ml. Incubations took place at 25’C for 1 hr.
334
KUHR
AND
DAVIS
minor importance in housefly microsomes (15), blowfly fat body (16), and silkworms (17). Gut tissues produced more 5,6-dihyHomogenates drodihydroxy and 5-hydroxy carbaryl, indicating that although qualitatively the Percent of total ethermetabolism was the same among the soluble radioactivity’ tissues, quantitative differences did exist between fat and gut. No desmethyl or Geneva Valley 7-hydroxy carbaryl could be detected in Gut Fat Gut Fat extracts from any tissue. body Carbaryl metabolite body That metabolism was catalyzed by a mixed-function oxidase is evident from 5,&Dihydrodiresults in Table 4. Activity was reduced on hydroxy 19 11 13 6 58 74 52 70 N-Hydroxymethyl omission of oxygen or NADPH, although 2 6 4 9 4-Hydroxy some endogenous cofactors must have been 5-Hydroxy 15 4 18 9 present since total inhibition did not occur Unknowns 6 5 13 6 in their absence. Marked reduction in activity occurred in the presence of carbon (1 Each result is the average of eight experiments monoxide and all tissue metabolism was with four different tissue homogenates. inhibited by piperonyl butoxide (molar 150 values about 2 X 10-j). Substitution of (gut) = 1.67 rug, and for a Valley larva this is 1.56 /*g (fat) + 0.19 pg (gut) = 1.75 NADPH by NADH, FMN or FAD was pg. Therefore, it would appear that borers not successful, and the latter two inhibited from both strains should be able to metabolism even in the presence of NADPH metabolize about the same amount of (Table 4). This is quite often the case with insecticide which agrees with their similar insect oxidases (11). EDTA, KCN and carbaryl in vivo half-lives described earlier. GSH all stimulated gut activity but had However, one hour after injection of 1 pg of little effect on fat body. This appears to be carbaryl, less than 0.5 pg of toxicant had the first instance where EDTA has been been metabolized, indicating that the shown to stimulate insecticide oxidation by substrate was not as available to the insect preparations. In mammalian liver oxidases of whole in vivo tissues as it was to microsomes, EDTA often stimulates subin vitro enzymes from homogenized tissues. strate oxidation by blocking a competitive The specific activities for borer gut and lipid peroxidation system (18). However, fat body were considerably lower than the peroxidation system is also inhibited by those from the most carbaryl-susceptible Mn2+ and Co2+ which was not the case for cabbage looper (1). Nevertheless, the the borer gut preparations. metabolic pattern was similar. Fat body Stimulation by KCN and GSH is also homogenates yielded an average of 30y0 difficult to interpret. When studying metabof the metabolites as water-soluble products olism which is catalyzed by a glutathione while guts averaged 40% water-solubles. transferase, it is often necessary to add Tentatively identified ether-soluble prod- GSH. With whole tissue homogenates, such as borer guts, it would seem that sufficient ucts were divided as listed in Table 3. In all tissues, hydroxymethyl carbaryl was endogenous GSH would be present for the the major metabolite. Detoxification by transferase to operate. Also, judging from N-methyl hydroxylation was also found to the nature of the metabolites, it is hard to be of primary importance in cabbage understand how GSH could be involved looper tissues (2) and possibly bollworms directly. Stimulation of insect oxidases and boll weevils (14), although only of with cyanide has been explained as an TABLE
3
Tentative Identity of Ether-Soluble Rfetabolites from Corn Borer Gut and Fat Body
METABOLISM
OF
CARBARYL TABLE
The
Efleet
of Various
Materials
IN
CORN BORER
335
4
on the Metabolism of Carbaryl and Fat Body Honkogenotes
by Fifth-Zn’nstar
Percent
Corn
Rarer
activityb
Geneva Incubation
medium additions or omissions0
None - Oxygen - NADP - NADP + NADH + FMN (10-W) + FAD (IOPM) + EDTA (lo-3Jf) + KCN (10-W) + GSH (10-3hf) + Nicotinamide (10WM) + Carbon monoxide (80%)
Fat
body
(1 Incubation medium as described in Table 2. b Each result is the average of at least four experiments activity is based on the amount of carbaryl metabolized/mg
inhibition of a tyrosinase system the products of which inhibit the oxidases operating on the insecticide substrate (19). If tyrosinase itself was using carbaryl as a substrate, then KCN would inhibit rather than stimulate metabolism. A previous study with housefly microsomes indicated that insect tyrosinase was not able to metabolize carbaryl (14). But even though the reasons for stimulation by these materials is unknown, what is significant is that the stimulation only took place with gut homogenates and not fat body homogenates. The effect of metal ions on insect oxidases shows no consistent patterns except that Cu2+, Cu+ and Zn2+ generally are potent inhibitors (11). This was also found to be true for the two copper ions when added to borer gut and fat body homogenates (Table 5). The only other ion tested which gave a pronounced effect on metabolism was Hg’+ which yielded inhibition equal to that of copper. Similar to results from cabbage loopers (1, 2), there were some differences in response of gut and fat body tissues to
Valley Gut
100 15 88 82 51 76 85 93 101 75 17
Gut
Fat
100 34 66 77 47 64 163 150 142 78 38
body
Gut
100 29 82 74 44 67 87 99 97 80 22
with four different protein/hr.
tissue
100 54 74 66 85 63 195 169 134 96 36
preparations.
Percent
some ions. For example, Co2+ had no effect on gut activity but slightly depressed fat body activity in both strains. On the other TABLE The Effect Corn Metal ion added to incubation mediuma -__ None cocu+2 Hgcz Fe+2 Mn+= Mg+2 Ni+2 Fe+3 Cu”
5
of itlelal Ions on the AGtivity of Fifth-Znstar Borer Gut and Fat Body Homogenates Percent
activityb
Geneva Fat body 100 82 3 2 101 104 95 80 101 17
Valley Gut
100 98 10 8 67 106 81 59 123 14
Fat body 100 83 5 4 87 97 98 70 103 18
a Incubation medium as described in Table ions at lO+M. 6 Percent activity as described in Table 4.
Gut
100 102 45 24 98 121 85 77 146 48 2. All
336
KUNR
9ND
hand, Fe3+ had no effect on fat body but enhanced metabolism by gut. One factor which would appear to speak against involvement of a mixed function oxidase in carbaryl metabolism by the borers is the appearance of 5,6-dihydrodihydroxy carbaryl and possibly other dihydrodihydroxy derivatives. Although formation of these products could conceivably be catalyzed by a different type of oxidase, their formation is inhibited by t,he absence of NADPH or OS and by the presence of carbon monoxide or piperonyl butoxide. Similar results with carbaryl and other homogenate or microsome preparations suggest that the dihydrodihydroxy mctabelites are a result of spontaneous degradation of epoxide intermediates (20). Thus, all of the tentatively identified metabolites probably originate from the mixed function oxidase catalyzed insertion of a single atom of oxygen into the carbamate substrate. CONCLUSIONS
On the whole, the laboratory data do not support any large differences in the toxicity or metabolism of carbaryl by the two corn borer strains. The most notable variation between borers was the specific activity reversal of gut and fat body, although total metabolism by whole larvae was the same. In fact, the differences found in metabolite pattern and responseto cofactors and metal ions between the gut and fat body within one strain were greater than any interstrain differences. Thus, although a mixed-function oxidase system was responsible for carbaryl metabolism in both tissues, there must exist some differences in the nature of the enzyme complex. The fact that the insects were able to oxidativcly metabolize carbaryl with a reasonable level of competence would seem to allow for the development of resistance by the borers. However, after repeated use of carbaryl for many years in the field, very little, if any, tolerance seemsto have been acquired. Under laboratory pressure
DAVIS
with carbaryl or DDT, some resistance to the compound developed in 11-12 generations (21). However, the selection also yielded reduced egg numbers and egg viability and reduced weights of larvae and pupae. Thus, selection for insecticide resistance, although the metabolic capacity is there, also may lead to selection of deleterious physiological factors which could continue to suppress any large resistance buildup. AC!KNOWLEDGMENTS
We thank Murphy for R. W. Straub
C. W. Hessney, A. A. Pace and L. C. their skilled technical assistance and for helpful suggestions.
REFERENCES
1. R. J. Kuhr, Comparative metabolism of carbaryl by resistant and susceptible strains of t,he cabbage looper, J. Econ. Entomol. 64, 1373 (1971). 2. Ii. J. Kuhr, Metabolism of carbamate insecticide chemicals in plants and insects, J. Agr. Food Chem. 18, 1023 (1970). 3. G. M. Chippendale and S. D. Beck, Nutrition of the European corn borer, Osttinia n&Z&s (Hbn.). V. Ascorbic acid as the corn leaf factor, Entomol. Ezp. Appl. 7, 241 (1964). 4. J. C. Miles, Laboratory and field studies on the European corn borer, Ostrinia nubilalis (Hbn.), (Lepidoptera: Pyralidae) in Sout,hwestern Ontario, M. SC. Dissertation, University of Guelph, 1970. 5. L. B. Brattsten and R. L. Metcalf, The synergistic ratio of carbaryl with piperonyl butoxide as an indicator of the distribution of multifunction oxidases in the Insecta, J. Econ. Entomd. 63, 101 (1970). 6. W. P. Kerr and J. R. Brazzel, Laboratory tests of insecticides against eggs and larvae of the cabbage looper, J. Econ. Entomol. 53, 991 (1960). 7. C. R. Harris and H. J. Svec, Laboratory studies of the contact toxicity of some insecticides to cabbage loopers, J. Econ. Entomol. 62, 1013 (1969). 8. C. It. Harris and H. J. Svec, Toxicological studies on cutworms. I. Laboratory studies on the toxicity of insecticides to the darksided cutworm, J. Ewn. Entomol. 61, 788 (1968). 9. C. It. Harris and H. J. Svec, Toxicological studies on cutworms. III. Laboratory investi-
METABOLISM
10.
11.
12.
13.
14.
1.5.
OF
CARBARYL
gations on the toxicity of insecticides t.o the black cutworm, with special reference t,o the influence of soil type, soil moisture, method of application, and formulation on insecticide activif!y, J. Econ,. Entomol. 61, 866 (1968). C. R. Harris and F. Gore, Toxicological studies on cutworms. VIII. Toxicity of three insecticides to the various stages in the development of the darksided cutworm, J. Econ. Entomol. 64, 1049 (1971). C. F. Wilkinson and L. B. Bra&ten, Microsomal drug metabolizing enzymes in insects, Drug Mctab. Rev. 1, 153 (1972). R. I. Krieger, Microsomal oxidases in select,ed lepidopterous larvae, primarily the sout,hern armyworm Prodenia eridania, Ph.D. Dissertation, Cornell University, 1970. R. I. Krieger and P. W. Lee, Properties of the aldrin epoxidase system in the gut and fat body of a caddisfly larva, J. Ewn. Entomol. 66, 1 (1973). N. R. Andrawes and H. W. Dorough, Metabolic fate of carbaryl-naphthyl-Cl4 in boll weevils and bollworms, J. Econ. Entomol. 60, 433 (1967). R. J. Kuhr, Possible role of tyrosinase and cytochrome P-450 in the metabolism of I-naphthyl mebhylcarbamate (carbaryl) and
IN
16.
17.
18.
19.
20.
21.
CORN
BORER
337
phenyl methylcarbamate by houseflies, J. Agr. Food Chem. 17, 112 (1969). G. M. Price and R. J. Kuhr, The metabolism of t.he insecticide carbaryl (1-naphthyl-N-methylcarbamate) by fat body of the blowfly larva Calliphora erythrocephala, Biochem. J. 112, 133 (1969). H. Moriyama, H. Sugiyama and H. Shigematsu, A hydroxy metabolite derived from carbaryl in the silkworm, Bombys muri, Pest. Hiochvm. Physiol. 2, 1 (1972). 8. E. Lewis, C. F. Wilkinson and J. W. Ray, The relationship between microsomal epoxidation and lipid peroxidation in houseflies and pig liver and the inhibit,ory effect of derivatives of 1,3-benzodioxole (methylenedioxybenzene), Biochem. Pharmacol. 16, 1195 (1967). J. W. Ray, The expoxidation of aldrin by housefly microsomes and its inhibition by carbon monoxide, Biochem. Phurmawl. 16,99 (1967). 1~. J. Kuhr. The formation and importance of carbamat,e insecticide metabolites as terminal residues, Pure Appl. Chem. Suppl., 199 (1971). J. A. Harding and 11. C. Uyar. Resistance induced in European corn borers in the laboratory by exposing successive generations to DDT, diazinon, or carbaryl, J. Ewn. Entontot. 63, 250 (1970).
BIOCHEMISTRY
Toxicity
AND
PHYSIOLOQY
and Metabolism
5,
of Entomology, Received
(1975)
of Carbaryl R. J.
Department
330-337
New
KUHR
York
August
in the European
AND
A. C.
State Agriculturul 27, 1974;
accepted
Corn
Borer’
DAVIS
Experiment January
Station,
Geneva,
New
York
17, 1975
Larvae from two strains of the European corn borer, Ostriniu nubilalis (Hiibner), were compared for differences in their tolerance and metabolism of carbaryl (I-naphthyl N-methylcarbamate). The Geneva strain was about twice as susceptible to carbaryl, but both Valley and Geneva borers converted carbaryl to oxidative metabolites at similar rates in viva and in vi&o. Maximum carbaryl-metabolizing activity WB present in last-instar larvae, particularly in the fat body and gut tissues. However, the specific activity of gut homogenates was highest in the Geneva strain and the specific activity of fat body was highest in the Valley strain. Other differences in the mixed-function oxidase systems of gut and fat body were also found. The major metabolite in vivo and in vitro was hydroxymethyl carbaril. INTRODUCTION
The European corn borer, Ostrinia nubilalis (Hiibner), is a pest of fresh and processing sweet corn as well as many other crops. In most parts of New York, this insect has been effectively controlled for many years with DDT, and more recently with carbaryl without showing any indications of resistance. However, borer control with carbaryl has not always been optimum. In the Hudson Valley region, where both broods of the borer must be controlled on fresh-market sweet corn, it is not unusual to use S-10 applications of carbaryl during a season at 3-5 day intervals for suppression of corn borer and corn earworm. Near Geneva, later plantings of processing corn require control measures for only the second brood of borers with three applications of carbaryl recommended but seldom used. Thus, because of greater insecticide pressure through continuous use, development of carbaryl resistance by 1 Supported search Project
in part 53.
by
Northeast
Regional
Re-
330 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in my form reserved.
borers in the Valley would appear to be more likely than by Geneva borers. Therefore, a thorough study of carbaryl toxicity and metabolism was conducted with laboratory strains of borers collected from the two areas of New York. Such instances, where insects have been treated year after year with the same insecticide with little or no resistance buildup, have not been given a great deal of attention with respect to toxicity and metabolism. MATERIALS
AND
METHODS
Chemicals. Technical samples of carbaryl and its metabolites were supplied by Union Carbide Corp., New York, N. Y. The C’4C]carbonyl insecticide (26.4 mCi/mmol) was purchased from Amersham/Searle Corp., Des Plaines, IL. Acetone solutions were prepared from stock by diluting with unlabeled carbaryl to give 55,000 cpm/5 pg of carbaryl. Ring-labeled insecticide was synthesized as before (1) and diluted to give 30,000 cpm/pg of carbaryl. The sources for all other chemicals and bio-
METABOLISM
OF
CARBARYL
IN
TABLE Toxicity
Instar
Average weight (mg/larvae)
Third Fourth Fifth
6.0 f 0.3 20 fl 64 f3
a Values
include
standard
of Carbaryl Ingested
(ppm
Geneva 66f 3 179 f 12 524 xk 50
331
BORER
1
to Di$erent LCsa
CORN
Instars
of Corn
Borersa
in diet)
Topical
Valley 90f 4 364 f 84 832 zk 96
L&o
(Mg/larvae)
Geneva 3.4 f 10 * 198 f
Valley 0.5 0.9 125
3.6 f 33 f 323 f
0.5 5 62
error.
chemicals used in these studies have been published (1, 2). Insects. Two laboratory strains of borers were started in 1971 from field collections taken from Geneva and from a concentrated sweet corn area in the Hudson Valley. Larvae were reared on a diet proposed by Chippendale and Beck (3) with modifications described by Miles (4). Both strains completed about 12 generations a year in the laboratory with a relative humidity of 70$& at 26°C and 16 hr of light. Toxicity tests. The toxicity of ingested carbaryl was determined by placing larvae on diet medium which had been prepared with water containing various concentrations of 80% WP carbaryl. After 48 hr, mortality counts were taken. Using 6 replicates of 15 insects at 5 dosage levels, LCSO values were calculated via probit analysis. Tests were conducted on third, fourth and fifth instars from each strain. Evaluation of contact toxicity was accomplished by topically treating third and fourth instars with 0.5 ~1 of acetone containing various amounts of technical carbaryl. Last-instar larvae were treated with 1 ~1 of acetone-insecticide solutions. Probit analysis of 4%hr mortalities from 6 replicates of 10 insects at each dose gave the reported LDso values. Metabolism studies. The rate of carbaryl metabolism in vivo was determined by injecting 1 Mg of ring-labeled carbaryl dissolved in 1 ~1 acetone into individual fifth-instar larvae and tracing the disappearance of parent compound by extrac-
tion, chromatography and radioassay as previous1.y described (1). The method also allowed for the tentative identification by cochromatography of ether-soluble metabolites with authentic standards. Watersoluble products were partially characterized after acidic and enzymic hydrolysis (1). The distribution of metabolic act’ivity was measured by incubating carbaryl with isolated whole tissues or tissue homogenates. Borers were dissected in ice-cold 1.15% KC1 and the isolated tissues were homogenized as described for cabbage loopers (2). Whole tissues or whole homogenates were placed in IO-ml beakers together with 5 fig of carbonyl-labeled carbaryl, phosphate buffer (5.0 X 1OF M), glucose-6-phosphate (2.4 X 10v3 M), glucose-6-phosphate dehydrogenase (1 unit), NADP (1.18 X 1O-4 M), KC1 (3.86 X 1O-3 M) and water in a final volume of 2 ml. Unless stated otherwise, the buffer pH was 7.4, fifth-instar larvae were used, and incubations took place with shaking at 25°C for 1 hr. Extraction of the reaction mixture with ether, chromatography and radioautography of the extracts, protein determinations, and other methods were identical to those already reported (2). RESULTS
AND
DISCUSSION
Toxicity. There was little difference in toxicity between the strains, although Valley borers generally tolerated about twice as much insecticide in each instar
332
KUHR
AND
DAVIS
lepidoptera such as the cabbage looper (6, 7) and various cutworms (S-10). In viva metabolism. The half-lives for injected carbaryl were 86 min and 96 min for the Valley and Geneva strains, respectively. This indicates very little difference in the rate at which borers from the two strains metabolized carbaryl in vivo. Not only were the rates similar, but the metabolic pathways were almost identical with each strain producing about 45y0 of the metabolites as ether-soluble materials (mainly hydroxymethyl carbaryl with some 5,6-dihydrodihydroxy carbaryl). The watersoluble products were cleaved by 55y0 with p-glucosidase or glusulase, 41% with Bglucuronidase-sulfatase, and 757, with heat carcms silk glandr fat body molp. tubules plus acid. Enzymic hydrolysis released gut primarily hydroxymethyl, 4-hydroxy and FIG. 1. Metabolism of mrbaryl by tissue homog5-hydroxy carbaryl, together with a mixture enates from jifth-instar European corn borer larvae. of dihydrodihydroxy derivatives and some Values are averages of three experiments with three unknown materials. Because of instability, cliffrent tissue preparations. the dihydroxy compounds were found in only minor quantities after acid treatment. (Table 1). When carbaryl was incorporated In all instances, from lo-25ye of t#he into the diet, younger larvae died at lower ether-extractable hydrolysatcs consisted of concentrations as expected. But when the 1-naphthol. body weight of each instar was considered, The rate of metabolism was considerably ingested carbaryl was almost equitoxie to slower than that reported for cabbage all three larval stages. On the other hand, loopers under identical conditions (1). carbaryl was considerably less toxic to Depending on the looper strain, half-lives fifth instars than to third or fourth instars for 1 pg of injected carbaryl ranged from 3 following topical application, even after to 13 min. This very active oxidative allowing for weight differences. metabolism of carbaryl in the loopers is A recent study comparing carbaryl partially responsible for carbaryl’s ineffectoxicity among 54 species of insects showed tiveness against this insect. Conversely, the that the European corn borer was one of slower metabolic rate in the borer may allow the insects most susceptible to carbaryl for greater toxicity. poisoning (5). However, the reported In vitro nletabolim. The in vitro distributopical LDS, for the borer larvae (12.3 tion of carbaryl metabolic activity among pg/g) was ca. 40X lower than that found different tissues from 5th-instar borers is in our studies with New York borers of shown in Fig. 1. When compared on a similar weight (fourth instars). With larger per-tissue basis, the fat body had the fifth-instar borers, the LD,o value fell in greatest capacity for carbaryl metabolism the range of the insects most tolerant to and, together with the gut, was probably carbaryl. Thus, the field-success of carbaryl responsible for over 95yo of the in vivo metabolism of carbaryl by borers from both for corn borer control must lie in its ability strains. Similar results were obtained when to kill early instars. This change in toxicity with ane with other~~ the tissues were homogenized or incubated -0 has been demonstrated
METABOLIRhl
Geneva
INSTAR
OF’
CARBARYL
Strain
PREPUPA
FIG. 2. Metabolism of curbaryl by gut and fat body homogenates from various developmental stages of European corn borers. Values are average of six experiments with six different tissue preparations.
as whole tissues. However, homogenization, which resulted in some loss of activity, allowed for quantitation on a protein basis and greater consistency from experiment to experiment. Thus, if specific activities are compared, the fat body of the Valley borers had the greatest metabolic capacity while the gut tissue from Geneva borers was most active (Fig. 1). The distribution of carbaryl metabolic activity in the Valley borer tissues was much like that found for cabbage loopers (2). On the other hand, high oxidase activity in the gut of the Geneva borers was like that reported for most other lepidoptera (11, 12). These differences between borer tissues emphasize the great variation that can exist among insects, not only between orders and species, but even between strains of the same species. With respect to age, fifth-instar gut and fat body from both strains were most active, with decreased metabolism evident in earlier instars and prepupae (Fig. 2). This bell-shaped curve of activity through the different developmental stages is wellestablished, particularly for holomctabolous species (11). On a per-insect basis the fat body was the most active tissue in all stages of both strains. The increase in specific activity with larval age, however, indicated
IN
CORN
333
BORER
that changes in metabolic activity were not only due to changes in size of tissues. Further comparative studies between 5th-instar larvae from the two corn borer strains yielded average specific activities for gut, and fat body homogenates as shown in Table 2. Maximum metabolism occurred at pH 7.2-7.4 after tissue dissection and homogenization in 1.15% KCl. Changing to a Tris buffer for incubation or a phosphate buffer for tissue dissection and homogenization did not alter activity significantly. All tissues gave linear reaction rates for at least 1 hr with 0.5 ml or less of homogenate. Boiled preparations were inactive and storage at 0°C also resulted in decreased act’ivity. The temperature optimum was bet’ween 20 and 3°C for all tissues. A similar range has been reported for cabbage looper tissues (2) and caddisfl larval tissues (13). Interestingly, the specific activities of the gut and fat body were almost exactly reversed in t’he two strains (Table 2). Considering that these tissues together probably comprised the principal sites of carbaryl metabolism in the insects (Fig. l), and using the average protein content found for each tissue, it is possible to multiply specific activity times weight expressed as mg protein to get an idea of the maximum possible metabolism in an individual borer. Thus, for a Geneva larva this amounts to 1.15 pg (fat) + 0.52 pg TABLE Speci’c
Actiwity enates from ~__-__
2
of Gut and Fat Hody HomogFifth-Instar Corn Borers PR carbaryl metzabolizedjmg protein/hr f SEa
Strain
Fat
Geneva Valley
0.44 f 0.77 f
Gut,
body 0.04 0.05
0.74 0.36
f 0.08 It 0.04
(2 Incubation medium contained phosphate buffer, KCl, NADPH generator, tissue homogenate, carbaryl, and water to tot,al2 ml. Incubations took place at 25’C for 1 hr.
334
KUHR
AND
DAVIS
minor importance in housefly microsomes (15), blowfly fat body (16), and silkworms (17). Gut tissues produced more 5,6-dihyHomogenates drodihydroxy and 5-hydroxy carbaryl, indicating that although qualitatively the Percent of total ethermetabolism was the same among the soluble radioactivity’ tissues, quantitative differences did exist between fat and gut. No desmethyl or Geneva Valley 7-hydroxy carbaryl could be detected in Gut Fat Gut Fat extracts from any tissue. body Carbaryl metabolite body That metabolism was catalyzed by a mixed-function oxidase is evident from 5,&Dihydrodiresults in Table 4. Activity was reduced on hydroxy 19 11 13 6 58 74 52 70 N-Hydroxymethyl omission of oxygen or NADPH, although 2 6 4 9 4-Hydroxy some endogenous cofactors must have been 5-Hydroxy 15 4 18 9 present since total inhibition did not occur Unknowns 6 5 13 6 in their absence. Marked reduction in activity occurred in the presence of carbon (1 Each result is the average of eight experiments monoxide and all tissue metabolism was with four different tissue homogenates. inhibited by piperonyl butoxide (molar 150 values about 2 X 10-j). Substitution of (gut) = 1.67 rug, and for a Valley larva this is 1.56 /*g (fat) + 0.19 pg (gut) = 1.75 NADPH by NADH, FMN or FAD was pg. Therefore, it would appear that borers not successful, and the latter two inhibited from both strains should be able to metabolism even in the presence of NADPH metabolize about the same amount of (Table 4). This is quite often the case with insecticide which agrees with their similar insect oxidases (11). EDTA, KCN and carbaryl in vivo half-lives described earlier. GSH all stimulated gut activity but had However, one hour after injection of 1 pg of little effect on fat body. This appears to be carbaryl, less than 0.5 pg of toxicant had the first instance where EDTA has been been metabolized, indicating that the shown to stimulate insecticide oxidation by substrate was not as available to the insect preparations. In mammalian liver oxidases of whole in vivo tissues as it was to microsomes, EDTA often stimulates subin vitro enzymes from homogenized tissues. strate oxidation by blocking a competitive The specific activities for borer gut and lipid peroxidation system (18). However, fat body were considerably lower than the peroxidation system is also inhibited by those from the most carbaryl-susceptible Mn2+ and Co2+ which was not the case for cabbage looper (1). Nevertheless, the the borer gut preparations. metabolic pattern was similar. Fat body Stimulation by KCN and GSH is also homogenates yielded an average of 30y0 difficult to interpret. When studying metabof the metabolites as water-soluble products olism which is catalyzed by a glutathione while guts averaged 40% water-solubles. transferase, it is often necessary to add Tentatively identified ether-soluble prod- GSH. With whole tissue homogenates, such as borer guts, it would seem that sufficient ucts were divided as listed in Table 3. In all tissues, hydroxymethyl carbaryl was endogenous GSH would be present for the the major metabolite. Detoxification by transferase to operate. Also, judging from N-methyl hydroxylation was also found to the nature of the metabolites, it is hard to be of primary importance in cabbage understand how GSH could be involved looper tissues (2) and possibly bollworms directly. Stimulation of insect oxidases and boll weevils (14), although only of with cyanide has been explained as an TABLE
3
Tentative Identity of Ether-Soluble Rfetabolites from Corn Borer Gut and Fat Body
METABOLISM
OF
CARBARYL TABLE
The
Efleet
of Various
Materials
IN
CORN BORER
335
4
on the Metabolism of Carbaryl and Fat Body Honkogenotes
by Fifth-Zn’nstar
Percent
Corn
Rarer
activityb
Geneva Incubation
medium additions or omissions0
None - Oxygen - NADP - NADP + NADH + FMN (10-W) + FAD (IOPM) + EDTA (lo-3Jf) + KCN (10-W) + GSH (10-3hf) + Nicotinamide (10WM) + Carbon monoxide (80%)
Fat
body
(1 Incubation medium as described in Table 2. b Each result is the average of at least four experiments activity is based on the amount of carbaryl metabolized/mg
inhibition of a tyrosinase system the products of which inhibit the oxidases operating on the insecticide substrate (19). If tyrosinase itself was using carbaryl as a substrate, then KCN would inhibit rather than stimulate metabolism. A previous study with housefly microsomes indicated that insect tyrosinase was not able to metabolize carbaryl (14). But even though the reasons for stimulation by these materials is unknown, what is significant is that the stimulation only took place with gut homogenates and not fat body homogenates. The effect of metal ions on insect oxidases shows no consistent patterns except that Cu2+, Cu+ and Zn2+ generally are potent inhibitors (11). This was also found to be true for the two copper ions when added to borer gut and fat body homogenates (Table 5). The only other ion tested which gave a pronounced effect on metabolism was Hg’+ which yielded inhibition equal to that of copper. Similar to results from cabbage loopers (1, 2), there were some differences in response of gut and fat body tissues to
Valley Gut
100 15 88 82 51 76 85 93 101 75 17
Gut
Fat
100 34 66 77 47 64 163 150 142 78 38
body
Gut
100 29 82 74 44 67 87 99 97 80 22
with four different protein/hr.
tissue
100 54 74 66 85 63 195 169 134 96 36
preparations.
Percent
some ions. For example, Co2+ had no effect on gut activity but slightly depressed fat body activity in both strains. On the other TABLE The Effect Corn Metal ion added to incubation mediuma -__ None cocu+2 Hgcz Fe+2 Mn+= Mg+2 Ni+2 Fe+3 Cu”
5
of itlelal Ions on the AGtivity of Fifth-Znstar Borer Gut and Fat Body Homogenates Percent
activityb
Geneva Fat body 100 82 3 2 101 104 95 80 101 17
Valley Gut
100 98 10 8 67 106 81 59 123 14
Fat body 100 83 5 4 87 97 98 70 103 18
a Incubation medium as described in Table ions at lO+M. 6 Percent activity as described in Table 4.
Gut
100 102 45 24 98 121 85 77 146 48 2. All
336
KUNR
9ND
hand, Fe3+ had no effect on fat body but enhanced metabolism by gut. One factor which would appear to speak against involvement of a mixed function oxidase in carbaryl metabolism by the borers is the appearance of 5,6-dihydrodihydroxy carbaryl and possibly other dihydrodihydroxy derivatives. Although formation of these products could conceivably be catalyzed by a different type of oxidase, their formation is inhibited by t,he absence of NADPH or OS and by the presence of carbon monoxide or piperonyl butoxide. Similar results with carbaryl and other homogenate or microsome preparations suggest that the dihydrodihydroxy mctabelites are a result of spontaneous degradation of epoxide intermediates (20). Thus, all of the tentatively identified metabolites probably originate from the mixed function oxidase catalyzed insertion of a single atom of oxygen into the carbamate substrate. CONCLUSIONS
On the whole, the laboratory data do not support any large differences in the toxicity or metabolism of carbaryl by the two corn borer strains. The most notable variation between borers was the specific activity reversal of gut and fat body, although total metabolism by whole larvae was the same. In fact, the differences found in metabolite pattern and responseto cofactors and metal ions between the gut and fat body within one strain were greater than any interstrain differences. Thus, although a mixed-function oxidase system was responsible for carbaryl metabolism in both tissues, there must exist some differences in the nature of the enzyme complex. The fact that the insects were able to oxidativcly metabolize carbaryl with a reasonable level of competence would seem to allow for the development of resistance by the borers. However, after repeated use of carbaryl for many years in the field, very little, if any, tolerance seemsto have been acquired. Under laboratory pressure
DAVIS
with carbaryl or DDT, some resistance to the compound developed in 11-12 generations (21). However, the selection also yielded reduced egg numbers and egg viability and reduced weights of larvae and pupae. Thus, selection for insecticide resistance, although the metabolic capacity is there, also may lead to selection of deleterious physiological factors which could continue to suppress any large resistance buildup. AC!KNOWLEDGMENTS
We thank Murphy for R. W. Straub
C. W. Hessney, A. A. Pace and L. C. their skilled technical assistance and for helpful suggestions.
REFERENCES
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METABOLISM
10.
11.
12.
13.
14.
1.5.
OF
CARBARYL
gations on the toxicity of insecticides t.o the black cutworm, with special reference t,o the influence of soil type, soil moisture, method of application, and formulation on insecticide activif!y, J. Econ,. Entomol. 61, 866 (1968). C. R. Harris and F. Gore, Toxicological studies on cutworms. VIII. Toxicity of three insecticides to the various stages in the development of the darksided cutworm, J. Econ. Entomol. 64, 1049 (1971). C. F. Wilkinson and L. B. Bra&ten, Microsomal drug metabolizing enzymes in insects, Drug Mctab. Rev. 1, 153 (1972). R. I. Krieger, Microsomal oxidases in select,ed lepidopterous larvae, primarily the sout,hern armyworm Prodenia eridania, Ph.D. Dissertation, Cornell University, 1970. R. I. Krieger and P. W. Lee, Properties of the aldrin epoxidase system in the gut and fat body of a caddisfly larva, J. Ewn. Entomol. 66, 1 (1973). N. R. Andrawes and H. W. Dorough, Metabolic fate of carbaryl-naphthyl-Cl4 in boll weevils and bollworms, J. Econ. Entomol. 60, 433 (1967). R. J. Kuhr, Possible role of tyrosinase and cytochrome P-450 in the metabolism of I-naphthyl mebhylcarbamate (carbaryl) and
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BORER
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phenyl methylcarbamate by houseflies, J. Agr. Food Chem. 17, 112 (1969). G. M. Price and R. J. Kuhr, The metabolism of t.he insecticide carbaryl (1-naphthyl-N-methylcarbamate) by fat body of the blowfly larva Calliphora erythrocephala, Biochem. J. 112, 133 (1969). H. Moriyama, H. Sugiyama and H. Shigematsu, A hydroxy metabolite derived from carbaryl in the silkworm, Bombys muri, Pest. Hiochvm. Physiol. 2, 1 (1972). 8. E. Lewis, C. F. Wilkinson and J. W. Ray, The relationship between microsomal epoxidation and lipid peroxidation in houseflies and pig liver and the inhibit,ory effect of derivatives of 1,3-benzodioxole (methylenedioxybenzene), Biochem. Pharmacol. 16, 1195 (1967). J. W. Ray, The expoxidation of aldrin by housefly microsomes and its inhibition by carbon monoxide, Biochem. Phurmawl. 16,99 (1967). 1~. J. Kuhr. The formation and importance of carbamat,e insecticide metabolites as terminal residues, Pure Appl. Chem. Suppl., 199 (1971). J. A. Harding and 11. C. Uyar. Resistance induced in European corn borers in the laboratory by exposing successive generations to DDT, diazinon, or carbaryl, J. Ewn. Entontot. 63, 250 (1970).