Inhibitor Report for: Fenoxycarb

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (k(cat)/K(M) ratio) and V(max) values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE.

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.

We studied the relationship between fenoxycarb levels and general esterase (GE) activity, juvenile hormone esterase (JHE) activity, and induction of permanent fifth instar (dauer) larvae in Bombyx mori. Fenoxycarb (1 ug/animal) was topically applied to B. mori at 0 h of the fifth instar. Hemolymph fenoxycarb levels, as determined by an ELISA, reached a peak within 1 h, and then decreased to undetectably low at 42 h. After 48 h, the feces of these larvae contained 99% of the applied fenoxycarb dose. The above fenoxycarb treatment induces dauer larvae with persistently increased hemolymph GE and JHE activities for the rest of the fifth instar. Application of fenoxycarb later in the fifth instar (48 h), when it still could induce dauer larvae, resulted in a similar pattern of elimination from the hemolymph and produced steadily elevated hemolymph GE and JHE activities for the rest of the fifth instar. Application of fenoxycarb late in the fifth instar (132 h) did not prevent pupal ecdysis; it increased hemolymph JHE activity, but not GE activity. The latter treatment prevented the JHE activity decrease observed in control larvae before pupal ecdysis. In vivo, in control animals, the highest hemolymph JHE activity was observed 12 h after pupal ecdysis. At that developmental time, fenoxycarb inhibited this enzyme in vitro IC50=2uM . The combined results suggest: (1) fenoxycarb is rapidly excreted from the larval body; (2) induction of dauer larvae and increased hemolymph GE and JHE activities are not directly associated with its presence in the larval body of B. mori.

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (k(cat)/K(M) ratio) and V(max) values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE.

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.

This study assessed the effects of exposure to IGRs on the long-term development of the honeybee colony, viability of queens and sperm production in drones and integrated the data into a honeybee population model. Colonies treated with diflubenzuron resulted in a short-term reduction in the numbers of adult bees and brood. Colonies treated with fenoxycarb declined during the season earlier and started the season slower. The number of queens that successfully mated and laid eggs was affected in the fenoxycarb treatment group but there were no significant differences in the drone sperm counts between the colonies. An existing honeybee population model was modified to include exposure to IGRs. In the model, fenoxycarb reduced the winter size of the colony, with the greatest effects following a June or an August application. Assuming a 'larvae per nurse bee' ratio of 1.5 for brood rearing capability, the reduction in winter size of a colony following a fenoxycarb application was at its worst about 8%. However, even if only those bees reared within 2 weeks of the IGR being applied are subject to premature ageing, this might significantly reduce the size of over-wintering colonies, and increase the chance of the bee population dwindling and dying in late winter or early spring.

Grass shrimp (Palaemonetes pugio) were reared separately through both embryonic and total larval development during exposure to fenoxycarb at measured concentrations of <2.2 to 888 microg L(-1). A fenoxycarb concentration of 888 microg L(-1) significantly (p < 0.05) inhibited embryonic development to larval hatching and extended the embryonic developmental period from 11.9 to 12.7 days. Exposure to fenoxycarb concentrations < or = 502 microg L(-1) had no significant (p > 0.05) effect on complete embryonic development. Significantly fewer shrimp successfully metamorphosed to postlarvae when exposed through complete larval development to fenoxycarb concentrations > or = 4 microg L(-1). Larval development of grass shrimp was therefore >2 orders of magnitude more sensitive to this juvenile hormone agonist than was embryonic development. Viability of larvae developing in fenoxycarb was concentration dependent. Development beyond third zoeal stage was significantly inhibited at fenoxycarb concentrations > or = 190 microg L(-1), whereas development beyond fourth zoeal stage was inhibited by a concentration of > or = 45 microg L(-1). Fenoxycarb exposure of developing larvae did not alter either the duration of total larval development or the total number of larval stages before metamorphosis. Rearing of fenoxycarb-exposed embryos through larval development without further exposure had no significant effect on number of larval stages, larval development rate, or metamorphic success of larvae. Similarities in the sensitivity of grass shrimp larvae and mosquito larvae to fenoxycarb suggests that the use of a bioassay protocol measuring the metamorphic success of crustacean larvae would be a valuable adjunct to the hazard assessment of newly developed pesticides that target endocrine control of metamorphosis in insects and possibly other endocrine-disrupting xenobiotics as well.

The mechanism by which carbamate pesticides inhibit rat alpha4beta2 nicotinic acetylcholine (ACh) receptors (nAChRs) expressed in Xenopus laevis oocytes has been investigated using the two-electrode voltage clamp technique. Carbaryl, S-ethyl N,N-dipropylthiocarbamate (EPTC), and fenoxycarb inhibit ACh-induced ion currents in a concentration-dependent way. EPTC and fenoxycarb inhibit ion currents induced by 1 mM ACh with 3-fold to 5-fold higher potency than ion currents induced by 1 microM ACh. The potency of carbaryl appears to be independent of ACh concentration. Fenoxycarb displaces (3)H-epibatidine bound to alpha4beta2 (nAChRs) with a K(i) of 750 microM, which is much higher than the functional IC(50) of 2.3-11 microM. This shows that the inhibition of ion current by the carbamate is a noncompetitive effect. Inhibition by fenoxycarb is independent of the state of the ion channel. The rate of onset of inhibition is enhanced, and the rate of reversal of inhibition is reduced, when the concentration of fenoxycarb is increased. The rate of reversal of inhibition is also reduced when the period of exposure to fenoxycarb is increased. The time- and concentration-dependent inhibition of nAChR-mediated ion current by fenoxycarb is accounted for by a two-step mechanism involving a rapid blocked state and a sequential more stably blocked or desensitized state.

Vitellin (Vn) and vitellogenin (Vg) of the beet armyworm, Spodoptera exigua, were identified and characterized. Both were similar in molecular size (180 kDa) and showed cross-immune reactivity. Vn was purified by ammonium sulfate (25-50%), size-exclusion chromatography (G-100), ion-exchange chromatography (DEAE), and affinity chromatography (concanavalin-A). The purified Vn was used for raising its antibody. The raised Vn-antiserum reacted specifically with Vn and Vg proteins in S. exigua, but had a slight cross-reactivity with P220, probably lipophorin large subunit. The Vn-antiserum did not react to Vns of Bombyx mori, Plutella xylostella, and Drosophila melanogaster. Using this antiserum, Vg biosynthesis could be assessed with adult development of S. exiua. Vg could be detected in the hemolymph and the fat body as early as 5h before adult emergence. Fenoxycarb, a juvenile hormone analog, could induce Vg biosynthesis.

Effects of commonly used carbamate pesticides on rat neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. The potencies of these effects have been compared to the potencies of the carbamates to inhibit rat brain acetylcholinesterase. The potency order of six carbamates to inhibit alpha4beta4 nicotinic receptors is fenoxycarb > EPTC > carbaryl, bendiocarb > propoxur > aldicarb with IC50 values ranging from 3 microM for fenoxycarb to 165 microM for propoxur and >1 mM for aldicarb. Conversely, the potency order of these carbamates to inhibit rat brain acetylcholinesterase is bendiocarb > propoxur, aldicarb > carbaryl > EPTC, fenoxycarb with IC50 values ranging from 1 microM for bendiocarb to 17 microM for carbaryl and > mM for EPTC and fenoxycarb. The alpha4beta2, alpha3beta4, and alpha3beta2 nicotinic acetylcholine receptors are inhibited by fenoxycarb, EPTC, and carbaryl with potency orders similar to that for alpha4beta4 receptors. Comparing the potencies of inhibition of the distinct subtypes of nicotinic acetylcholine receptors shows that the alpha3beta2 receptor is less sensitive to inhibition by fenoxycarb and EPTC. The potency of inhibition depends on the carbamate as well as on a combination of alpha and beta subunit properties. It is concluded that carbamate pesticides affect different subtypes of neuronal nicotinic receptors independently of acetylcholinesterase inhibition. This implicates that neuronal nicotinic receptors are additional targets for some carbamate pesticides and that these receptors may contribute to carbamate pesticide toxicology, especially after long-term exposure.

We studied the relationship between fenoxycarb levels and general esterase (GE) activity, juvenile hormone esterase (JHE) activity, and induction of permanent fifth instar (dauer) larvae in Bombyx mori. Fenoxycarb (1 ug/animal) was topically applied to B. mori at 0 h of the fifth instar. Hemolymph fenoxycarb levels, as determined by an ELISA, reached a peak within 1 h, and then decreased to undetectably low at 42 h. After 48 h, the feces of these larvae contained 99% of the applied fenoxycarb dose. The above fenoxycarb treatment induces dauer larvae with persistently increased hemolymph GE and JHE activities for the rest of the fifth instar. Application of fenoxycarb later in the fifth instar (48 h), when it still could induce dauer larvae, resulted in a similar pattern of elimination from the hemolymph and produced steadily elevated hemolymph GE and JHE activities for the rest of the fifth instar. Application of fenoxycarb late in the fifth instar (132 h) did not prevent pupal ecdysis; it increased hemolymph JHE activity, but not GE activity. The latter treatment prevented the JHE activity decrease observed in control larvae before pupal ecdysis. In vivo, in control animals, the highest hemolymph JHE activity was observed 12 h after pupal ecdysis. At that developmental time, fenoxycarb inhibited this enzyme in vitro IC50=2uM . The combined results suggest: (1) fenoxycarb is rapidly excreted from the larval body; (2) induction of dauer larvae and increased hemolymph GE and JHE activities are not directly associated with its presence in the larval body of B. mori.

Cross-resistance of the obliquebanded leafroller, Choristoneura rosaceana (Harris), to tebufenozide was reported from laboratory studies before it had been used in commercial orchards in New York State. Bioassays with obliquebanded leafroller larvae from tebufenozide and organophosphate susceptible and resistant colonies were conducted with chlorfenapyr, emamectin benzoate, fenoxycarb, fipronil, spinosad, and tebufenozide to determine if cross-resistance was present before these new insecticides were introduced into commercial orchards. Resistance ratios ranged from 1.1 to 3.2 for all insecticides except tebufenozide (12.8). Significant differences between susceptible and resistant colonies were found with emamectin benzoate, fenoxycarb, and fipronil. The effect of the metabolic synergists piperonyl butoxide (PBO) and diethyl maleate (DEM) on tebufenozide toxicity was examined to determine mechanisms for obliquebanded leafroller resistance to tebufenozide and potential mechanisms for other new insecticides. At a concentration of 20 ppm, PBO and DEM significantly synergized the toxicity of tebufenozide in resistant and susceptible colonies (three- to fourfold). Obliquebanded leafrollers may be resistant to new insecticides with distinct modes of action even if these compounds have not been previously used in commercial orchards.