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Residual effects of Bacillus thuringiensis and chemical insecticide treatments on spruce budworm (Choristoneura fumiferana Clemens)
CROP PROTECTION (1983) 2 (2), 225-230
R e s i d u a l effects o f Bacillus thuringiensis and c h e m i c a l i n s e c t i c i d e t r e a t m e n t s on spruce b u d w o r m (Choristoneura fumiferana C l e m e n s ) W. A. SMIRNOFF
Laurentian Forest Research Centre, Canadian Forestry Service, Environment Canada, Sainte Foy, Quebec, Canada ABSTRACT. Biochemical analysis has shown that populations of Choristoneurafumiferana Clemens (Lepidoptera: Tortricidae) which have survived treatment with chemical insecticides (organophosphates and carbamates) have greater energy reserves and vigour than those from untreated areas: conversely, populations surviving treatment with Bacillus thuringiensis (B.t.) are considerably less vigorous. Surviving pupae from untreated, organophosphate-treated and B.t.-treated areas gave, respectively, the following results on biochemical analysis: average weight, 69-5, 82.0 and 46"9 mg; Ca ++, 80.0, 114.5 and 54"0 mg/kg; total proteins, 27.6, 36"3 and 19-2 mg/kg; 3-hydroxybutyrate dehydrogenase, 1004, 1091 and 200 mU/g; alkaline phosphatase, 541,580 and 251 mU/g. These analyses confirmed that B.t. treatments had a detrimental effect on survivors whereas chemical insecticides encouraged the resurgence of vigorous populations of C.fumiferana as a result of stimulation of these insects by sub-lethal dosages (hormoligosis).
Introduction T h e spruce b u d w o r m (Choristoneurafumiferana Clemens) (Lepidoptera: Tortricidae) is the most important defoliator of coniferous forests in north-eastern areas of N o r t h America. Periodic outbreaks of this insect cause major losses of timber over millions o f hectares. At present, chemical insecticides (previously D D T and currently organophosphates and carbamates) are being used to control such outbreaks. Protection of the environment necessitates the development of safe control methods. One such method is biological control by means of the bacterium Bacillus thuringiensis serotype 3a3b (B.t.) which is pathogenic specifically to lepidopterous larvae and has no effect on other insects, plants, animals and man. Furthermore, B.t. has no known noxious effects on water, air or the environment. F o r more than ten years the Insect Pathology Unit of the Laurentian Forest
o261-2194/83/o21o225-o65o3.oo0 1983Butterworth& Co (Publishers)Ltd
226
Residual effects of control treatments on spruce budworm
Research Centre, Quebec (Environment Canada) has been investigating the efficacy of B.t. against spruce budworm. Concentrated formulations of B.t. providing an aerial dispersion of 20 × 109 International Units of B.t. in final liquid volumes of 4-7 and 2.5 f/ha are now available commercially. These formulations also contain traces ofchitinase, an enzyme which hydrolyses the chitin contained in the intestinal walls of larvae of C. fumiferana, thereby increasing the septicaemic potential of B.t. (Smirnoff, 1971; Smirnoff and Val&o, 1972). The results of aerial applications carried out so far have shown that B.t. chitinase complex is necessary for an effective control of C. fumiferana (Smirnoff, Juneau and Val~ro, 1972; Smirnoff, Larsen, Juneau and Val~ro, 1974; Smirnoff, 1976) at least under certain conditions. The potential activity of C. fumiferana populations has been assessed by biochemical analysis of various substances which are present in the pupa and which have key roles in insect metabolism. Such substances include calcium and total proteins which, together with weight, provide a measure of the energy reserves available to the organism: in addition, determination of the activity of various enzymes sheds light on the type of metabolic processes taking place. Such assessments indicate not only whether the overall activity of an insect is increasing or decreasing, but also the possible type, feasibility and impact of various control measures, and the vigour of surviving insect populations. Previous studies have shown that residual populations of C. fumiferana in areas treated with chemical insecticides are more vigorous than those in untreated areas, whereas those from areas treated with B.t. are much less vigorous than either the chemically treated or untreated survivors (Smirnoff et al., 1972; Smirnoff, 1973, 1976, 1978). To determine the source of this higher potential activity of residual populations in areas treated with chemical insecticides, pupae from untreated, chemical-treated and B.t.-treated areas were subjected to biochemical analysis.
Materials and methods The dosage of chemical insecticide used consisted of two annual treatments with 85 g of fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) or of phosphamidon (2-chloro-2-diethylcarbamoyl-l-methylvinyldimethylphosphate) in 1.2 f of oil with a final volume of 2.4 E/ha per year. B.t. was applied in the spring and pupae were collected when they formed at the end of the feeding season. They were kept cold (4°C) and analysed within 3-5 days. Analyses were also carried out on pupae formed by larvae reared in the laboratory and which received non-lethal dosages of fenitrothion. One hundred female and 100 male pupae were analysed each year for each of the treated or untreated groups listed below: 1. 2.
3. .
Pupae from the Province of Quebec, Canada, 1976-81. (a) untreated territories; (b) territories treated with B. thuringiensis. Pupae from New Brunswick, Canada. (a) untreated territories; (b) territories treated with organophosphates (fenitrothion and phosphamidon). Pupae from the Province of Quebec, Canada, 1976-81. (a) untreated territories; (b) territories treated in 1977, 1978, 1979, 1980 with fenitrothion. Pupae formed by larvae reared individually in the laboratory and which received
W. A. SMIRNOFF
5.
227
a non-lethal dosage of fenitrothion, 1 x 10 3 of the formulation mentioned above. The solvent used for preparing dilutions was identical with that used for field application of fenitrothion. The dosages required for each larva were administered by means of a calibrated microsyringe. Pupae from untreated larvae individually reared in the laboratory.
After pupae had been weighed individually they were allocated to groups according to their origin or experimental conditions and were homogenized in batches of five for biochemical analysis. Amounts of calcium and total proteins were determined and the activities of 3-hydroxybutyrate dehydrogenase (1.1.1.30; ~-HBDH), alanine aminotransferase (2.6.1.2.; glutamic-pyruvic transaminase (GPT)) and alkaline phosphatase (3.1.3.1) were assessed using the methods described previously (Smirnoff, 1973) which essentially are modifications of methods proposed for the use of the bichromatic analyser ABA-50 (Abbott Laboratories) and UV spectrophotometer (Boehringer Mannheim Co.). Calcium forms a coloured chromophore with o-cresolphthalein complex ion in alkaline solution and the absorbance is measured against distilled water with a 550/650 nm filter. Total proteins were estimated by biuret reagent. ~ - H B D H catalyses the reduction of ~-ketobutyrate to ~-hydroxybutyrate by N A D H and its activity is determined by the rate of decrease in absorbance at 340 nm. Alkaline phosphatase splits p-nitrophenyl phosphate into organic phosphate and p-nitrophenol and this reaction may be followed by measuring the increase in absorbance at 415 nm. G P T converts L-alanine and ~-ketoglutarate into glutamate and pyruvate, and enzyme activity is measured by the rate of decrease in absorbance of NADH. The reagents recommended by the above-mentioned companies for clinical chemistry analyses were used. R e s u l t s a n d discussion The results of the various analyses, summarized in Tables 1 and 2, showed that residual populations of C. fumiferana surviving in B.t.-treated areas have greatly reduced potential activity compared with that of pupae from untreated areas, as shown by their weights, calcium levels and total protein content as well as by their TABLE1. Biochemical analysis of Choristoneurafumiferana pupae from all the areas treated with Bacillus thuringiensis and organophosphates in 1981 and untreated areas. Two hundred pupae were analysed in each lot
Treatment Untreated Treated with B. thuringiensis Treated with organophosphates
Average weight Ca +- Total proteins ~-HBDH Phosphatase (mg) (mg/kg) (g/kg) (mU/g)t (mU/g)t 69.5
80"0
27"6
1004"0
541"0
46"9
54"0
19"2
200-0
251"0
82-0
114"5
36"3
1091"0
580"0
t 1 mU/g=
1 m# mole of converted substrate minx g
Treatment
Untreated Treated each year with organophosphates Untreated Quebec Treated with organophosphates in 1977, 78, 79, 80 Laboratory Untreated larvae Larvae treated with sub-lethal doses of fenitrothion, 1 × 10-3 normal dose
New Brunswick
Area/Source
32-4+6-7
T Standard deviation
min ×
g
(~
927-0+18.5 536'0+12.8 695"0+12"8 948"0+23"0446.0+20.0426.0+19.0
28"0+3"4 1012.0+32.0 1075.0+21.0 430"0+20"0 398-0+20-0
t 1 m U / g - 1 m/~ mole of converted substrate
94.0+25.0
~
966'5_+20'0 471.6___11.7 416-0_+12.0
~'
GPT (mU/g)t
43"7+6"3 1109-0+20'0 1074"0_+20'0 377"4+6"6 450"4+7.0 26'4+4"9 983"0+19"0 1039'0+22.0 464"0+11"2 527.6+12.0
33"0+6"0 46"0+5.0 1519"0+23-0 36"2+11"5 21.2+5-6 1082"0+40-0
28.9+6.1 20"4+4.8
62"0+14.0 105"0+35"0
93'6+9"8 70"0+12.0
123'0+9.2 90.8+10.2
78.0 + 20.0
2
60"0+7.8 104.0+11.1 60"0+20"0 90"0+15.0
8
90"8 + 9'0 70.0__+20.0~
~
66.2+10.1 106-0+10.0 46"2+7"5 70.1+7.1
(~
98"1 +8"1 60.8 + 10.0
q
~-HBDH (mU/g)t
59-9_+8"7 56"4_+6.8 103"6___8"9 25-8-+5"0 29-4-+5.1 1042"0+20.0
8
Total proteins (g/kg fresh weight)
1979, and 1980 and of pupae from larvae treated with a sub-lethal dose
79'2_+9"0T
~
Ca + + (mg/kg)
C.fumiferana pupae collected in 1978,
Weight (mg)
TABLE 2. Biochemical analysis of surviving of fenitrothion in the laboratory
9
tJ t~
W. A. SMIRNOFF
229
c¢-HBDH and phosphatase activities. The decrease in these levels was greater in female pupae than in males (Table 2). Conversely, the weights of calcium and total proteins of pupae from areas treated with chemical insecticides are higher than those in untreated pupae. The ~-HBDH level is higher (Table 1) and that of G P T is lower (Table 2). These differences are significant when evaluating energy potential because c~-HBDH is of great importance during metamorphosis and adult development (Florkin and Jeuniaux, 1964), whereas G P T is only an intracellular enzyme and an index of cytolysis. Again, these differences were more marked in the female pupae (Table 2). It thus appears that the organophosphate treatments encourage the survival of a vigorous C. fumiferana residual population with a high potential activity (Table 2); in addition, such treatment ensures that more foliage is available for the survivors the following year. Sub-lethal dosages of organophosphates appeared to stimulate the metabolism of larvae. Female pupae formed from larvae treated in the laboratory with non-lethal dosages of fenitrothion weighed more and contained more calcium than untreated pupae (Table 2). This stimulant effect of sub-lethal dosages of fenitrothion corroborated the results obtained by Luckey (1968) who observed that non-lethal dosages of chemical insecticides induced hormoligosis in house crickets (hormoligosis is the stimulation of an organism by means of minute quantities of stressor). In current experiments this phenomenon is being investigated further in C.fumiferana, together with the interaction of sub-lethal dosages of chemical insecticides with different inhibitors and stimulants of the nervous system, including sodium bromide, potassium bromide, atropine, caffeine, chlordiazepoxide and methylphenidate. Preliminary results suggest that inhibitors of the nervous system increase the toxic effect of fenitrothion on C. fumiferana, whereas stimulants reduce the toxicity. Sub-lethal dosages which stimulate C. fumiferana by hormoligosis considerably reduce the toxic action of concentrated solutions of NaBr or KBr. Our investigations therefore confirm that surviving populations of C.fumiferana from areas treated with organophosphate insecticides contain the necessary components of high potential activity and that non-lethal dosages of fenitrothion stimulate the metabolism of this insect by hormoligosis. These results have extremely important implications when assessing the efficiency and validity of organophosphates in control of C. fumiferana. The use of these insecticides is severely restricted because they are toxic inhibitors of cholinesterase enzyme activities. Consequently, for the control of spruce budworm, they have to be used at the lowest dosage possible, two or even three times a year over the same area. The treatments do not always produce a marked reduction in insect populations and hormoligosis can occur in larvae receiving sublethal dosages oforganophosphates. In turn, these larvae form pupae with a higher biochemical potential than the untreated ones. The emergence of very vigorous populations of C.fumiferana could contribute to the formation of permanent spruce budworm epidemics in areas repeatedly treated with chemical insecticides, as suggested by the epidemiological studies conducted by Blais (1974). On the other hand, B. thuringiensis has a remanance effect on the surviving C. fumiferana populations, involving a marked decline in the energy potential which is seen in practice as a reduction in the reproductive ability of the pest population (Smirnoff, 1976). We must demonstrate that biological control with B. thuringiensis, which is safer for the environment than insecticidal sprays, provides an efficient control of C.
230
Residual effects of control treatments on spruce budrporm
f u m f e r a n a . S o m e formulations which have already been developed have been s h o w n to p r o v i d e a d e q u a t e control ( S m i r n o f f e t al., 1972, Smirnoff, 1973, 1976).
References BLAIS,J.R. (1974). The policy of keeping trees alive via spray operations may hasten the recurrence of spruce budworm outbreaks. Forestry Chronicle 50, 19-21. FLORKIN,M. ANDJEUNIAUX,C.H. (1964). In: The Physiology oflnsecta, vol. III, p. 146. (ed. by M. Rockstein). New York: Academic Press. LUClCEY,T.D. (1968). Insecticide horrnoligosis. Journal of Economic Entomology 61, 7-12. SMIRNOFF, W.A. (1971). Effect of chitinase on the action of Bacillus thuringiensis. Canadian Entomologist 103, 1829-1831. SMIRNOFr, W.A. (1973). Biochemical exploration in insect pathology. Current Topics in Comparative Pathobiology 2, 89-106. SMmNOFF,W.A. (1976). In: Proceedings of the First International Colloquium on Invertebrate Pathology, Queen's University at Kingston, Canada, pp. 339-340. SMmNOFF,W.A. (1978). Impact des traitements bact~riologiques et des traitements chimiques sur une ~pid6mie de tordeuses de bourgeons de l'~pinette. In: Rksum~ des communications 4C congr~s de rACFAS, p. 68. SMIRNOFF,W.A. ANDVALI~RO,J.R. (1972). Perturbations m~taboliques chez Choristoneurafumiferana Clemens au cours de l'infection par Bacillus thuringiensis seul ou en presence de chitinase. Revue Canadienne de Biologie 31, 163-169. SMIRNOFF, W.A., JUNEAU,A. AND VALI~RO,J.R. (1972). Results of experimental aerial sprayings of Bacillus thuringiensis against spruce budworm larvae. Bi-Monthly Research Notes, Canadian Department of the Environment, Canadian Forestry Service, Ottawa 28, (1), 1-2. SMIRNOFF,W.A., LARSEN,L.V., JUNEAU,A. ANDVALERO,J.R. (1974). Test with a highly concentrated lowZvolume formulation of Bacillus thuringiensis against spruce budworm. Bi-Monthly Research Notes, Canadian Department of the Environment, Canadian Forestry Service, Ottawa 30, 9. Accepted 20 August 1982
R e s i d u a l effects o f Bacillus thuringiensis and c h e m i c a l i n s e c t i c i d e t r e a t m e n t s on spruce b u d w o r m (Choristoneura fumiferana C l e m e n s ) W. A. SMIRNOFF
Laurentian Forest Research Centre, Canadian Forestry Service, Environment Canada, Sainte Foy, Quebec, Canada ABSTRACT. Biochemical analysis has shown that populations of Choristoneurafumiferana Clemens (Lepidoptera: Tortricidae) which have survived treatment with chemical insecticides (organophosphates and carbamates) have greater energy reserves and vigour than those from untreated areas: conversely, populations surviving treatment with Bacillus thuringiensis (B.t.) are considerably less vigorous. Surviving pupae from untreated, organophosphate-treated and B.t.-treated areas gave, respectively, the following results on biochemical analysis: average weight, 69-5, 82.0 and 46"9 mg; Ca ++, 80.0, 114.5 and 54"0 mg/kg; total proteins, 27.6, 36"3 and 19-2 mg/kg; 3-hydroxybutyrate dehydrogenase, 1004, 1091 and 200 mU/g; alkaline phosphatase, 541,580 and 251 mU/g. These analyses confirmed that B.t. treatments had a detrimental effect on survivors whereas chemical insecticides encouraged the resurgence of vigorous populations of C.fumiferana as a result of stimulation of these insects by sub-lethal dosages (hormoligosis).
Introduction T h e spruce b u d w o r m (Choristoneurafumiferana Clemens) (Lepidoptera: Tortricidae) is the most important defoliator of coniferous forests in north-eastern areas of N o r t h America. Periodic outbreaks of this insect cause major losses of timber over millions o f hectares. At present, chemical insecticides (previously D D T and currently organophosphates and carbamates) are being used to control such outbreaks. Protection of the environment necessitates the development of safe control methods. One such method is biological control by means of the bacterium Bacillus thuringiensis serotype 3a3b (B.t.) which is pathogenic specifically to lepidopterous larvae and has no effect on other insects, plants, animals and man. Furthermore, B.t. has no known noxious effects on water, air or the environment. F o r more than ten years the Insect Pathology Unit of the Laurentian Forest
o261-2194/83/o21o225-o65o3.oo0 1983Butterworth& Co (Publishers)Ltd
226
Residual effects of control treatments on spruce budworm
Research Centre, Quebec (Environment Canada) has been investigating the efficacy of B.t. against spruce budworm. Concentrated formulations of B.t. providing an aerial dispersion of 20 × 109 International Units of B.t. in final liquid volumes of 4-7 and 2.5 f/ha are now available commercially. These formulations also contain traces ofchitinase, an enzyme which hydrolyses the chitin contained in the intestinal walls of larvae of C. fumiferana, thereby increasing the septicaemic potential of B.t. (Smirnoff, 1971; Smirnoff and Val&o, 1972). The results of aerial applications carried out so far have shown that B.t. chitinase complex is necessary for an effective control of C. fumiferana (Smirnoff, Juneau and Val~ro, 1972; Smirnoff, Larsen, Juneau and Val~ro, 1974; Smirnoff, 1976) at least under certain conditions. The potential activity of C. fumiferana populations has been assessed by biochemical analysis of various substances which are present in the pupa and which have key roles in insect metabolism. Such substances include calcium and total proteins which, together with weight, provide a measure of the energy reserves available to the organism: in addition, determination of the activity of various enzymes sheds light on the type of metabolic processes taking place. Such assessments indicate not only whether the overall activity of an insect is increasing or decreasing, but also the possible type, feasibility and impact of various control measures, and the vigour of surviving insect populations. Previous studies have shown that residual populations of C. fumiferana in areas treated with chemical insecticides are more vigorous than those in untreated areas, whereas those from areas treated with B.t. are much less vigorous than either the chemically treated or untreated survivors (Smirnoff et al., 1972; Smirnoff, 1973, 1976, 1978). To determine the source of this higher potential activity of residual populations in areas treated with chemical insecticides, pupae from untreated, chemical-treated and B.t.-treated areas were subjected to biochemical analysis.
Materials and methods The dosage of chemical insecticide used consisted of two annual treatments with 85 g of fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) or of phosphamidon (2-chloro-2-diethylcarbamoyl-l-methylvinyldimethylphosphate) in 1.2 f of oil with a final volume of 2.4 E/ha per year. B.t. was applied in the spring and pupae were collected when they formed at the end of the feeding season. They were kept cold (4°C) and analysed within 3-5 days. Analyses were also carried out on pupae formed by larvae reared in the laboratory and which received non-lethal dosages of fenitrothion. One hundred female and 100 male pupae were analysed each year for each of the treated or untreated groups listed below: 1. 2.
3. .
Pupae from the Province of Quebec, Canada, 1976-81. (a) untreated territories; (b) territories treated with B. thuringiensis. Pupae from New Brunswick, Canada. (a) untreated territories; (b) territories treated with organophosphates (fenitrothion and phosphamidon). Pupae from the Province of Quebec, Canada, 1976-81. (a) untreated territories; (b) territories treated in 1977, 1978, 1979, 1980 with fenitrothion. Pupae formed by larvae reared individually in the laboratory and which received
W. A. SMIRNOFF
5.
227
a non-lethal dosage of fenitrothion, 1 x 10 3 of the formulation mentioned above. The solvent used for preparing dilutions was identical with that used for field application of fenitrothion. The dosages required for each larva were administered by means of a calibrated microsyringe. Pupae from untreated larvae individually reared in the laboratory.
After pupae had been weighed individually they were allocated to groups according to their origin or experimental conditions and were homogenized in batches of five for biochemical analysis. Amounts of calcium and total proteins were determined and the activities of 3-hydroxybutyrate dehydrogenase (1.1.1.30; ~-HBDH), alanine aminotransferase (2.6.1.2.; glutamic-pyruvic transaminase (GPT)) and alkaline phosphatase (3.1.3.1) were assessed using the methods described previously (Smirnoff, 1973) which essentially are modifications of methods proposed for the use of the bichromatic analyser ABA-50 (Abbott Laboratories) and UV spectrophotometer (Boehringer Mannheim Co.). Calcium forms a coloured chromophore with o-cresolphthalein complex ion in alkaline solution and the absorbance is measured against distilled water with a 550/650 nm filter. Total proteins were estimated by biuret reagent. ~ - H B D H catalyses the reduction of ~-ketobutyrate to ~-hydroxybutyrate by N A D H and its activity is determined by the rate of decrease in absorbance at 340 nm. Alkaline phosphatase splits p-nitrophenyl phosphate into organic phosphate and p-nitrophenol and this reaction may be followed by measuring the increase in absorbance at 415 nm. G P T converts L-alanine and ~-ketoglutarate into glutamate and pyruvate, and enzyme activity is measured by the rate of decrease in absorbance of NADH. The reagents recommended by the above-mentioned companies for clinical chemistry analyses were used. R e s u l t s a n d discussion The results of the various analyses, summarized in Tables 1 and 2, showed that residual populations of C. fumiferana surviving in B.t.-treated areas have greatly reduced potential activity compared with that of pupae from untreated areas, as shown by their weights, calcium levels and total protein content as well as by their TABLE1. Biochemical analysis of Choristoneurafumiferana pupae from all the areas treated with Bacillus thuringiensis and organophosphates in 1981 and untreated areas. Two hundred pupae were analysed in each lot
Treatment Untreated Treated with B. thuringiensis Treated with organophosphates
Average weight Ca +- Total proteins ~-HBDH Phosphatase (mg) (mg/kg) (g/kg) (mU/g)t (mU/g)t 69.5
80"0
27"6
1004"0
541"0
46"9
54"0
19"2
200-0
251"0
82-0
114"5
36"3
1091"0
580"0
t 1 mU/g=
1 m# mole of converted substrate minx g
Treatment
Untreated Treated each year with organophosphates Untreated Quebec Treated with organophosphates in 1977, 78, 79, 80 Laboratory Untreated larvae Larvae treated with sub-lethal doses of fenitrothion, 1 × 10-3 normal dose
New Brunswick
Area/Source
32-4+6-7
T Standard deviation
min ×
g
(~
927-0+18.5 536'0+12.8 695"0+12"8 948"0+23"0446.0+20.0426.0+19.0
28"0+3"4 1012.0+32.0 1075.0+21.0 430"0+20"0 398-0+20-0
t 1 m U / g - 1 m/~ mole of converted substrate
94.0+25.0
~
966'5_+20'0 471.6___11.7 416-0_+12.0
~'
GPT (mU/g)t
43"7+6"3 1109-0+20'0 1074"0_+20'0 377"4+6"6 450"4+7.0 26'4+4"9 983"0+19"0 1039'0+22.0 464"0+11"2 527.6+12.0
33"0+6"0 46"0+5.0 1519"0+23-0 36"2+11"5 21.2+5-6 1082"0+40-0
28.9+6.1 20"4+4.8
62"0+14.0 105"0+35"0
93'6+9"8 70"0+12.0
123'0+9.2 90.8+10.2
78.0 + 20.0
2
60"0+7.8 104.0+11.1 60"0+20"0 90"0+15.0
8
90"8 + 9'0 70.0__+20.0~
~
66.2+10.1 106-0+10.0 46"2+7"5 70.1+7.1
(~
98"1 +8"1 60.8 + 10.0
q
~-HBDH (mU/g)t
59-9_+8"7 56"4_+6.8 103"6___8"9 25-8-+5"0 29-4-+5.1 1042"0+20.0
8
Total proteins (g/kg fresh weight)
1979, and 1980 and of pupae from larvae treated with a sub-lethal dose
79'2_+9"0T
~
Ca + + (mg/kg)
C.fumiferana pupae collected in 1978,
Weight (mg)
TABLE 2. Biochemical analysis of surviving of fenitrothion in the laboratory
9
tJ t~
W. A. SMIRNOFF
229
c¢-HBDH and phosphatase activities. The decrease in these levels was greater in female pupae than in males (Table 2). Conversely, the weights of calcium and total proteins of pupae from areas treated with chemical insecticides are higher than those in untreated pupae. The ~-HBDH level is higher (Table 1) and that of G P T is lower (Table 2). These differences are significant when evaluating energy potential because c~-HBDH is of great importance during metamorphosis and adult development (Florkin and Jeuniaux, 1964), whereas G P T is only an intracellular enzyme and an index of cytolysis. Again, these differences were more marked in the female pupae (Table 2). It thus appears that the organophosphate treatments encourage the survival of a vigorous C. fumiferana residual population with a high potential activity (Table 2); in addition, such treatment ensures that more foliage is available for the survivors the following year. Sub-lethal dosages of organophosphates appeared to stimulate the metabolism of larvae. Female pupae formed from larvae treated in the laboratory with non-lethal dosages of fenitrothion weighed more and contained more calcium than untreated pupae (Table 2). This stimulant effect of sub-lethal dosages of fenitrothion corroborated the results obtained by Luckey (1968) who observed that non-lethal dosages of chemical insecticides induced hormoligosis in house crickets (hormoligosis is the stimulation of an organism by means of minute quantities of stressor). In current experiments this phenomenon is being investigated further in C.fumiferana, together with the interaction of sub-lethal dosages of chemical insecticides with different inhibitors and stimulants of the nervous system, including sodium bromide, potassium bromide, atropine, caffeine, chlordiazepoxide and methylphenidate. Preliminary results suggest that inhibitors of the nervous system increase the toxic effect of fenitrothion on C. fumiferana, whereas stimulants reduce the toxicity. Sub-lethal dosages which stimulate C. fumiferana by hormoligosis considerably reduce the toxic action of concentrated solutions of NaBr or KBr. Our investigations therefore confirm that surviving populations of C.fumiferana from areas treated with organophosphate insecticides contain the necessary components of high potential activity and that non-lethal dosages of fenitrothion stimulate the metabolism of this insect by hormoligosis. These results have extremely important implications when assessing the efficiency and validity of organophosphates in control of C. fumiferana. The use of these insecticides is severely restricted because they are toxic inhibitors of cholinesterase enzyme activities. Consequently, for the control of spruce budworm, they have to be used at the lowest dosage possible, two or even three times a year over the same area. The treatments do not always produce a marked reduction in insect populations and hormoligosis can occur in larvae receiving sublethal dosages oforganophosphates. In turn, these larvae form pupae with a higher biochemical potential than the untreated ones. The emergence of very vigorous populations of C.fumiferana could contribute to the formation of permanent spruce budworm epidemics in areas repeatedly treated with chemical insecticides, as suggested by the epidemiological studies conducted by Blais (1974). On the other hand, B. thuringiensis has a remanance effect on the surviving C. fumiferana populations, involving a marked decline in the energy potential which is seen in practice as a reduction in the reproductive ability of the pest population (Smirnoff, 1976). We must demonstrate that biological control with B. thuringiensis, which is safer for the environment than insecticidal sprays, provides an efficient control of C.
230
Residual effects of control treatments on spruce budrporm
f u m f e r a n a . S o m e formulations which have already been developed have been s h o w n to p r o v i d e a d e q u a t e control ( S m i r n o f f e t al., 1972, Smirnoff, 1973, 1976).
References BLAIS,J.R. (1974). The policy of keeping trees alive via spray operations may hasten the recurrence of spruce budworm outbreaks. Forestry Chronicle 50, 19-21. FLORKIN,M. ANDJEUNIAUX,C.H. (1964). In: The Physiology oflnsecta, vol. III, p. 146. (ed. by M. Rockstein). New York: Academic Press. LUClCEY,T.D. (1968). Insecticide horrnoligosis. Journal of Economic Entomology 61, 7-12. SMIRNOFF, W.A. (1971). Effect of chitinase on the action of Bacillus thuringiensis. Canadian Entomologist 103, 1829-1831. SMIRNOFr, W.A. (1973). Biochemical exploration in insect pathology. Current Topics in Comparative Pathobiology 2, 89-106. SMmNOFF,W.A. (1976). In: Proceedings of the First International Colloquium on Invertebrate Pathology, Queen's University at Kingston, Canada, pp. 339-340. SMmNOFF,W.A. (1978). Impact des traitements bact~riologiques et des traitements chimiques sur une ~pid6mie de tordeuses de bourgeons de l'~pinette. In: Rksum~ des communications 4C congr~s de rACFAS, p. 68. SMIRNOFF,W.A. ANDVALI~RO,J.R. (1972). Perturbations m~taboliques chez Choristoneurafumiferana Clemens au cours de l'infection par Bacillus thuringiensis seul ou en presence de chitinase. Revue Canadienne de Biologie 31, 163-169. SMIRNOFF, W.A., JUNEAU,A. AND VALI~RO,J.R. (1972). Results of experimental aerial sprayings of Bacillus thuringiensis against spruce budworm larvae. Bi-Monthly Research Notes, Canadian Department of the Environment, Canadian Forestry Service, Ottawa 28, (1), 1-2. SMIRNOFF,W.A., LARSEN,L.V., JUNEAU,A. ANDVALERO,J.R. (1974). Test with a highly concentrated lowZvolume formulation of Bacillus thuringiensis against spruce budworm. Bi-Monthly Research Notes, Canadian Department of the Environment, Canadian Forestry Service, Ottawa 30, 9. Accepted 20 August 1982