Heliothis punctigera Wallengren and Heliothis armigera Hübner are important pests of cotton and of other economic crops in Australia. The development of resistance to DDT in H. armigera together with the hazard posed to the environment by a number of insecticides, indicates a need for replacement, non-persistent insecticides for use in pest management programmes. Juvenile hormone analogues "Third Generation Pesticides" of Professor Carroll Williams - represent an entirely new class of compounds with a unique mode of action.
This thesis reports work to evaluate the activity of three juvenile hormone analogues (JHAs) of differing chemical structure - hydroprene (ethyl-3,7,ll-trimethyl-2,4-dodecadienoate), RO 20-3600 (6,7-epoxy-3,7-dimethyl-l-3,4-(methylene dioxy) phenoxy-2-nonene), and R 20458 (l (4-ethyl-phenoxy)-6,7-epoxy-3,7-dimethyl-2-octene) - to both H. punctigera and resistant and susceptible strains of H. armigera. The colony of H. punctigera was established from one maintained by the Queensland Department of Primary Industries. A presumed susceptible (SM) and DDT-resistant strain (RM) of H. armigera was established from material collected on tobacco at Mareeba, north Queensland, while a further DDT-resistant strain (RN) was obtained from cotton at Narrabri, N.S.W.
Sixth instar larvae and pupae of H. punctigera were used to assay the effects of the 3 JHAs, The sensitive period of 6th instar larvae, assessed with RO 20-3600, was maximal during the first 54-56 h after ecdysis after which it declined rapidly. The sensitive period of pupae, assessed with hydroprene, was maximal during the first 18-22 h after pupation and then gradually declined. After 73-77 h virtually no effect was obtained. On the basis of these results, all the JHAs were applied to 6th instar larvae and pupae when 4-12 h old.
Overall when tested on 6th instar larvae and pupae, RO 20-3600 was more effective than hydroprene which was more effective than R 20458 with both the quantal and graded scoring procedures. Pupae were more sensitive than 6th instar larvae. When larvae were treated, emergence of normal adults from normal pupae which formed was inversely related to the dose applied; as the dose was increased the percent emergence of normal adults decreased.
No clear relationship between either species or DDT susceptibility and the activity of the three JHAs was evident. With RO 20-3600 H. armigera SM larvae were more susceptible than H. punctigera but the reverse was true with hydroprene. Overall both the DDT-resistant strains (RM and RN) of H. armigera were less susceptible to RO 20-3600 and hydroprene i.e. these strains exhibited cross-resistance. R 20458 was relatively inactive.
The effect of applying of piperonyl butoxide (PB) with the JHA was variable. In general with larvae PB increased the toxicity of hydroprene. It decreased the toxicity of RO 20-3600 with H. punctigera and H. armigera SM and slightly increased toxicity with the RM and RN strains of H. armigera. PB produced variable and minor changes in the toxicity of R 20458 except with H. armigera RN where toxicity was considerably decreased. With pupae PB increased the toxicity of the three JHAs with all strains.
The metabolism of hydroprene was studied and an extraction procedure was developed which was simple, virtually eliminated lipid interference with thin-layer chromatography, and which gave high percent recovery of the applied 14C-hydroprene (80.42 percent). The metabolites of hydroprene were separated on GF 254 silica gel thin-layer plates and a developing solvent system of benzene:ethyl acetate: acetic acid (110:10:1) Autoradiography, followed by liquid scintillation counting of active regions of the TLC plate were used to identify and quantify metabolites.
There were only minor differences between larvae of H. punctigera and of three strains of H. armigera in the absorption of 14C-hydroprene. In all cases absorption was rapid, and a similar situation was found with pupae. H. armigera RN larvae excreted considerably more radioactivity than the other H. armigera strains and H. punctigera in which excreted radioactivity after 48 h was relatively low.
Absorbed 14C-hydroprene was almost completely degraded by larvae within 48 h to eight distinguishable metabolites (hydroprene acid, unknowns A-G) and a large quantity of polar origin material. In larvae the major metabolic products were hydroprene acid, unknowns A-D and polar origin material. In pupae degradation of 14C-hydroprene was less extensive than in larvae, with 48-66 percent of the absorbed dose being metabolised. The major metabolites were unknowns A-C and polar origin material; the latter constituting 53-79 percent of the total metabolites. Polar origin material was appreciable in H. armigera RM. After enzymatic cleavage of the polar origin material, 86 percent of the activity was extractible with ether and when re-chromatographed, primary metabolites A-F were detected with metabolites A, B and C being most abundant, but 42 percent of the activity still resided at the origin. Most of the metabolites found in the internal fraction were also detected in the external and excretion fractions.
When PB was applied to 6th instar Heliothis larvae together with 14C-hydroprene, there was a reduction of the polar origin material and unknowns C and D in the internal fraction after 48 h. After 24 h there was a considerable reduction in unknown A. Metabolites C and D might represent the epoxide and epoxy acid.
Larvae of H. punctigera had much higher amounts of unknown C in the internal fraction, and considerably more unchanged hydroprene and hydroprene acid than did the strains of H. armigera from Mareeba (even though these were of differing DDT susceptibility) and had an increased amount of polar origin material in the internal fraction. While toxicologically the strains of Heliothis varied in susceptibility to hydroprene, no clear correlation between the metabolic data and the toxicological data could be discerned. One correlation appeared meaningful with the pupal data - the RM strain of H. armigera was the least susceptible, and degradation of hydroprene was greatest in that strain. While PB greatly increased the activity of hydroprene with H. armigera SM it had virtually no effect on H. punctigera. Subsequently the effect of PB on the metabolism of hydroprene was investigated in these two species. In both, absorption was reduced and excretion was increased 24 h post-treatment. In the internal fraction, while PB also reduced the metabolism of hydroprene, the degree of reduction was greater in H. armigera SM.
The DDT susceptibility status of the test strains was established, LD50 data (0.263µg/larva) for H. punctigera corresponded to that reported in the literature and showed that the colony of this species was susceptible to DDT, The 'susceptible' strain of H. armigera from Mareeba, QLD had a low level of resistance, shown by both a slightly elevated LD50 (3.89µg/larva) and an ld-p line of low slope. The two resistant strains were highly resistant; the LD50 values were greater than 100µg/larva and the slopes of the ld-p lines were very low. Since it has not been reported previously, the metabolism of DDT in these species was investigated.
Absorption of DDT in H. punctigera was much greater than in the susceptible H. armigera SM strain at 12 h and this differential was maintained up to 48 h post treatment. While there was no difference in DDT absorption between the H. armigera SM and H. armigera RM strains, absorption was less in the H. armigera RN strain.
Extraction clean-up, and analyses of 14C-DDT metabolites were the same as for hydroprene, and recovery of radioactivity averaged 86.43 percent. Metabolism of DDT was similar in both H. punctigera and the H. armigera strains with metabolites DDE, DDA, DDD and dicofol being identified in addition to 5 unknown metabolites and polar origin material. DDE was the major metabolite in both species comprising approximately 60 percent of the total metabolic products. While the metabolism of DDT in H. armigera and H. punctigera was qualitatively similar, it was quantitatively different. Degradation to polar compounds was more extensive in resistant strains of H. armigera than in the susceptible H. armigera strain and H. punctigera, and increased with increasing post-treatment period in all strains. Both the susceptible and resistant strains of H. armigera had a greater dehydrochlorinating ability than did H. punctigera. Most of the metabolites found in the internal fraction were also detected in the external and excretion fractions.
Piperonyl butoxide applied with DDT had no effect on the toxicity of DOT to H. armigera SM but reduced the toxicity of DDT to H. armigera RM. When assessed 12 h after treatment absorption of DDT was little affected by PB but excretion was enhanced, particularly in the RM strain, DDE production was reduced in both strains, but the DDE/DDT ratio in internal extract was higher in the RM strain than in the SM strain. Absorption and excretion of DDT in the presence and absence of PB were investigated further sequentially over a 12 h period to obtain data on the rate of penetration and absorption, PB increased penetration in H. armigera RM and increased both penetration and excretion in H. armigera RM2.
The effect of PB and 3 other synergists (sulfoxide, tropital, SKP) on feeding and excretion of 6th instar H. punctigera larvae was investigated. All synergists decreased feeding, particularly in the 24 h following treatment, and rate of excretion increased during the 48 h following treatment. This action of synergists does not appear to have been recorded previously.