Some physiological aspects of the insecticidal action of diflubenzuron, an inhibitor of chitin synthesis

Diflubenzuron is the common name for 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea, the active ingredient of the insecticide Dimilin.Diflubenzuron was discovered in 1971 as a larvicide. Evidence was provided by several authors that the larvicidal effect of this compound was caused by its interference with chitin synthesis. Chitin is one of the main components of the insect cuticle. As a result, treatment of larvae with diflubenzuron prevents normal growth of the cuticle in many cases. In most susceptible insect species this effect expresses itself as an incapacity of larvae to moult.During the development of diflubenzuron it was discovered that in some species the compound additionally showed an interesting ovicidal activity, caused either by treatment of females (papers I and II), or by direct contact with eggs (paper II).In paper I is shown that in the housefly (Musca domestica) diflubenzuron can evoke ovicidal effects by topical application to adult females, by oral uptake or by injection of the compound. It is remarkable, however, that in all cases of ovicidal activity the embryo inside the egg appears to develop normally. However, the apparently full-grown embryo turned out to be unable to hatch.Treatment with diflubenzuron did not influence the fecundity of the females. The compound also had no effect on fertilization of the eggs.In order to study the role of male insects in the ovicidal phenomenon, adult males of the housefly (paper I) or of the Colorado potato beetle, Leptinotarsa decemlineata (paper II) were injected with 5 μg of diflubenzuron before they were allowed to mate with untreated females. In this type of experiment no effect on fertilization or on egg mortality was observed. It was therefore concluded that diflubenzuron does not exert its ovicidal activity through the males of these two insect species.Based upon the above mentioned experiments it was concluded that the activity of diflubenzuron on eggs of insects could best be described as an ovicidal effect of the compound rather than a type of chemosterilization.To finally prove that chemosterilization plays no role whatsoever in the mode of action of diflubenzuron, embryos from eggs laid by treated females of the Colorado potato beetle were examined under the electron microscope (paper III, figures 3 and 4). It was found that instead of the normal lamellate cuticle deposition patterns that could be observed in untreated embryos, the diflubenzuron treatment resulted in an amorphous cuticle. Again, these observations suggest an identical mode of action of diflubenzuron on larvae and embryos. As a result, by virtue of a lack of rigidity in the embryonic cuticle, treated embryos cannot use their muscles in the process of egg hatching.We also found that after termination of adult treatment with diflubenzuron, the percentage of egg eclosion again increases. The degree of this reversibility depends a.o. on the concentration of diflubenzuron during the previous treatment (paper I).Many factors appeared to influence the ovicidal activity of diflubenzuron in the case of direct contact activity on susceptible eggs. From the literature there was already evidence that the particle size of the active ingredient plays an important role. In paper II is shown that the susceptibility of eggs of the large cabbage white (Pieris brassicae) decreased with increasing age. With eggs of the Colorado potato beetle we also observed that egg mortality increased with increasing relative humidity. The addition of surfactants to the spray liquid also proved to be necessary sometimes to obtain a sufficient coverage of the eggs.With eggs of the leaf miner Leucoptera scitella we found that the concentration of diflubenzuron in the spray liquid influenced the moment mortality occurred. Generally, at rates of diflubenzuron between 100 and 1 mg a.i./l, mortality occurred either in the egg stage or in the early larval stages. However, at lower concentrations the contributions of the ovicidal mortality to the overall activity of diflubenzuron decreased.Several aspects of the larvicidal activity of diflubenzuron such as feeding and contact activity, larval behaviour after treatment, and differences in susceptibility are described in paper III. This article also deals with the resumption of normal cuticle deposition in larvae of the Colorado potato beetle, after termination of diflubenzuron treatment, which is illustrated with histological observations.In paper IV a number of benzoylureas, with variations in lipophilic properties were tested for their larvicidal and ovicidal activities on the housefly. In larvicidal tests the compounds were incorporated into the larval culture medium. Ovicidal activities were assessed on female adults either by feeding or by injection.Correlation of biological activities by means of a physical chemical parameter revealed that the ovicidal activity after injection was the basic activity in both the ovicidal activity by adult feeding and in the larvicidal activity. However, in either case this activity was coupled to a lipophilic parameter, which was expressed as πin the octanol/water system. Optimum πvalues for the ovicidal activity after adult feeding and for the larvicidal activity were 1.0 and 1.5, respectively.It is likely that at increasing lipophilicity of the compounds above the optimum πvalues, the gut wall becomes an important barrier. Though penetration also decreases when the lipophilicity decreases to below the optimum πvalue, it is possible that the activity at the target site is too low to allow the gut barrier to play an important role.The properties of diflubenzuron in connection with cross-resistance and with the development of resistance in the housefly are described in paper V. Upon selection with diflubenzuron, incorporated into the larval culture medium, development of both larvicidal and ovicidal resistance was studied in a susceptible laboratory strain (S) and in a multiresistant strain (Nic). Due to the decrease in reproductive potential upon progressive selection, the breeding of strain Nic had to be determined in the F32. Observations for this study with strain S were discontinued in the F40.The larval resistance against diflubenzuron in both strains increased slowly. The final assessment of larval resistance in strain Nic, which was made in the F26, showed a 13.6-fold decrease in susceptibility when based on LC 50 values. In the F40 of strain S this factor was 40-fold.With respect to the egg resistance against diflubenzuron in the F40 of strain S we measured a similar factor as compared to the larval resistance development. However, in strain Nic the ovicidal resistance proved to increase so rapidly that in the adult feeding experiments in the F26 even the highest test concentration of 3000 mg a.i./l, of diflubenzuron in the milk only resulted in an ovicidal activity of 26%.In two field-collected strains of the housefly, cross-resistance factors for the larvicidal effect were either absent or low (viz. 5). The ovicidal cross'resistance factors for the two field strains were found to be 8.4 and 87.5 respectively. Furthermore, the level of ovicidal cross-resistance could probably be related to the resistance of the adults to some standard insecticides tested.Chitin synthesis in the integument not only occurs in embryos and larvae, but also in adult insects during a restricted period after adult emergence. On feeding diflubenzuron to newly hatched adults of the Colorado potato beetle we observed that the elytra remained weak (paper VI). For that end the stiffness of the elytra was measured mechanically as the resistance of the elytra to being punctured. In untreated beetles the mechanical penetrability of the elytra decreases up to about 10 days after adult emergence. At any time during this period we found that this decrease in penetrability could be blocked by administering diflubenzuron to the adults. Also of interest is the fact that concomitant with the effect of diflubenzuron on the penetrability of the elytra, a strong inhibition of chitin formation could be measured.Histological observations of elytra of the Colorado potato beetle revealed several types of mesocuticles. Treatment with diflubenzuron induces characteristic distortions in each of them. In the elytra of treated beetles the thickness of the cuticle, surprisingly, continued to increase. We explained this observation as an indication that protein deposition was not affected by diflubenzuron (paper VI).Quantative data in addition to the histological observations are presented in paper VII. From the experimental data we furthermore concluded that the level of ketocatechols, which parameter can be related to the degree of tanning of the structural proteins, is only partially inhibited by diflubenzuron. However, we observed that, upon feeding adults with diflubenzuron, inhibition of chitin synthesis took place at lower concentrations than its effect on the level of ketocatechols. This might indicate that the interference of diflubenzuron with the tanning process is only of a secondary nature.Based upon experiences with both larvicidal and ovicidal applications, the practical possibilities of diflubenzuron for insect pest control are evaluated in paper III, section 6