Microsomal metabolism of juvenile hormone analogs in the house fly,Musca domestica L.

Of six juvenile hormone analogs of the alkyl 3,7,11-trimethyl-2,4-dodecadienate type, only the isopropyl ester was strongly morphogenic in the house fly, Musca domestica L. In vitro assays revealed that house fly microsomes contain B-esterases as well as oxidases which metabolize such analogs. However, these esterases did not hydrolyze the isopropyl ester, ZR-515. Enzymes prepared from larvae, pupae, and adults were all active and there was evidence that in the late larval stage the esterase activity was cyclic, showing a minimum in the early third instar and a maximum a few hours later. When microsomes from two susceptible and two resistant house fly strains were compared for metabolic activity against the juvenile hormone analogs, those from the resistant strains were 1.3 to 20 × higher in oxidase activity but there was no difference in esterase activity. The oxidative metabolism of two analogs ZR-515 and 512 was greatly enhanced when the flies were induced with phenobarbital but there was no enhancement in metabolism of three of the remaining analogs and only a slight enhancement of a fourth. It is concluded that the insecticidal action of ZR-515 is largely due to its stability in the presence of the house fly esterases.     

The joint action of insect growth regulators and insecticides on strains of Spodoptera littoralis Boisd. That are susceptible or resistant to aminocarb

An investigation was made of the action of the juvenile hormone analogues (JHAs) methoprene (ZR-515) and 3-[5-(4-ethylphenoxy)-3-methylpent-3-enyl]-2, 2-dimethyloxirane (R-20458), and the insecticides chlorpyrifos and methomyl, when applied, separately or in combination, to the late insect stages of susceptible (S) and aminocarb-resistant (R_m) strains of Spodoptera littoralis Boisd. Sixth-instar larvae of the R_m strain showed detectable levels of cross-resistance to chlorpyrifos, methomyl and R-20458. However, cross-resistance to methoprene was less pronounced in the R_m strain. Treatment of the same instar with the ED₅₀ of methoprene produced an appreciable level of sterility in the S strain, but this level decreased in the R_m strain. Similar treatment with R-20458 caused a lower level of sterility in the S strain and the R_m strain was less affected. In the S strain, the ED₂₅ of either chlorpyrifos or methomyl when applied simultaneously with the ED₂₅ of R-20458, produced an antagonistic effect and the R_m strain was more capable of resisting the joint action of these compounds. The treatment, ED₂₅ chlorpyrifos + ED₂₅ methoprene produced an additive effect on the S strain, while the R_m strain tolerated their combined action. Nevertheless, the treatment, ED₂₅ methomyl + ED₂₅ methoprene produced additive effects on both the S and R_m strains. The prepupae of the R_m strain tolerated the action of the insecticides methomyl and chlorpyrifos. A similar pattern of cross-resistance was also detected against the action of the two hormones at the ED₂₅ level, while at the ED₂₅ level, both the S and R_m strains were almost equally sensitive to the action of the two juvenoids. Chlorpyrifos-JHA combinations produced additive effects on prepupae of the S strain while the R_m strain completely resisted their joint action. Methomyl-JHA combinations produced high potentiation in the S strain, but the R_m strain remained insensitive to their joint action. Detectable levels of tolerance to the action of chlorpyrifos and methomyl at the ED₂₅ and ED₅₀ levels were indicated in 2-day-old pupae of the R_m strain. This was less evident in the case of JHAs, particularly methoprene, which was to some extent equally effective on both strains; the reproductive ability of the S and R_m strains was highly affected by this compound. The pupae of the R_m strains were equally as affected as those of the S strain by the combination ED₂₅ chlorpyrifos+ ED₂₅ R-20458. Nevertheless, the two strains showed antagonism to the action of chlorpyrifos with methoprene. A high level of potentiation was produced in the S strain to the combination of methomyl and R-20458 but the R_m strain was able to withstand their combined action. Of interest in this respect was the action of the combination of methomyl and methoprene, for which high levels of potentiation were detected in pupae of the S and R_m strains. This combination also Produced a high percentage of sterility in mated females of the R_m strain.     

Studies on the metabolism of vamidothion and its thioanalog in insecticide-resistant and susceptible house fly strains

The in vivo and in vitro metabolism of vamidothion [O,O-dimethyl S-[2-(1-methylcarbamoyl)-ethylthio] ethylphosphorothiolate] as well as the in vitro metabolism of thiovamidothion [O,O-dimethyl S-[2-(1-methylcarbamoyl)ethylthio] ethylphosphorodithioate] was investigated in insecticide-resistant and susceptible house fly strains. Vamidothion was converted in vivo to the sulfoxide, the principle metabolite, and subsequently to the sulfone at a slower rate. Vamidothion and vamidothion sulfoxide were hydrolyzed at the PS and SC bond. The resulting primary alcohol metabolite was further oxidized to a carboxylic acid followed by decarboxylation. No metabolism of vamidothion or thiovamidothion occurred in vitro without the addition of NADPH. The addition of NADPH resulted in rapid conversion of vamidothion to the sulfoxide, and thiovamidothion was oxidatively metabolized to six metabolic products. No qualitative differences were found between resistant and susceptible strains, but there were signficant quantitative differences. The metabolism was highest in the Rutgers strain followed by Cornell-R, Hirokawa, and then CSMA strain. The route of vamidothion and thiovamidothion metabolism was via the cytochrome P-450-dependent monooxygenase system, and none of the resistant strains showed glutathione S-transferase activity toward vamidothion or thiovamidothion. No further oxidation of vamidothion sulfoxide to the sulfone was observed and also no hydrolysis products were formed, in vitro.     

Metabolism of juvenile hormone I by microsomal oxidase,esterase, and epoxide hydrase of Musca domestica and some comparisons with Phormia regina and Sarcophaga bullata

House fly (Musca domestica L.) microsomes prepared from larvae, pupae, or adults contain three enzyme system which can metabolize juvenile hormone I: an esterase, an oxidase, and epoxide hydrase. The presence of the oxidase is indicated by the increased metabolism when microsomes are supplemented with NADPH and by the occurrence of additional metabolites tentatively identified as products arising from oxidation of the 6, 7 double bond. Additional evidence of the activity of the oxidase system is the increased metabolism of juvenile hormone I by the NADPH-dependent system from phenobarbital-induced insects, by inhibition of the oxidation by piperonyl butoxide and carbon monoxide, and by the greater metabolism of the hormone by microsomes from insecticide-resistant (high oxidase) strains. In vivo studies of house fly adults treated with ³H-labeled juvenile hormone I reveal a pattern of metabolism similar to that seen during NADPH-supplemented in vitro metabolism. The three enzymes have somewhat different patterns of activity during the larval stage of the house fly, juvenile hormone esterase and epoxide hydrase beginning at a high level of activity in the young larvae while the juvenile hormone oxidase is low at this stage. In the late larval stage all three enzymes show increased activity followed by declines during the pupal stage and further increases in the adult stage. Comparison of in vitro enzyme levels of the house fly, flesh fly (Sarcophaga bullata Parker), and blow fly [Phormia regina (Meigen)] showed that, although the enzymes were present in the latter two species, their activity on a per insect basis was considerably less than that of the house fly.     

Metabolism of fenitrothion by organophosphorus-resistant and -susceptible house flies, Musca domestica L

The metabolism of fenitrothion was investigated in highly resistant (Akita-f) and susceptible (SRS) strains of the house fly, Musca domestica L. The Akita-f strain was 3500 times more resistant to fenitrothion than the SRS strain. Fenitrothion, topically applied to the flies, was metabolized in vivo far faster in the Akita-f strain than in the SRS strain. In vitro studies revealed that fenitrothion was metabolized by a cytochrome P-450-dependent monooxygenase system and glutathione S-transferases. The former oxidase system metabolized fenitrothion in vitro into fenitrooxon and 3-methyl-4-nitrophenol as major metabolites, and into 3-hydroxymethyl-fenitrothion and 3-hydroxymethyl-fenitrooxon as minor metabolites. Glutathione S-transferases metabolized fenitrothion into desmethylfenitrothion. The cytochrome P-450-dependent monooxygenase system and glutathione S-transferases of the resistant Akita-f strain had 1.4 to 2.2 times and 9.7 times, respectively, as great activities as those of the susceptible SRS strain. These results suggest the importance of glutathione S-transferases in fenitrothion resistance in the Akita-f strain.     

Reproductive and developmental defects in a malathion-resistant,laboratory-selected population of Drosophila melanogaster

We have examined the chromosomal basis for reproductive and developmental defects that are associated with malathion resistance in a laboratory-selected population of Drosophila melanogaster. Strains homozygous for second or third chromosomes from this population were more resistant to malathion and had greater mixed-function oxidase activity, decreased fertility, and lower egg production when compared with first chromosome or susceptible strains. Some of the strains carrying resistant third chromosomes were developmentally delayed and required a significantly longer time to pupate. Delayed pupation was not associated with increased in vitro degradation of ecdysone by larvae having increased mixed-function oxidase activity, nor could it be reversed by feeding larvae ecdysone. Differences in mixed function oxidase activity among strains homozygous for second or third chromosomes were strongly correlated with malathion resistance but not with fitness. Although both second and third chromosome strains had high mixed-function oxidase activity, only fly extracts from the third chromosome strains oxidatively degraded [³H]juvenile hormone in vitro to a significant extent. A deficit of vitellogenic oocytes and increased egg laying by females in response to topically applied juvenile hormone-I supported the hypothesis that juvenile hormone titer was lower than normal in these strains. The results indicate that different polygenic systems control malathion resistance and associated fitness defects in this selected population of D. melanogaster. Although these systems are partly independent, they overlap due to pleiotropic effects of third chromosomal genes controlling mixed-function oxidase activity on female reproduction.     

Mechanisms of resistance to diflubenzuron in the house fly, Musca domestica (L.)

The mechanisms of resistance to the chitin synthesis inhibitor diflubenzuron were investigated in a diflubenzuron-selected strain of the house fly (Musca domestica L.) with > 1000 × resistance, and in an OMS-12-selected strain [O-ethyl O-(2,4-dichlorophenyl)phosphoramidothioate] with 380 × resistance to diflubenzuron. In agreement with the accepted mode of action of diflubenzuron, chitin synthesis was reduced less in larvae of the resistant (R) than of a susceptible (S) strain. Cuticular penetration of diflubenzuron into larvae of the R strains was about half that of the S. Both piperonyl butoxide and sesamex synergized diflubenzuron markedly in the R strains, indicating that mixed-function oxidase enzymes play a major role in resistance. Limited synergism by DEF (S,S,S-tributyl phosphorotrithioate) and diethylmaleate indicated that esterases and glutathione-dependent transferases play a relatively small role in resistance. Larvae of the S and R strains exhibited a similar pattern of in vivo cleavage of ³H- and ¹⁴C-labeled diflubenzuron at N₁C₂ and N₁C₁ bonds. However, there were marked differences in the amounts of major metabolites produced: R larvae metabolized diflubenzuron at considerably higher rates, resulting in 18-fold lower accumulation of unmetabolized diflubenzuron by comparison with S larvae. Polar metabolites were excreted at a 2-fold higher rate by R larvae. The high levels of resistance to diflubenzuron in R-Diflubenzuron and R-OMS-12 larvae are due to the combined effect of reduced cuticular penetration, increased metabolism, and rapid excretion of the chemical.     

An evaluation of the role of oxidative enzymes in Colorado potato beetle resistance to carbamate insecticides

Carbofuran and carbaryl LD₅₀ values were determined with and without piperonyl butoxide pretreatment for a resistant (New Jersey) and two susceptible (Utah and Netherland) populations of Colorado potato beetle larvae. Similar bioassays were conducted with carbofuran for resistant (Rutgers) and susceptible (NAIDM) adult house flies. The degree of resistance development by New Jersey Colorado potato beetles (RR = 848) was greater than that of the laboratory-selected colony of Rutgers house flies (RR = 583). Comparisons of synergist difference calculations including “percentage synergism” (%S), “log percentage synergism” (L%S), and “relative percentage synergism (R%S) for the resistant (R) and the susceptible (S) populations indicated the possibility that monooxygenases and other resistance mechanisms may be involved in Colorado potato beetle resistance to these carbamates. Monooxygenase involvement in resistance of Rutgers house flies was demonstrated in vitro by a 4-fold enhancement of p-nitroanisole O-demethylation over that of NAIDM house flies. O-demethylation of p-nitroanisole could not be demonstrated for potato beetle larvae. Colorado potato beetle resistance was associated with increases in microsomal levels of NADPH-cytochrome c reductase (ca. 2-fold) and NADPH oxidation (1.2-fold). The inability to measure O-demethylation in Colorado potato beetles may have been due to the solubilization of NADPH-cytochrome c reductase during microsomal preparation. Significant differences between resistant and susceptible Colorado potato beetle larvae were not observed in the penetration of [¹⁴C]carbaryl. Excretion of the radiocarbon may have been significantly greater in the resistant New Jersey population, but some of the insecticide may have also rubbed off the cuticle. This increased capacity for excretion, combined with increased levels of monooxygenase enzymes, could account for the high resistance level of this population.     

Biochemical genetics of oxidative resistance to diazinon in the house fly

The genetics and biochemistry of oxidative resistance to diazinon were investigated in a diazinon-resistant strain of the house fly, Musca domestica L. The resistant strain was crossed with a multimarker susceptible strain and substrains containing portions of the resistant strain genome were prepared. Resistance, microsomal oxidase, and cytochrome P-450 spectral characteristics were then compared in the different strains. The major gene for resistance to diazinon is semidominant and is located on chromosome II, 13 crossing over units from the recessive mutant stubby wing. Additional resistance genes occur on chromosome II and on other chromosomes as well. Resistance to diazinon was introduced into a susceptible mutant-marked strain via genetic crossing over. Increases in parathion oxidase, total and P-450-specific N- and O-demethylase activity, and resistant strain type I binding spectrum were introduced along with resistance, indicating genes controlling these parameters and resistance are either identical or closely linked. No increase in activity of cytochrome P-450 itself was introduced into the mutant strain. Additional genes controlling the amount of cytochrome P-450 and several spectral changes characteristic of the resistant strains are apparently controlled by genes located at different loci on chromosome II. Resistance factors on other chromosomes are also present, but were not characterized.     

The mechanisms of carbaryl resistance in the fall armyworm,Spodoptera frugiperda (J. E. Smith)

The physiological mechanisms of resistance to carbaryl were investigated in a carbaryl-resistant strain of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Piperonyl butoxide greatly reduced the resistance level from 90- to 6-fold, indicating that microsomal cytochrome P-450-dependent monooxygenases may play a major role in resistance. This finding is consistent with metabolic data in which the oxidative metabolism of carbaryl by midgut homogenates was five times more active in the resistant strain than in the susceptible strain. In addition, the resistant strain showed increased activities of microsomal hydroxylation and epoxidation compared to the susceptible strain. Cuticular penetration studies using [¹⁴C]carbaryl revealed that 55% of the applied radioactivity remained on the cuticle of resistant larvae while 32% remained on susceptible larvae 24 hr after topical treatment. The resistance appeared to be unrelated to target site insensitivity. It is concluded that the high level of resistance to carbaryl in this insect was mainly due to enhanced oxidative metabolism of the insecticide (via hydroxylation and epoxidation) with reduced cuticular penetration playing a very minor role, if any.     

Studies on the acetylcholinesterase of Anopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides

Acetylcholinesterase from fourth instar Anopheles albimanus larvae was studied in vitro. The acetylcholinesterase from both the resistant and susceptible strains behaved as a single enzyme “type,” with straight pseudo first-order insecticide inhibition lines which intersected the Y axis at 100%. The enzyme from resistant larvae was more slowly inhibited than the susceptible enzyme; bimolecular rate constants (k_i) differed by approximately 1.2- to 6-fold for a range of organophosphorous compounds and 17- to 1570-fold for the carbamates. There was a good correlation between the levels of resistance and the acetylcholinesterase inhibition rates.     

A simple bioassay for detecting and characterising insecticide resistance

A simple and rapid paralysis assay was developed to detect and characterise knockdown resistance in larvae of the house fly and pink bollworm. Pyrethroidtreated larvae unable to perform a stereotypic curling movement when probed with a warm needle, 10 min (house fly) or 60 min (pink bollworm) after treatment, were considered to be paralysed. Responses in a susceptible strain of house fly were compared with those of the pyrethroid-resistant strains kdr and super-kdr. In this assay, the kdr strain displayed over 28 fold resistance to deltamethrin, and super-kdr larvae were unaffected by doses up to 100μg. These results are in good qualitative agreement with previous studies. The assay detected no significant fenvalerate resistance in pink bollworm larvae collected from the field; this was consistent with mortality bioassays performed on wild adult males. The limitations and potential utility of the paralysis bioassay in resistance screening are discussed.     

In-vivo and in-vitro studies on DDT uptake and metabolism in susceptible and resistant strains of the mosquito Aedes aegypti L.

Twelve strains of Aedes aegypti have been compared for resistance to pp′-DDT, uptake of pp′-DDT, and dehydrochlorination of pp′-DDT to pp′-DDE in vivo and in vitro both at the larval and adult stages. Resistant larvae were shown to contain significantly more pp′-DDE than susceptible larvae after a standard exposure to pp′-DDT but also substantially more pp′-DDT in an unmetabolised state. There was a small increase in the percentage dehydrochlorinated in vivo in the resistant strains compared with the susceptible strains, but this was not correlated with the level of resistance nor with dehydrochlorination in vitro. However, dehydrochlorination in vitro was correlated with resistance. Adult resistance was correlated positively with dehydrochlorination, both in vivo and in vitro, but the resistant adults did not contain increased levels of unmetabolised DDT. By comparing resistance levels at the two stages, it was found that there were two kinds of resistant strain: four strains of Asian origin and one from West Africa were highly resistant as larvae but showed almost no resistance as adults; five strains from Central and South America were highly resistant at both stages. The different mechanisms of resistance in adults and larvae are discussed in relation to genetic studies.     

Insensitive acetylcholinesterase,high glutathione-S-transferase,and hydrolytic activity as resistance factors in a tetrachlorvinphos-resistant strain of house fly

The gene producing insensitive acetylcholinesterase in a tetrachlorvinphos-resistant strain of house fly was introduced into the genome of a susceptible strain. The resistance to a number of organophosphorus insecticides, which was high in the original strain (234- and more than 800-fold for paraoxon and tetrachlorvinphos, respectively), was much lower in the resulting strain, ranging from 53-fold for paraoxon to 3.9-fold for tetrachlorvinphos. Synergists further decreased these factors. The reduction in sensitivity of the acetylcholinesterase in vitro was about 20-fold for both inhibitors. The Michaelis parameters for hydrolysis of acetylcholine and acetylthiocholine of the mutant and normal acetylcholinesterase showed only minor differences. An increased rate of metabolism of the insecticides was found in the original strain. In vitro, glutathione-dependent detoxication was from 9- to 120-fold that of a susceptible strain, depending on the insecticide and the conditions used. The enzyme de-alkylated parathion and methylparaoxon, but there was no de-arylation of parathion, although it is important in other strains. In vitro, paraoxon and methylparaoxon were also hydrolysed, and this is not so in the susceptible strain. The high resistance present in the original strain seems to be due to joint action of at least three mechanisms.     

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.     

Comparison of insecticide absorption and detoxification in larvae of the bollworm,Heliothis zea,and the tobacco budworm,H. virescens

Rates and nature of metabolism of several insecticides were compared in larvae of the bollworm, Heliothis zea (Boddie), and the tobacco budworm, H virescens (F.). Both species are resistant to organochlorine compounds. In addition, H. virescens is resistant to carbamate and organophosphate insecticides. All test insecticides were metabolized by larvae of both species under in vivo conditions. In most instances, metabolism was more rapid in H. virescens larvae.A series of in vivo and in vitro assays implicated high levels of NADPH-dependent oxidative enzymes as a major metabolic factor in resistance. Other work demonstrated that H. virescens larvae absorbed insecticides at a slower rate than did H. zea larvae. High resistance to organophosphates present in H. virescens may be due to interaction of factors for increased rate of detoxification and decreased rate of absorption.     

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.     

CNS insensitivity to pyrethroids in the resistant kdr strain of house flies

Solutions of tetramethrin, RU 11679, or cismethrin caused uncoupled convulsions in 30–40 min in exposed thoracic ganglia from S_NAIDM house flies at concentrations down to 10^?10M: whereas these same compounds at 10^?6M concentrations failed to produce poisoning symptoms when perfused onto the exposed ganglia of the kdr strain of house fly. The pyrethroid analogs examined had a negative temperature coefficient of action on the exposed thoracic ganglia from S_NAIDM flies. DDT and GH-74 possessed positive temperature coefficients of action on the exposed thoracic ganglion of susceptible house flies. It is concluded that the central nervous system of the kdr strain of house fly is resistant to pyrethroid action; furthermore, the resistance appears to be widespread throughout the house fly nervous system, involving sensory, motor, and central neural elements.     

The mechanism of resistance to lindane and hexadeuterated lindane in the Third Yumenoshima strain of house fly

The factors which cause lindane resistance in the Third Yumenoshima strain, a strain of house flies highly resistant to insecticides, were studied using hexadeuterated lindane. Hexadeuterated lindane has the same physicochemical properties as lindane, but the former is much less biodegradable than the latter. The LD₅₀ ratio of lindane to hexadeuterated lindane in this strain, deuterium isotope effect on LD₅₀ values, was larger than that in S_NAIDM, a susceptible (nonresistant) strain. The penetration rates of labeled and nonlabeled lindane through the insect cuticle were about the same for both strains. Thus, penetration rate does not cause resistance. The metabolic degradation of lindane in the resistant strain in vivo occurred much faster than in the susceptible strain. This was also the case for lindane degradation processes in vitro such as microsomal oxidation and glutathione conjugation. In both strains, significant isotope effects were observed in the degradation rates in vitro of labeled and nonlabeled lindane. Therefore, principal biodegradation and detoxication pathways should include reactions which cleave the CH bonds. When the much less biodegradable d₆ counterpart of lindane was applied to both strains, the susceptible strain became much more highly intoxicated than the other within 20 to 30 min. This indicates that a combination of both greater degradability and probably lower sensitivity at the action site are the main factors underlying resistance in the Third Yumenoshima strain.     

Possible involvement of herbicide sequestration in the resistance to diclofop-methyl in Italian biotypes of Lolium spp.

The mechanism of resistance to diclofop-methyl in three Italian populations of Lolium spp. (two resistant and one susceptible) was investigated. The major proportion of R-1 (Tuscania 1997) and R-2 (Roma 1994) plants (approximately 80%) survived after herbicide treatment by emitting new tillers from the crown. Both resistant (R-1 and R-2) and susceptible (Vetralla 1994) Lolium spp. populations were target-site sensitive. No difference in diclofop-methyl absorption by shoots of resistant and susceptible biotypes was observed. At the dose corresponding to 1× the recommended field rate, a relatively higher metabolism was found in R-2 biotype. In contrast, at the doses 2× and 10× the field rate no difference in herbicide metabolism between susceptible and resistant biotypes was observed. At all the three herbicide doses (1×, 2×, and 10× the field rate) 48 h after the treatment (HAT), the total amount of metabolites produced by wheat was more than three times higher than that produced by resistant and susceptible ryegrass biotypes. At the doses 1× and 2× the field rate, the herbicide translocation was different in the susceptible biotypes compared to resistant biotypes. The total amount of the radiolabel found 48 HAT in culm and root was approximately twice in susceptible biotype than in resistant biotypes. Susceptible and resistant ryegrass biotypes differed in the capability of their roots to acidify the external medium. Susceptible biotype acidified the external solution at approximately 6 times the rates of the resistant biotypes. In the present study, the mechanism responsible for resistance in the investigated resistant biotypes was not univocally identified. Indirect evidence supports the possible involvement of herbicide sequestration or immobilization.