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Abstract The purpose of our study was to further investigate the immediate and delayed toxicity of abamectin (Avermectin B1) and thuringiensin (13- exotoxin of Bacillus thuringiensis), representing novel classes of unconventional insecticides, on :both laboratory and field strains of cotton leafworm, Spodoptera littoralis (Boisd.), as an aid to the field use of such compounds in pest management programmes. This work included fourth parts summarized as follows: Part I ”Biological Activity of Abamectin and Thuringiensin against Cotton Lcafworm, Spodoptera littoralis (Bois d.}” , In this part we evaluated abamectin and thuringiensin as well as diflubenzuron to determine their biological activity against laboratory and field strains of S. littoralis following larval feeding on leaves treated with sublethal concencentrations of such compounds. Fourth-instar larvae were fed, for 72h, castor bean leaves treated with sublethal concentrations, causing ca. 20-60% mortality 3 days after treatment, of each compound tested. The survivors were allowed to complete their development until pupation. The delayed effects of these chemicals on pupation, emergence. and fecundity were determined for both strains tested . ._--’- - -- - _.- - 1.19 The results obtained from this work can be summarized as follows : 1- The larval mortality following 72h-feeding on leaves treated with sublethal concentrations of test compounds reached 20-57% by day 3 after treatment. It seems that abamectin and thuringiensin were similar to diflubenzuron indicating slow acting toxicity at which the latent toxicity was consistent with a disruption of the molting process from fourth to fifth ins tar. Thuringienzin appeared to be more effective than other compounds, at tested concentrations. 2- Percent pupation reported for surviving larvae, following feeding on treatd leaves, demonstrated that all sublethal concentrations of any tested compounds significantly decreased pupation percentages compared to those reported for the untreated control. For example, in the field strain, pupation percents reported at concentration of 0.1 ppm were 9.5, 16.7, and 12.9%, for abamectin, thuringiensin, and diflubenzuron, respectively, compared to those reported for the untreated control (- 70.8 %). 3- The mean weight of pupae recorded at test concentrations, III susceptible strain, was generally not significantly different than that in the control treatment. However, the mean weight of pupae recorded in case of field strain was clearly decreased in all insecticide treatments, especially abamectin and thuringiensin, compared to those of the untreated control. For example, the mean weights reported, at concentration of 0.001 ppm were 183.3, 177.9,and 209.7 mg for treatments of abamectin, thuringiensin, and diflubenzuron , 1 20 respectively, compared to that of control (.....231.7 mg). It seems that reduction in pupal weight was proportional to concentration. 4- All insecticide treatments resulted in decreasing adult emergence than that of the control. In general, thuringiensin treatments, at any of the tested concentrations exhibited clearly decreasing in adult emergence, either in susceptible or field strain, compared to the other compounds. For example, in the field strain, emergence percentages recorded at concentration of 0.001 ppm were 83.3, 53.3, and 77.5%, for treatments of abamectin, thuringiensin, and diflubenzuron, respectively. Reduction in adult emergence seems to be proportional to concentration. 5- It was obvious, also, that all compounds tested, especially thuringiensin, caused clearly reduction in the fecundity of moths developing from larvae fed treated leaves compared to those developing from larvae fed untreated leaves. The reduction in fecundity of moths in the field strain was much more happened in the susceptible strain. For example, in the field strain, the mean numbers of eggs deposited, at 0.01 ppm, were 520, 202, and 362/0 , for treatments of abamectin, thuringiensin, and diflubenzuron, respectively, compared to that of the control(l840 eggs/ 0 ). It seems also that reduction in fecundity was proportional to concentration. PART II Histopathological Effects of Abamectin and Thuringiensin on The Cotton Leafworm, Spodoptera littoralis ----- - - ---- -- -_.----- 121 histopathological changes in the midgut of cotton leafworm, S. littoralis, larvae resulted from feeding on abamectin-, thuringiensin-, or diflubenzuron- treated leaves; in an attempt to understand the nature and significance of such changes. The results obtained in this study can be summarized as follows: 1. The midgut of a normal 4lh.-instar larva consists of a single cellular layer resting upon a basement membrane which is surrounded externally first by circular then longitudinal muscles. The cellular layer, the epithelium, consists of columnar-cells, each with a large granular nucleus, which are interspersed with cluster of small regenerative cells, also resting upon the basement membrane. The lumen is surrounded by a peri trophic membrane. 2. Feeding of 4th-instar larvae on abamectin-treated leaves (0.01 ppm), for 72h, ’caused an obvious cytological changes in the midgut. The epithelial cells showed marked disintegration and disorganization. A few scattered groups of the epithelial cells were separated from their inner ends, and were sloughed into’ the lumen of the gut. The cell walls appeared to be indistinct and destroyed in some instances. The histopathological symptoms caused in the midgut of larvae fed leaves treated with the lower concentration tested (0.001 ppm) of abamectin were somewhat less than those caused by the higher concentration used. However, the epithelial cells showed marked degeneration, the cell walls were indistinct, and the cells appeared to be elongated. 3. Strong histopathological symptoms were clearly shown in the midgut of larvae fed leaves treated with thuringiensin, at 0.0 1 ppm. The --~_.------ 1 22 epi thelial cells of the midgut in most areas showed marked generation. Cell boundaries disappeared, and scattered groups of the epithelial cells were sloughed off into the lumen. Also, the basement membrane and surrounding muscles detached from the epithelium in some areas. It was evident that the lower concentration tested, 0.00 1 ppm, had also some histological changes in the midgut. Where, the epithelial cells showed marked degeneration, and elongated throughout the whole section. There was fading of cellular boundaries. 4. The histopathological symptoms in the midgut of larvae after feeding on diflubenzuron-treated leaves, at 0.01 ppm, were less pronounced than those caused by either abamectin or thuringiensin. The epithelial cells of the midgut, in some areas, were disorganized and disintegrated. There was elongation of epithelial cells and fading of their boundaries. In addition. a few scattered groups of epithelial cells were sloughed off into the lumen or III the space between the epithelium and the peritrophic membrane. Vacuolations were observed in the epithelium in some areas, and also the basement membrane and surrounding muscle layer, in some regions. showed marke degeneration. The degeneration of midgut epithelium caused by the lower concentration tested, 0.001 ppm, of diflubenzuron was unlike that caused by concentration of 0.01 ppm, in that the cells did not slough off, as well as the basement membrane and surrounding muscle layer unaffected. -~ -_ .. -----.- -~----_._---- 123 that caused by concentration of 0.01 ppm, in that the cells did not slough off, as well as the basement membrane and surrounding muscle layer unaffected. PART III nBiochemical Effects of Abamectin and Thuringiensin on The Cotton Leafworm, Spodoptera Littoralis” - In this part of our present study an attempt was made to elucidate the possibility of biochemical effects of abamectin and thuringiensin on the activity of digestive enzymes and also of non-specific esterases in S. _littoralis larvae. The fifth-instar larvae were fed, for only 1 day, on leaves treated with sublethal concentrations, ranged from 5 to 200 ppm, through three successive generations. Larvae, then, were allowed to feed untreated leaves for 2 successive days. The activity of both digestive enzymes, i.e. invertase, amylase, and trehalase, and non-specific esterases, i.e. a-esterase and I}-esterase.was determined during the 3-day feeding period, compared to that of control where larvae were fed on untreated leaves. The results of our present study can be summarized as follows: 1. It was evident that feeding of larvae on abamectin-treated leaves, at 5 ppm, through F1-generation resulted in a suppression of the activity ---- ---- --- - - --- --------- 124 On the other hand, the weight of larvae fed on treated leaves decreased continuously; this decrease, in most cases, seemed to be directly proportional to the lower enzymatic activity. where the larval weights for the control larvae were gradually increased reaching the maximum on the third day. 2. At F3-generation, where larvae fed on abamectin-treated leaves, at 200 _ppm, the inhibitory effects were reported for only trehalase activity which decreased by 60% than that of control, at I-day period after trearment. Moreover, amylase activity was reduced by 21 and 46%, at 2-and 3-day periods, respectively, compared to that of control.The larval weights showed a slight decrease during 3-day feeding period compared to that of control. 3. Similar inhibitory effects of thuringiensin on larval digestive enzymes were also obtained, at Fl-generaltion, where larvae were fed on thuringiensin-treated leaves, at 5 ppm. Larval amylase activity was reduced by 63, 40 and 77%, at 1-, 2-, and 3-day period, respectively, compared to that of control. Invertase activity was also decreased by 54 and 76%, at 2- and 3-period. However, the trehalase activity was less sensitive than amylase and invertase. The decrease occurred in the weights of larvae fed treated leaves was generally proportional to the lowered enzymatic activity. 4. At F3-generation, where larvae fed thuringiensin-treated leaves, at 200 ppm, the inhibitory effects of thuringiensin on the activity of ._--- - ----_._-- ----------------- 12’5 digestive enzyme was clearly shown at 2- and 3-period days. The trehalase activity was affected to a lesser extent. 5. The acnvity of both a-esterase and (3-esterase in larvae fed on abamectin-treated leaves, at 5 ppm, through Pj-generation, was reduced by 28 and 30%, respectively, than that of control, at l-day period after treatment. The reduction caused in enzyme activities was also reported at 3-day period, though larvae were allowed to feed on untreated leaves. Similar inhibitory effects on enzyme activities were also observed when larvae were fed leaves treated with 200 ppm, through. the F3-generation. However, the reduction caused in ((- esterase or B-esterase activity, at 1-day period, was much more than that occurred in larvae fed on leaves treated with 5 ppm, indicating only 42 and 34%, respectively, of that of control. 6. The inhbitory effects of thuringiensin on the activity of both cesterase and B-esterase was relatively less than that caused by abamectin. For example, the activity of a-esterase and B-estcrase in larvae fed on treated leaves, at 5 ppm, uhrough the FI -generation, was decreased by only 19 and 20%, respectively than that of control. Similar inhibitory effects were observed at 3-day period. However, the inhibitory effects of thuringiensin on such enzyme activities were more affected, when larvae were allowed to feed leaves treated with the higher concentration of 200 ppm~ through the F3 -generation. For example, at l-day period, the activity of a-esterase and b-esterase was decreased by 38 and 41%, respectively, than that of control. - ---- _. - --. _.--- 126 PART IV If Joint Action of Abamectin or Thuringiensin with : Organophosphorus Carbamate, and Pyrethroid Insecticides on Spodoptera littoralis (Boisd.) Larvae” In t.his part, we evaluated the joint action of binary mixtures of either abamectin (Avermectin Bj ) or thuringiensin (B-exotoxin of Bacillus thuringiensis) with certain insecticides represented the main chemical groups of pesticides, i.e. organophosphates, carbamates, and synthetic pyrethroids, on laboratory-reared larvae of the cotton Ieafworm, Spodoptera littoralis (Boisd.), to determine whether the binary mixtures exhibited synergistic activity against this insect. The influence of different insecticide : abamectin (or thuringiensin) ratios on t.he synergistic activity and toxicity of the mixtures was examined. Mixing ratios (insecticide: abamectin [or thuringiensinl) tested were 99:1, 19:1,9:1,4:1,1.5:1, and 1:1, respectively. These ratios were used to produce a series of binary mixtures (insecticide + abamectin [or thuringiensinl) that contained 99 + 1%, 95 + 5%, 90 +10%, 80 + 20%, 60 + 40%, 50 + 50% respectively. Dosage-mortality data were obtained for all mixtures, where the method of Sun & Johnson (1960) was used to test for synergism and to compute the joint toxicity of the binary mixtures. The joint toxicity coefficients of the binary mixtures tested were calculated on basis of LCso values, at 48 and 72h after treatment, against fourth-instar S. littoralis larvae. The results obtained in this study can be summarized as follows: .- -- ._. -- .- -- -- ’-.- ----- _. -~ --- - ----_.--_._------ 127 reported that, with the exception of chlorpyrifos + abamectin mixture, at ratio of 1:1, all other mixing ratios tested exhibited clearly antagonistic action. Data also reported, at 72h after treatment, for the profenfos + abamectin mixtures, indicated that most mixing ratios tested exhibited synergistic activity, especially ratios of 99:1,1:1, and 9:1 indicating CC with ca. 468, 227, and 181, respectively. The mixtures of pirimi phos-ethyl+abamecti n, especially those combined at ratio of 99: I, were 5A-fold more toxic to the larvae, at 48h after treatment, than insecticides alone; whereas most other mixing ratios resulted in antagonistic action. Low synergistic activity was obtained by the mixtures of acephate + abamectin, at.ratios of 4: 1 and 1.5: 1, indicating CC=111.9 and 140.9, respectively, however, antagonistic action was reported by the other tested ratios. The mixtures of methamidophos + abamectin exhibited synergistic activity, at 72h after treatment, at ratios of 9:1, 19:1,99:1, and 1:1, indicating CC= 310.3, 292 , 9, 179,1, and 141,3, respectively. 2. Data concerned the joint action and toxicity of carbamate insecticides, methomyl and thiodicarb, with abamectin showed that all tested mixtures tested of methomyl + abamectin,· at 72h after treatment,· resulted in antagonistic activity compared to the insecticides alone. Howvere, the toxicity data, at 72h, indicated that all mixtures of thiodicarb + abamectin exhibited low synergistic activity indicating CC ranged from 118.6 (at ratio of 1:1) to 150.4 (at ratio of 9: 1). 3. The results of joint action and toxicity of the mixtures of pyrethroid insecticides plus abamectin indicated that all mixtures of ------------------------------------------------ 128 3. The results of joint action and toxicity of the mixtures of pyrethroid insecticides plus abamectin indicated that all mixtures of deltamethrin+abamectin exhibited synergistic activity, indicatig CC ranged from 109.8 (at ratio of insecticides alone. However, strong antagonsitic action was reported by all mixtures of fenpropathrin + abarnectin. The data demonstrated also that all mixtures of fenvalerate + abamectin, with the execption of ratio of 1:1, exhibited low synergistic activity indicating CC ranged from 106.1 (at ratio of 9:1) to 166.2 (at ratio or 1.5:1). Similar results were obtained when cypermethrin was combined with abamectin at tested ratios. 4. Concern for the joint action and toxicity of the mixtures of . organophosphorus insecticides plus· thuringiensin, our results demonstrated that the chlorpyrifos + thuringiensin mixtures, at ratios of 99:1 , 19:1 , 4:1 , and 1.5:1 , were 1.3-, 1.4- , 1.5-, and 1.5- fold. respetively, more toxic to S. littoralis larvae, at 48h after treatment, than insecticides alone. Similar results were shown by the mixtures of profenofos + thuringiensin, in which mixing . ratios of 99: 1, 9: 1, and 19: 1 exhibited synergistic activity indicating cotoxicity coefficients (CC) by ca. 170, 170, and 110. Synergistic activity reported for such mixtures, especially at ratios of 99: 1 and 9: I, was increased, at 7211 after treatment. Also, all tested mixtures of pirimiphos-ethy1 + thuringiensin showed synergistic activity, especially those combined at ratios of 99:1, 4:1, and 1:1 indicating CC = 475.4, 465.4, and 245.8, respectively , at 48h after treatment. _.- .-- --- --- ._- ..~------ 129 Moreover, all mixtures of methamidophos + thuringiensin exhibited clearly synergistic activity, especially those combined at ratios of 19:1, 4:1, and 99:1 indicating CC = 769.1, 220.0, and 195.9 , respectively at 48h after treatment. However, all mixtures of accephate + thuringiensin cleraly showed antagonistic activity (CC < 100), compared to insecticides alone. 5. Regarding the joint action between carbamate insecticides used, i.e. methomyl and thiodicarb, and thuringiensin, our data showed that methomyl + thuringiensin mixtures combined at ratios of 9: 1 and 4:1 exhibited synergistic activity ,at 48h after treatment, indicating CC= 142.6 and 196.1, respectively. However, antagonistic action was shown when methomyl was combined with thuringiensin at ratios of 1:1.5 and 1:1. However, strong antagonism was shown when thiodicarb was combined with thuringiensin at most mixing ratios tested, whereas ratios of 9: 1 and 4: 1 resulted in a reasonable synergism, compared to insecticides alone. 6. Concern to the joint action and toxicity of the mixtures of pyrethroid insecticides plus thuringiensin, the data of cotoxicity coefficients (CC) revealed that slight synergistic activity was reported for mixtures of deltamethrin + thuringiensin, especially those combined at ratios of 99: 1, 9: 1, and 4:1, indicating CC = 174.2, 136.3, and 109.9, respectively. All mixtures of cypermethrin + thuringiensin combined at tested ratios, with the exception of ratio of 1.5:1, exhibited synergistic activity with coefficients ranged from 125.2 (at ratio of 1:1) to 253.1 (at ratio of 19:1). Similar results were reported for the ~._- - - - --- ---- .. _.- ..--_ ....------- 130 mixtures, of fenvalreate + thuringiensin, at which any of the tested mixing ratios were usually more toxic to the larvae than insecticides alone. the cotoxicity coefficients reported ranged from 111.0 (at ratio of 99:1) to 126.7 (at ratio of 4:1). However, antagonistic action was reported for all tested mixtures of fenpropathrin + thuringiensin indicating coefficients < 100. 7. The data also indicated , generally, that mean weight of larval body estimated at LCso, 48h after treatment. for most tested mixtures was decreased clearly than that for control larvae. In general it seems that most mixtures of tested organophosphorus insecticides with thuringiensin exhibited greater synergistic activity than mixtures of such insecticides with abamectin. However, the synergistic activity and toxicity of the mixtures of tested synthetic pyrethroids with abamectin were markedly greater than the synergistic activity and toxicity of the mixtures of tested organophosphorus insecticides with abamectin. |