The cuticle,growth and moulting in insects: The essential background to the action of acylurea insecticides

The functions, structure and biochemistry of the insect cuticle in relation to the moulting cycle are briefly reviewed as an introduction to the actions of insecticides that act on the cuticle, particularly acylureas. The symptoms of poisoning with diflubenzuron (DFB) and other acylureas are consistent with ultra-structural and biochemical evidence that these insecticides inhibit the formation of chitin microfibrils in newly synthesised cuticle. It is probable that DFB acts at a late stage in chitin biosynthesis, perhaps inhibiting chitin synthase (CS) itself. However, the results of studies using cell-free preparations of CS have not, on the whole, supported this hypothesis. A number of alternative suggestions as to the mode of action of DFB are reviewed. Among the most attractive of these is the possibility that DFB may inhibit the transmembrane transport of chitin synthesis precursors from their site of production within the epidermal cells to the site of the final poly condensation reaction, presumably at the apical membrane of the epidermal microvilli.     

Inhibition of Tribolium gut chitin synthetase

The chitin synthetase (CS) of Tribolium castaneum gut is inhibited 50% by 0.02 μM nikkomycin and 4 μM polyoxin D, two pyrimidine nucleoside fungicides, in in vitro assays with 10-min preincubation of enzyme and inhibitor prior to substrate addition. Tribolium CS is also sensitive to inhibition by the pyrimidine nucleotides uridine and cytidine di- and triphosphates. Captan, a known inhibitor of insect chitin synthesis, and the related fungicides captafol and dichlofluanid are highly potent inhibitors of Tribolium CS. Moderately active CS inhibitors are the acaricide oxythioquinox and the herbicide barban. One phenylcarbamate insect growth reatardant, H-24108, is weakly active in inhibiting Tribolium gut CS, as are three of its analogs but not 26 others. Many triazines are not inhibitory including several herbicides and an azido derivative, CGA 19255, which is active in blocking insect growth and chitin synthesis. Although the benzoylphenyl urea insecticides diflubenzuron and SIR 8514 are potent in vivo inhibitors of the polymerization step in insect chitin synthesis, they do not affect T. castaneum gut CS activity in vitro and greatly stimulate Tribolium brevicornis gut CS activity in vivo. These studies and preliminary findings on an integumental enzyme indicate that CS of these tissues is not sensitive to the direct action of benzoylphenyl ureas. This leads to speculation that the benzoylphenyl ureas act either as CS inhibitors via active metabolites formed in the integument or as blocking agents by direct binding to non-CS sites important in chitin polymerization and fibrillogenesis.     

1-(2,6-disubstituted benzoyl)-3-phenylurea insecticides: Inhibitors of chitin synthesis

The mode of action has been investigated of the 1-(2,6-dichlorobenzoyl)-3-phenyl ureas, a new type of insecticide. A microautoradiographical study was made of the incorporation of glucose, tyrosine, and proline in endocuticle of fifth-instar larvae of Pieris brassicae L., both normal and treated with 1-(2,6-dichlorobenzoyl)-3-(3,4-dichlorophenyl)-urea (DU 19111). The results of this work clearly indicate that DU 19111 blocks the synthesis of cuticular chitin. The nature of this inhibition was investigated by comparison of the rates of incorporation of [¹⁴C]glucose into the ultimate chitin precursor, uridine diphosphate N-acetyl glucosamine (UDPAG), in both normal and DU 19111-treated Pieris larvae. It was found that these levels did not differ significantly.The finding that only in DU 19111-treated Pieris larvae were substantial amounts of labeled N-acetyl glucosamine present 1 hr after the injection of [¹⁴C]glucose is considered as a clue to the mechanism by which this insecticide inhibits chitin synthesis. Apparently the coupling of UDPAG to the chitin synthetase still proceeds, but the function of connecting N-acetyl glucosamine moieties to the chitin chain is disrupted.Tentative results with a structural analog of DU 19111 suggest that this compound induces accumulation of UDPAG, but not of N-acetyl glucosamine. This would imply that in the latter case the polycondensing enzyme is completely blocked.     

Cytochemical demonstration of the effects of the acylureas flufenoxuron and diflubenzuron on the incorporation of chitin into insect cuticle

Flufenoxuron (“Cascade”) is a novel acylurea with acaricidal and insecticidal properties. It acts in a similar manner to diflubenzuron (DFB) by impairing chitin incorporation into insect cuticle. Chitin can be localised cytochemically using the lectin, wheat germ agglutinin (WGA), which binds specifically to N-acetylglucosamine-containing polymers. WGA adsorbed to monodisperse colloidal gold (WGA-gold) was used as an electron-dense marker for localising chitin in the cuticle of sixth-instar Spodoptera littoralis. In the cuticle of control insects, the deposition zone and endocuticle were heavily labelled. In flufenoxuron- and DFB-treated insects, only the endocuticle (formed before treatment) was labelled, the deposition zone being devoid of label. These results show that flufenoxuron and DFB act in similar manner in reducing chitin incorporation in the cuticle of S. littoralis.     

Inhibition of chitin synthesis by two 1-(2,6-disubstituted benzoyl)-3-phenylurea insecticides. II

The knowledge of the biochemical mode of action of 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea (diflubenzuron) is presented, explaining the insecticidal effect. Like its structural analog, 1-(2,6-dichlorobenzyl)-3-(3,4-dichlorophenyl)urea (Du 19111), it inhibits chitin synthesis in the cuticle of larvae. Virtually complete inhibition was demonstrable 15 min after the application of diflubenzuron. Neither diflubenzuron nor Du 19111 has any effect upon chitinase activity either in vivo or in vitro. The insecticidal effect upon the cuticle, therefore, must be explained as an inhibition of chitin synthesis and not as an activation of chitin degradation. In contrast to the action of Du 19111, no accumulation of N-acetylglucosamine occurs upon treatment of larvae with diflubenzuron. Similarities and differences in the mode of action of both compounds are discussed, together with other effects reported in the literature.     

Mode of action of insect growth regulators in lepidopteran tissue culture

Insect growth regulators (IGRs) have been proposed as agents for the control of insect pests. These compounds disrupt the normal development of insects by mimicking juvenile hormone and the molting hormone, 20-hydroxyecdsyone, or by interfering with chitin synthesis. The effectiveness and selectivity of IGRs provide new tools for integrated pest management. The simultaneous advances in the chemistry of IGRs and the ability to study insect tissues in culture, have led to research on the mode of action of IGRs in vitro. Plodia interpunctella and Spodoptera frugiperda have been used to examine the effects of IGRs on wing imaginal discs in organ culture, as well as in hormonally responsive cell lines established from wing imaginal discs of these species. Our research has focused on the action of ecdysteroid mimics, chitin synthesis inhibitors and juvenile hormone mimics. The effects of the IGRs on chitin synthesis, uptake of amino-sugars, and cellular proliferation were studied in tissue culture. The results demonstrate the effectiveness of using organ cultures and hormonally responsive cell lines for investigating IGRs at the cellular and tissue level. © 1998 Society of Chemical Industry     

Effects of diflubenzuron on the pupal-adult development of Tenebrio molitor L. (Coleoptera,Tenebrionidae): Growth and development,cuticle secretion,epidermal cell density,and DNA synthesis

Diflubenzuron (DFB), applied by dipping on newly emerged pupae of Tenebrio molitor L., disturbs the pupal-adult development. Four main types of treated insects were obtained according to external morphology: blocked pupae, adults unable to ecdyse, adults partially ecdysed, and adults completely ecdysed. The proportion of these four types varied with the time of treatment during the pupal life. When DFB was administered by dipping at a 10 g/liter concentration to newly emerged pupae, HPLC measurements showed that the different responses were due to a difference in the levels actually incorporated. Histological and ultrastructural observations of sternal cuticles revealed that DFB disturbs the cuticle deposition: reduction of its thickness and modifications of its architecture. Tritiated thymidine incorporation and cell density measurements in sternal epidermis show that DFB affects both mitoses and DNA synthesis in blocked pupae. These findings suggest that DFB interferes with other biochemical processes besides the chitin biosynthesis; whether these effects are primary or secondary remain to be determined.     

Insecticidal and neuroexciting actions of DDT analogs

The neuroexciting activity of DDT and its analogs to produce repetitive responses on the nerve cord of Periplaneta americana was determined using the extracellular electrode method. The convulsive activity on P. americana and the insecticidal effect on Callosobruchus chinensis were also examined. It was found that the convulsive and insecticidal activities increase almost proportionally with increase in the neuroexciting activity within a set of p,p′-substituted DDT analogs. The intimate connections among these biological effects suggest that symptoms such as convulsion and death caused by DDT analogs are closely related with their neuroexcitory effect and there is a common mode of action in spite of differences in insect species.     

Effects of nereistoxin on the neuroexcitatory action of insecticides

Nereistoxin suppressed the excitatory actions of α- and γ-BHC and HEOD in the central nervous system of Periplaneta americana (L.). However, it did not affect the neurotoxic action of DDT and DBrDT. Since nereistoxin is an acetylcholine antagonist, it is postulated that BHC and HEOD act to produce high level of acetylcholine and the resulting after-discharges, while DDT and DBrDT seem to attack the noncholinergic region of insect nervous system.     

Properties of acetolactate synthase from sulfonylurea-resistant Scirpus juncoides Roxb. var. ohwianus T. Koyama

Properties of acetolactate synthase (EC 4.1.3.18; ALS) from sulfonylurea-resistant (SUR) Scirpus juncoides Roxb. var. ohwianus T. Koyama were studied biochemically and physiologically in comparison with those from sulfonylurea-susceptible weed (SUS). GR₅₀ values for growth inhibition and I₅₀ values for ALS inhibition by imazosulfuron were determined for both SUR and SUS. Imazosulfuron controlled the SUS above 80% at the dosage more than 10 g a.i./ha but did not control the SUR at the even great dosage of 1000 g a.i./ha. The rates required for 50% growth inhibition of the SUR relative to the SUS (R/S ratio) were 271-fold. The I₅₀ value for inhibition of ALS from the SUS was 15 nM, compared to I₅₀ of >3000 nM for inhibition of ALS from the SUR. These results suggest that a resistance may due to an altered ALS that is insensitive to imazosulfuron. The K_m (pyruvate) value of ALS from the SUR was similar to the K_m for ALS from the SUS, suggesting that a mutation resulting in resistance does not change the affinity of the enzyme for pyruvate. The specific activity of the SUR ALS was similar to that of the SUS ALS, which indicates that resistance is not an over-expression of the enzyme. ALS activity from both biotypes was inhibited by isoleucine, valine, and leucine in this order. However, the SUR ALS was less sensitive to inhibition by valine than the SUS ALS.     

Action of avermectin B1a on the leg muscles and the nervous system of the American cockroach

The action of avermectin was studied in the leg muscle and the central nervous system of the American cockroach, Periplanata americana L. Avermectin at a low concentration (10^?7M) causes a failure of the leg muscles to respond to external stimuli within 30 min without affecting the magnitude of contraction. Avermectin was found to stimulate Cl^? uptake by the leg muscles within 4 min at 10^?7M. The threshold concentration to cause such stimulation was on the order of 10^?8M. This stimulatory action could be antagonized by picrotoxinin (10^?4M) and to a lesser extent by bicuculline methiodide (10^?4M). The phenomenon is observable under both Na⁺-free and K⁺-free conditions. It was concluded that the action of avermectin is to open the chloride channel on the plasma membrane. This action of avermectin does not seem to be mediated through GABA, GABA receptors, diazepine receptors, or picrotoxinin receptor in this insect species, and therefore suggests that avermectin directly attacks the chloride channel proper both in the central nervous and the neuromuscular systems.     

Acetylcholinesterase inhibition by nootkatone and carvacrol in arthropods

The essential oils from many botanicals have been screened for insecticidal activity. Two constituents of the Alaskan yellow cedar tree, the monoterpenoid carvacrol and the sesquiterpenoid nootkatone, both are toxic against several arthropods. The mode of action through which nootkatone and carvacrol exert their insecticidal activity remains uncertain. It has been hypothesized that they may inhibit acetylcholinesterase enzyme activity. The degree of acetylcholinesterase inhibition of carvacrol and nootkatone was compared to that of carbaryl, a known acetylcholinesterase inhibitor, in the house fly (Musca domestica), yellow fever mosquito (Aedes aegypti), American dog tick (Dermacentor variabilis) and American cockroach (Periplaneta americana). The concentration of carbaryl, at which 50% of the acetylcholinesterase activity was inhibited (IC₅₀), was less than 2 μM in all four arthropod models. Carvacrol was observed to cause slight inhibition of the acetylcholinesterase enzyme in house flies, ticks and cockroaches, but it did not inhibit the mosquito acetylcholinesterase enzyme. Nootkatone did not inhibit the acetylcholinesterase enzyme in any of the four arthropod models tested. From this study, we conclude that the acetylcholinesterase inhibition is not likely the primary mode of action for insecticidal activity by nootkatone or carvacrol.     

MoTlg2, a t-SNARE component is important for formation of the Spitzenkörper and polar deposition of chitin in Magnaporthe oryzae

T-SNAREs are a family of conserved proteins involved in intracellular transport of membrane-coated cargo among subcellular compartments. In this study, we identified a putative t-SNARE gene, MoTLG2, in Magnaporthe oryzae via insertion mutagenesis. Deletion of MoTLG2 resulted in slower vegetative growth and less conidiation relative to the wild-type strain, but the ΔMotlg2 null mutant was as virulent as the wild-type strain. MoTlg2 has 30% overall amino acid identity with Saccharomyces cerevisiae Tlg2, and rescued the defect of monensin de-sensitivity in the yeast strain where TLG2 had been deleted. More importantly, apical regions of the hyphae of the ΔMotlg2 null mutant were only weakly stained by FM4-64, which was reported as an excellent vesicle tracer, suggesting that the Spitzenkörper was not well formed in the ΔMotlg2 null mutant. In addition, more uneven lateral deposition of chitin was observed in the cell wall of vegetative hyphae of the ΔMotlg2 null mutant. Taken together, this study shows that the t-SNARE Tlg2 is important for both vegetative hyphal growth and conidiation, but dispensable for plant infection in filamentous fungi, and suggests that Tlg2 is important for formation of the Spitzenkörper and polar distribution of chitin.     

Anthranilic diamides: A new class of insecticides with a novel mode of action, ryanodine receptor activation

Development of insecticides with unique modes of action is necessary to combat widespread insecticide resistance. A new class of insecticides has been discovered, the anthranilic diamides, that provides exceptional control through action on a novel target, the ryanodine receptor. Anthranilic diamides potently activate this receptor, releasing stored calcium from the sarcoendoplasmic reticulum causing impaired regulation of muscle contraction. Expression of a recombinant Drosophila ryanodine receptor in a lepidopteran cell line confers sensitivity to anthranilic diamides similar to that observed with native receptors. Ligand-binding studies with radiolabeled ryanodine and radiolabeled anthranilic diamide in Periplaneta americana reveal a single, saturable binding site for this chemistry distinct from that of ryanodine. Further, calcium mobilization studies using mammalian cell lines indicate anthranilic diamides exhibit >500-fold differential selectivity toward insect, over mammalian, receptors. Consequently, anthranilic diamides offer a novel pharmacological tool for calcium signaling research in addition to a unique alternative to existing pest-management strategies.     

Effects of diflubenzuron on the integument of fifth instar<Emphasis Type="Italic">Galleria mellonella</Emphasis> larvae

Effects of the chitin synthesis inhibitor diflubenzuron (DFB) on the integument of 5th instarGalleria mellonella L. larvae were investigated. When larvae were fed with semi-artificial diets containing 250, 500 and 1000 ppm of the compound, DFB affected the integument. The affected larvae failed to ecdysis, their cuticle was ruptured, lost haemolymph and blackened. In treated larvae, cuticle deposition was disrupted and the cuticle thickness was decreased by ∼50% compared with the untreated control, particularly at day 3 1/2. However, statistically there was no significant difference among the three concentrations (P>0.05). This may indicate that all three concentrations are equally effective in decreasing level. DFB at 500 and 1000 ppm also affected the epidermal cells and caused the occurrence of vacuoles. http://www.phytoparasitica.org posting Dec. 16, 2003.     

The effect of diflubenzuron feeding on glycosaminoglycan and sulfhemoglobin biosynthesis in mice

Diflubenzuron (Dimilin), an inhibitor of insect chitin synthesis, was fed to mice in concentrations of 50 to 2000 ppm in the diet. The activity of the mammalian hexosamine transferases responsible for glycosaminoglycan (mucopolysaccharide) formation was monitored by measuring the in vivo rate of incorporation of a labeled precursor into hyaluronic acid and chondroitin sulfate of skin. No inhibition of biosynthesis was noted at any concentration; indeed the insecticide appeared to stimulate the production of these compounds. After 3 weeks on the diet, mice eating 1000 and 2000 ppm diflubenzuron showed obvious signs of cyanosis, and sulfhemoglobin was demonstrated in the blood of mice eating more than 200 ppm. The amount of sulfhemoglobin appeared to be related to the dietary insecticide content and the highest level seen was 13% of total hemoglobin in the 2000-ppm group after 31 days. These values returned to normal within 3 weeks when diflubenzuron was removed from the diet.     

Isoxaben analogs inhibit chitin synthesis in the cultured integument of the rice stem borer Chilo suppressalis

Benzoylphenylureas (BPUs) were discovered as novel type insecticides about a half century ago; many analogs have been launched as insecticides and acaricides. BPUs are known to inhibit chitin synthesis in insects and other arthropods, but they have no effect against microorganisms such as fungi. We designed new chitin synthesis inhibitors based on the hypothesis that biomolecules that play important roles in cellulose and chitin biosynthesis are similar. In the full automatic modeling system (FAMS), the cellulose synthase was selected as a template three-dimensional structure. Thus, we focused on the structure of cellulose synthase inhibitor, isoxaben, to develop new chemistry. The 1,1-diethylethyl [-C(CH₃)(CH₂CH₃)₂] group of isoxaben was changed to a 4-substituted phenyl group bearing Cl, Et, or Ph. These compounds significantly inhibited chitin synthesis in the cultured integument of the rice stem borer Chilo suppressalis. The activity of the 4-ethylphenyl analog was enhanced 30-fold by adding piperonyl butoxide to the culture medium.     

The effect of insect “Autoneurotoxin” on Periplaneta americana (L) and Schistocerca gregaria (Forskal) Malpighian tubules

The effect of insect “autoneurotoxin” upon Malpighian tubules of Periplaneta americana (L.) and Schistocerca gregaria (Forskal) was investigated in vitro. “Autoneurotoxin” collected under different stress conditions, caused an acceleration in the frequency of coiling. The increase of the observed coiling frequency was not solely due to 5HT.     

A comparative study of the metabolic effects of hypertrehalosemic hormone and 1,2,3,4,5,6-hexachlorocyclohexane (γ-HCH) in the American cockroach, Periplaneta americana

Several effects of hypertrehalosemic hormone (Peram-HTH) have been compared with the action of 1,2,3,4,5,6-hexachlorocyclohexane (γ-HCH) in the cockroach, Periplaneta americana. The data show that both agents stimulate respiration while maintaining an R.Q. of approximately 1. The action of γ-HCH differs from that of Peram-HTH-I in that it promotes oxidation of large amounts of soluble carbohydrate. Recent studies show that Peram-HTH-I has an equally potent stimulatory effect on lipid deposition in the fat body and on the release of trehalose from the same tissue. It is of interest that γ-HCH not only increases triacylglycerol accumulation in the fatγ body but that this effect is more pronounced if the γ-HCH is applied to the ventral surface of the mesothorax. γ-HCH also depleted whole body glycoprotein and glycolipid whereas Peram-HTH-I had no effect on glycoprotein but increased that of glycolipid. It is clear that γ-HCH causes complex disruptions to the metabolism of the insect, many of which are related to the release of hormones. The study supports the idea that certain actions of γ-HCH are mediated through the release of Peram-HTH-I.     

Effects of pyriproxyfen, a juvenile hormone analog, on development of the mealworm, Tenebrio molitor

Pyriproxyfen, an analog of the juvenile hormone (JH) in insects, has been evaluated in vivo and in vitro on an important stored product pest, the mealworm Tenebrio molitor. On newly ecdysed pupae, this insect growth regulator (IGR) was applied topically (at 0.10 and 0.20 μg/insect). Enzyme immunoassay measurements showed a decrease of the ecdysteroid titer in the hemolymph of treated specimens. In addition, the treatment induced an increase of the protein concentration in the hemolymph, however, there was no significant effect on the protein band pattern. Using in vitro pupal integument explant cultures, pyriproxyfen (at 1 and 10 μM in the medium) caused a significant inhibition of ecdysteroid amounts and of the thickness of new cuticle secreted. These new results are discussed in relation to the physiology of this IGR with a JH action.