The purification and characterization of esterases from insecticide-resistant and susceptible house flies

Four major esterases in one susceptible (CSMA) and two resistant (Hirokawa, E₁) house fly strains were separated by chromatofocusing. Of the four esterases, those with pI's of 5.1 and 5.3 accounted for 90% of the p-nitrophenyl butyrate hydrolyzing activity in the three house fly strains. They also accounted for 70% (Hirokawa, E₁) and 40% (CSMA) of the paraoxon-hydrolyzing activity as well as 87% (Hirokawa), 39% (E₁) and 66% (CSMA) of the malathion-hydrolyzing activity in microsomes as measured by esterase-antibody interaction. In the Hirokawa strain, the pI 5.1 esterase was the predominant esterase and was more active than that of the the CSMA strain. Different substrate specificities and a different K_m toward acetylthiocholine, as well as different rates of malathion and paraoxon hydrolysis between the Hirokawa and CSMA strains, suggest a qualitative difference in the pI 5.1 esterase. For the pI 5.1 esterase from the E₁ strain, a different substrate specificity, a different K_m for p-nitrophenyl butyrate, a different sensitivity to inhibitors, and a different rate of paraoxon hydrolysis suggest that it is a modified esterase. This esterase is not a phosphorotriester hydrolase, nor does it lack nonspecific esterase activity. It is a modified esterase which has a different substrate specificity when compared to the esterases from the other strains. The molecular weight of the esterases studied was approximately 220,000, with pH optima of about 7.0.The ratio of malathion α-monoacid to β-monoacid formation was about 9.0 for the pI 5.1 and 5.3 esterases and 1.5 for the pI 4.8 and 5.6 esterases. The existence of a higher $\text{α}\text{β}$ ratio for the pI 5.1 and 5.3 esterases and their significant rate of malathion hydrolysis in the Hirokawa strain indicate that an increase in the $\text{α}\text{β}$ ratio in house flies reported was due to the increase in the pI 5.1 esterase in the resistant strain.     

General esterase,malathion carboxylesterase,and malathion resistance in Culex tarsalis

The role of esterases in malathion resistance in Culex tarsalis has been investigated. When larvae of a resistant and a sensitive strain were placed in water containing [¹⁴C]malathion, malathion penetrated to give initially similar internal levels. With resistant mosquitoes, after 15 min the internal malathion concentration decreased to low levels while the monoacid degradation products accumulated in the larvae and were excreted into the surrounding water, whereas in susceptible larvae the internal malathion level stayed high and was lethal. It is suggested that the decrease in internal malathion and the resulting resistance were caused by an active malathion carboxylesterase in the resistant strain. A specific assay for malathion carboxylesterase with [¹⁴C]malathion showed 55 times more activity in resistant than in susceptible larvae, whereas when general esterase activity was assayed with α-naphthyl acetate only 1.7 times the activity was found. Analyses by starch gel electrophoresis showed a peak of malathion carboxylesterase, 60-fold higher from resistant than from susceptible larvae, in a gel zone which did not stain for general esterase activity. General esterases that did not hydrolyze malathion showed different electrophoretic patterns in the two populations, which are likely due to the nonisogenic character of the strains. These results show that use of a specific assay and the demonstration of degradation of malathion in vivo are essential for assessment of the contribution of esterase activity to the malathion-resistant phenotype in mosquito populations.     

Effect of a mixture of iprobenfos and malathion on the development of malathion resistance in the mosquito Culex pipiens pallens Coq

A malathion-resistant (RM) strain of Culex pipiens pallens Coq was obtained by successively selecting a field population with malathion in the laboratory. The synergistic effect of iprobenfos on malathion toxicity and alpha-naphthyl acetate (alpha-NA) esterase assay revealed that malathion resistance in the RM strain was associated with increased alpha-NA esterase activity and the synergism was mainly due to the inhibition by iprobenfos of this activity. There was no difference in alpha-NA esterase activity between the larvae and female adults in the susceptible (S) strain, but the activity in the adults was 13-fold higher than in the larvae of the RM strain. To understand the effect of the application of a mixture of iprobenfos and malathion on the evolution of malathion resistance, an artificial strain (Syn) was generated by mixing the RM and S strains with 0.1 frequency of the malathion-resistant individuals. The offspring of the Syn strain were divided into two sub-strains, Rm and Rm+ibp, which were successively treated with, respectively, malathion alone and malathion + iprobenfos (1:2) at LC70. In the mixture, the fungicide iprobenfos acted as a synergist of malathion. After treatment for 10 generations, the resistance level to malathion was 317.4-fold for the Rm sub-strain, whereas for the Rm+ibp sub-strain it was only 38.9-fold, compared with the Syn strain. Similar results were obtained by measurement of alpha-NA esterase activity from both larvae and female adults. The alpha-NA esterase activities in larvae and female adults at F10 generation were 2.6- and 10.9-fold from the Rm+ibp sub-strain and 5.7- and 98.5-fold from the Rm sub-strain, respectively, compared with the Syn strain. The above results suggested that iprobenfos, although it cannot completely stop or prevent the onset of malathion resistance, could dramatically delay its evolution.     

Esterase-mediated malathion resistance in the human head louse, Pediculus capitis (Anoplura: Pediculidae)

Resistance in a dual malathion- and permethrin-resistant head louse strain (BR-HL) was studied. BR-HL was 3.6- and 3.7-fold more resistant to malathion and permethrin, respectively, compared to insecticide-susceptible EC-HL. S,S,S-Tributylphosphorotrithioate synergized malathion toxicity by 2.1-fold but not permethrin toxicity in BR-HL. Piperonyl butoxide did not synergize malathion or permethrin toxicity. Malathion carboxylesterase (MCE) activity was 13.3-fold and general esterase activity was 3.9-fold higher in BR-HL versus EC-HL. There were no significant differences in phosphotriesterase, glutathione S-transferase, and acetylcholinesterase activities between strains. There was no differential sensitivity in acetylcholinesterase inhibition by malaoxon. Esterases from BR-HL had higher affinities and hydrolysis efficiencies versus EC-HL using various naphthyl-substituted esters. Protein content of BR-HL females and males was 1.6- and 1.3-fold higher, respectively, versus EC-HL adults. Electrophoresis revealed two esterases with increased intensity and a unique esterase associated with BR-HL. Thus, increased MCE activity and over-expressed esterases appear to be involved in malathion resistance in the head louse.     

Selective inhibition of separate esterases in rat and mouse liver microsomes hydrolyzing malathion,trans-permethrin,and cis-permethrin

Separate esterase activities of rat and mouse liver microsomes hydrolyzing malathion, trans-permethrin, and cis-permethrin were differentiated on the basis of their sensitivities to inhibition by paraoxon and α-naphthyl N-propylcarbamate (NPC). In rat liver microsomes, the malathionhydrolyzing activity was more sensitive to both inhibitors and showed a different time course of NPC inhibition than the activities hydrolyzing the permethrin isomers. Paraoxon completely inhibited trans-permethrin hydrolysis, but only partially inhibited that of cis-permethrin. The paraoxonsensitive trans- and cis-permethrin-hydrolyzing activities were not differentially inhibited, but separate inhibition curves were obtained for the inhibition of trans- and cis-permethrin hydrolysis by NPC. The mouse liver esterase activity hydrolyzing trans-permethrin showed a similar paraoxon sensitivity to that of rat liver, but that the paraoxon-sensitive portion of the cis-permethrinhydrolyzing activity was 5.5-fold less sensitive to paraoxon than the corresponding rat liver activity and was clearly differentiated from the mouse liver trans-permethrin-hydrolyzing activity. The mouse liver malathion-hydrolyzing activity was 100-fold less sensitive to paraoxon and 14-fold less sensitive to NPC than the corresponding rat liver activity. Rat and mouse liver esterase activities hydrolyzed trans- and cis-permethrin at similar rates under standard assay conditions, but mouse liver esterases were 10-fold less active in hydrolyzing malathion. The higher specific activity of rat liver malathion-hydrolyzing esterases resulted from the greater apparent affinity and maximum velocity for malathion hydrolysis. These results demonstrate that the hydrolysis of malathion, trans-permethrin, and cis-permethrin by rat and mouse liver microsomal preparations involves several esterases with differing substrate specificities and inhibitor sensitivities.     

Malathion carboxylesterase enzymes in Anopheles arabiensis from Sudan

Malathion resistance in Anopheles arabiensis from Sudan is monofactorially inherited and is expressed in the adults but not in the larvae. The resistance is suppressed by the esterase synergist, triphenylphosphate. Semipurification of the soluble esterase enzymes by Sephadex G-25 and Sephacryl S-200 gel filtration revealed no difference between the enzymes of the resistant and susceptible strains with α- or β-naphthylacetate (NA) with a fixed substrate concentration in either the adults or larvae. However, with the malathion-specific assay a second peak of activity was observed in the adult resistant strain which was not present in either the larvae of this strain or the larvae and adults of the susceptible strain. A corresponding threefold difference in the K_m value for α-NA was also observed in the resistant adults over the range of this second peak, but there was no change in the K_m with β-NA.     

The biochemical nature of malathion resistance in Anopheles stephensi from Pakistan

Malathion resistance in Anopheles stephensi from Pakistan was synergized by triphenyl phosphate, primarily a carboxylesterase inhibitor. There was a slight degree of antagonism with piperonyl butoxide. The major metabolite of malathion in larvae of both the resistant and susceptible strains was malathion monocarboxylic acid. Resistant larvae produced about twice as much of this product as the susceptible larvae. This suggests that a qualitative or a quantitative change in a carboxylesterase enzyme may be the basis of malathion resistance in this strain. Analysis of general esterase levels to α- and β-naphthyl acetate showed that there was no quantitative change in the amount of carboxylesterase enzyme present in the resistant strain as compared to the susceptible.     

The in vitro degradation of [14C]malathion by enzymatic extracts from resistant and susceptible strains of Drosophila melanogaster

Enzyme preparations from Drosophila melanogaster flies degraded [¹⁴C]malathion to α- and β-malathion monoacids and, hence, were considered to contain malathion carboxylesterase (ME) activity. Although ME- activity was stable during preincubation in the absence of malathion, it decreased dramatically during the course of the reaction, and could not be completely recovered by Sephadex G-25 chromatography. Furthermore, the protein fraction after chromatography still contained ¹⁴C, suggesting that the enzyme had become inhibited by a bound, ¹⁴C-labeled derivative. Extracts from a resistant (malathion-selected), an intermediate control, and the susceptible Canton S strains of D. melanogaster differed in the lability of ME activity during the reaction. This difference was partly attributed to the production of small amounts of malaoxon (2–8%) by the extracts from the more resistant strains. No consistent strain differences were found when the rate of malathion degradation was measured during the first minute of reaction, either with or without a microsomal oxidase inhibitor (metyrapone) present. These results, together with the cross-resistance of the malathion-selected strain to other insecticides and the lack of a synergistic effect of two carboxylesterase inhibitors (triphenyl phosphate and S,S,S-tributylphosphorotrithioate) suggested that malathion carboxylesterase does not contribute significantly to the observed differences in malathion resistance between strains.     

Detection of single-base substitution in an esterase gene and its linkage to malathion resistance in the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae)

Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae) is an important parasitoid of stored-grain insect pests. Partial cDNA sequences of an esterase-like enzyme have been obtained from a malathion-resistant (R) strain and a susceptible (S) strain of this wasp. A single-base substitution in the R strain has been confirmed by using PCR amplification of specific allele (PASA) to amplify genomic DNA extracted from individual resistant and susceptible parents, F₁ hybrids from double reciprocal crosses, and progeny from backcrosses. The R allele appeared to be inherited in a strict Mendelian fashion in both diploid female and haploid male progeny. The esterase fragment co-segregated with resistance in these crosses and backcrosses. Female wasps in a mixed population of A calandrae that survived a malathion screen carried the R allele for the esterase-like enzyme, while those wasps that died did not have the R allele. The single base-pair mutation, guanine in the R strain and thymine in the S strain, presumably results in a tryptophan-to-glycine amino acid substitution in the encoded protein. We do not know how these amino acid substitutions may relate to functional differences in the enzyme. However, this esterase gene or another linked esterase gene may encode the resistance-associated malathion detoxifying activity in the R strain. © 1999 Society of Chemical Industry     

Glutathione-dependent degradation of parathion and its significance for resistance in the housefly

Glutathione-dependent degradation of parathion was studied in six strains of houseflies to find out whether it might be important as a cause of resistance. When supernatant fractions of high-speed centrifuged homogenates were fortified with glutathione and incubated with parathion, water-soluble products were formed. The rate of parathion detoxication was highest in a malathion-resistant strain (c. 4 μg parathion degraded per abdomen per hour), lowest in a susceptible strain, and intermediate in some other organophosphate-resistant strains. In one of the latter strains, E₁, the gene for glutathione-dependent degradation is located on the second chromosome, closely linked with gene cm⁺. This is the same chromosome on which gene a for low ali-esterase activity and hydrolytic detoxication of paraoxon is located. It is not likely that the gene for glutathione-dependent degradation is identical with gene a, since it is also present in strain Nie which lacks gene a, and, therefore, the presence of a separate gene which is called gene g is postulated.     

Synergism of insecticidal action and effects on detoxifying enzymes in vivo of lambda-cyhalothrin and malathion by some nitrogen heterocycles in resistant and susceptible strains of Tribolium castaneum

The interactions of the synthetic pyrethroid, lambda-cyhalothrin and malathion were studied with purines, pyrimidines, caffeine and some other related nitrogenous compounds in resistant and susceptible strains of Triboliurn castaneum (Herbst.) The results were compared with those obtained with a known synergist, piperonyl butoxide (PBO) and precocene I. Adenine, cytosine, guanine, thymine and uracil synergised lambda-cyhalothrin, especially in the susceptible strain, with maximum effect at a 1:1 mass ratio, with the effect decreasing with increasing proportion of the heterocycle. The order of synergism of lambda-cyhalothrin was; precocene I > PBO > the nitrogenous compounds, in both resistant and susceptible strains. On the other hand, caffeine (lethal effect increased about twice), barbital (about twice), isobarbituric acid (less than twice) and bromacil (up to eight times) synergised malathion in malathion-resistant strains and antagonised in the susceptible strains. Total in-vivo esterases, carbox-ylesterases and cytochrome P₄₅₀ of susceptible and resistant strains showed significantly increased activity or content when treated with either insecticide plus a heterocyclic compound. Exceptions were with bromacil and malathion and for the malathion-specific strain, Kano-C with malathion and the N-heterocycles.     

微量滴度酶标板法测定棉蚜(Aphis gossypii Glover)酯酶特性与有机磷抗性的关系

通过生物测定表明高密棉蚜对有机磷的抗性高于北京棉蚜,用紫外分光光度计比色法(A法)及微量滴度酶标板法(B法 )测定高密棉蚜及北京棉蚜的α-乙酸萘酯酯酶活力和α-乙酸萘酯酯酶动力学。北京棉蚜和高密棉蚜的α- NA酯酶活力分别为 2.23、4.48(A法 )和1.13、3.30(B法)μmol·mg-1pro.·min-1,高密、北京棉蚜的酶活之比为 2 .00(A法 )、2 .92(B法) ;北京棉蚜、高密棉蚜的Km值分别为:6.06×10-5、7.51× 10-5(A法 )和 7.66×10-5、8.87×10-5 (B法) mol·L-1,Vmax值为2.53、5.82(A法)和1.28、3.61(B法)μmol·mg-1·min-1。比较紫外分光光度计比色法及微量滴度酶标板法的测定结果,表明微量滴度酶标板法的测定结果是可靠的。     

Esterases and resistance to synthetic pyrethroids in the Egyptian cotton leafworm

Esterases hydrolyzing α-naphthyl acetate (α-NA), β-naphthyl acetate (β-NA), and p-nitrophenyl acetate (p-NPA) were investigated colorimetrically in larval homogenates of synthetic pyrethroid susceptible (S) and resistant (R) strains of Spodoptera littoralis (Boised). The hydrolytic activity towards the three substrates in cybolt, decamethrin, and fenvalerate R strains were from 3 to 6.5 times as high as in the S strain. The increase in esterase activity was closely associated with the development of resistance in the R strains. DEF (S,S,S-tributyl phosphorotrithioate) proved to be an inhibitor for all esterases, with a particularly potent action on p-NPA-hydrolyzing enzymes. The inhibitory action was more pronounced in R strains than in the S strain. Pretreatment with DEF increased the toxicity of pyrethroid compounds in the R strains more than in the S strain and hence decreased the levels of resistance in these strains. This is evidence that the esterases contribute to the resistance against synthetic pyrethroids in S. littoralis larvae.     

Studies on hydrolases in various house fly strains and their role in malathion resistance

Aliesterase, carboxylesterase, and phosphorotriester hydrolase activities in six house fly strains were studied in relation to malathion resistance. Selection of two susceptible strains with malathion for three generations resulted in an increase in both carboxylesterase activity and LD₅₀ of malathion, indicating that the increased detoxication by the enzyme was the major mechanism selected for malathion resistance. With the highly resistant strains, however, the carboxylesterase activity alone was not sufficient to explain the resistance level, and the involvement of additional mechanisms, including phosphorotriester hydrolase activity, was suggested. The E₁ strain, which had high phosphorotriester hydrolase activity but normal or low carboxylesterase activity, showed a moderate level, i.e., sevenfold resistance. Upon DEAE-cellulose chromatography, two or three esterase peaks were resolved from susceptible, moderately resistant, and highly resistant strains. The substrate specificity, the sensitivity to paraoxon inhibition, and the $\text{α}\text{β}$ ratio of malathion hydrolysis were studied for each esterase peak from the different strains. The results suggested the existence of multiple forms of esterases with overlapping substrate specificity in the house fly.     

Dual role of esterases in insecticide resistance in the green rice leafhopper

The role of esterases as related to insecticide resistance was studied in an organophosphorus (OP)-resistant strain of the green rice leafhopper. As judged by p-nitrophenyl acetate hydrolysis, 21, 5, and 74% of the esterase activity was located in nuclei/mitochondria, microsomes, and the soluble fraction, respectively. All the fractions were active in hydrolyzing malathion, paraoxon, and fenvalerate. Hydrolysis of malathion and fenvalerate increased with time while that of paraoxon reached a plateau within 15 min. Since a considerable amount of p-nitrophenol was detected in the paraoxon reaction at 0°C and at zero time, the formation of p-nitrophenol may be due to phosphorylation of the esterases rather than phosphorotriesterase action. The results suggest a dual role for esterases in resistance mechanisms; a catalyst for hydrolysis of malathion and fenvalerate, and a binding protein for the oxygen analogs of other OP insecticides, both of which would protect the intrinsic target, acetylcholinesterase, from inhibition. Chromatofocusing of the soluble fraction resolved five esterase peaks, I–V. These esterases were active toward the three general substrates as well as for the three insecticides tested, except for Peak I in which the overall activity was too low. Thin-layer agar gel electrophoresis showed that the chromatofocusing peaks I–V corresponded to the electrophoretic bands E₁–E₅, some of which were previously shown to be associated with OP resistance. The dual role of these esterases may explain the cross-resistance between malathion and other OP insecticides as well as synergism between OP and carbamate insecticides.     

In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs

Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O-halogenated-phenyl-O-alkyl phenylphosphonates was studied in vitro. The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The pI₅₀ of O-(2,6-dichlorophenyl) O-methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy compounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than desbromoleptophosoxon. Within a like group of chlorinated phenylphosphonates, a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in vivo delayed neurotoxic potential by the corresponding phosphonothionate exists. In vivo inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in vivo inhibitor. Ethoxyleptophos and leptophos have about equal in vivo esterase inhibitory properties. For desbromoleptophos and leptophos there is good agreement between the minimum dose causing delayed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.     

Pyrethroid resistance and esterase activity in three strains of the cotton bollworm, Helicoverpa armigera (Hübner)

Microplate assay technique for estimation of esterase activity in a single insect was used in combination with dose mortality bioassays to detect pyrethroid resistance in three strains of Helicoverpa armigera and to study the frequency of pyrethroid resistant individuals within the population of the same strain at two larval stages, third and fifth instar. The third and fifth instar larvae of the field strains i.e., Nagpur strain and Delhi strain that displayed high degree of resistance towards deltamethrin, had higher esterase activity compared to a susceptible laboratory strain. The frequency distribution of individuals with elevated esterase activity above 1.00 absorbance unit was correlated with the resistance level of the strains. The frequency of resistant individuals in the third instar larvae of Nagpur strain and Delhi strain were 29% and 23%, respectively compared to 4% in the susceptible strain. The resistant individuals in the resistant strains have markedly increased in the fifth instar larvae with a frequency distribution of 63% and 90% in Delhi strain and Nagpur strain, respectively, while only 14% of individuals was found to have elevated esterase activity in the susceptible strain. The results demonstrated the role of esterase in pyrethroid resistance in H. armigera. Microplate assay proved to be a rapid and reliable biochemical technique for monitoring of pyrethroid resistance in H. armigera.     

Oxidative cleavage of a carboxyester bond as a mechanism of resistance to malaoxon in houseflies

The synergistic effect of triphenyl phosphate (a carboxyesterase inhibitor), sesamex (inhibitor of microsomal oxidation) and O,O-diethyl O-phenyl phosphorothioate on the toxicity of malathion and malaoxon for one susceptible and two resistant strains of housefly was studies. It was found that in the resistant strain G (characterized by high carboxyesterase activity) both malathion and malaoxon were synergized by triphenyl phosphate, but only malaoxon (and not malathion) by sesamex. The other resistant strain E 1, moderately tolerant for malathion but highly resistant to malaoxon, differed from strain G in that triphenyl phosphate had no effect; its response to sesamex was similar to that of strain G. The third synergist, O,O-diethyl O-phenyl phosphorothioate, combined the properties of triphenyl phosphate and sesamex. It was found to be the best of the three compounds used.Biochemical in vitro studies showed that both resistant strains could degrade malaoxon oxidatively at a rate at least 10 × higher than that of the susceptible strain. This oxidation could be inhibited by very low concentrations of the thiono analogue; a malaoxon to malathion ratio of 10:1 gave an inhibition of about 70% at a malaoxon concentration of 5 × 10^?6M. The product of this oxidation is malaoxon β-monocarboxylic acid. This metabolite was also found 1 hr after application of malaoxon in vivo.The results mentioned in this paper indicate that houseflies may become resistant to malaoxon by an increased rate of oxidative carboxyester bond cleavage.     

Evidence that the elevated carboxylesterase (esterase 2) in organophosphorus-resistant Myzus persicae (Sulz.) is identical with the organophosphate-hydrolyzing enzyme

An enzyme hydrolyzing methylparaoxon in vitro in an organophosphorus-resistant strain of the peach potato aphid (Myzus persicae Sulz.) is present in the same electrophoretic fraction as a carboxylesterase (esterase 2) which has previously been shown to have characteristically increased activity in organophosphorus resistant strains of this aphid.No in vitro organophosphate hydrolysis was found in a susceptible strain with low carboxyl-esterase-2 activity. Carboxylesterase-2 and the methylparaoxon-hydrolyzing enzyme are both inhibited by n-propylparaoxon but not by methyl- and ethylparaoxon. This indicates that the two enzymes are identical.     

Substituted thiotrifluoropropanones as potent selective inhibitors of juvenile hormone esterase

A series of 27 substituted thio-1,1,1-trifluoropropanones was synthesized by reacting the corresponding thiol with 1,1,1-trifluoro-3-bromopropanone. The resulting sulfides were screened as inhibitors of hemolymph juvenile hormone esterase and α-naphthyl acetate esterase activity of the cabbage looper, Trichoplusia ni, electric eel acetylcholinesterase, bovine trypsin, and bovine α-chymotrypsin. The presence of the sulfide bond increased the inhibitory potency on all of the enzymes tested when compared with compounds lacking the sulfide. In general, the compounds proved to be poor inhibitors of chymotrypsin and moderate inhibitors of trypsin. By varying the substituent on the sulfide, good inhibitory activity was obtained on α-naphthyl acetate esterase, acetylcholinesterase, while some of the compounds proved to be extremely powerful inhibitors of juvenile hormone esterase. The most powerful inhibitor tested was 3-octylthio-1,1,1-trifluoro-2-propanone, with an I₅₀ of 2.3 × 10^?9M on JH esterase. This compound showed a molar refractivity similar to that of the JH II backbone, was not toxic to T. ni, and was moderately toxic to mice, with a 48-hr LD₅₀ of >750 mg/kg. It effectively delayed pupation when applied to prewandering larvae of T. ni, as expected for a JH esterase inhibitor. Thus, some members of this series are promising for evaluating the role of JH esterase in insect development. The series also indicates that, by varying the substituent on the sulfide moiety, potent “transition-state” inhibitors can be developed for a wide variety of esterases and proteases.