Role of Carboxyamidase in the in vivo metabolism of Chlorfluazuron in the Black Cutworm , Agrotis ipsilon ( Hfn . ) ( Lepidoptera : Noctuidae )

Triphenyl phosphate (TPP), a carboxylesterase inhibitor, has synergized chlorfluazuron in the resistant strain of the black cutworm but not in the susceptible strain. In vivo metabolism of C – chlorfluazuron revealed that the amount of radiolabeled major metabolites (2,6 – difluorobenzoic acid and 2,6 – difluorobenzamide) recovered from resistant larvae was four times higher than susceptible larvae. Addition of TPP did not affect chlorfluazuron metabolism pattern in the susceptible insects but it caused a significant reduction of the amount of detected metabolites in the resistant insects. Cleavage of chlorfluazuron molecule took place at one or both of the urea C-NH-C bonds which suggests the responsibility of carboxyamidase enzyme for chlorfluazuron metabolism. In vitro assays revealed that carboxyamidase activity was about four times higher in the resistant larvae than the susceptible ones which supports the hypothesis of the involvement of carboxyamidase in chlorfluazuron resistance in this insect pest.


INTRODUCTION
The black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae) is a serious cosmopolitan pest of corn and several agricultural crops.This noctuid is almost polyphagous that attacks a large number of vegetable crops (Hill, 1983).The control of this pest has become a serious challenge facing applied entomologists nowadays regarding the widening circle of resistance and cross resistance to most available conventional insecticides, including organophosphorus and carbamate insecticides.
Chlorfluazuron, a benzoylphenyl urea compound, is a promising insecticide which is highly effective as a chitin synthesis inhibitor especially on lepidopterous and coleopterous larvae.It is characterized by its unique action, safety to mammals and lack of cross resistance with conventional insecticides (Ishaaya, 1992).Chlorfluazuron has a relatively long half-life in insect bodies with a slow metabolism and elimination rate (Fahmy andMiyata, 1992 andSammour et al., 2008) which render it as a candidate insecticide for better pest control strategies.
Xenobiotic resistance in insects has evolved predominantly by increasing the metabolic capability of detoxication systems.Carboxylesterases, one of these systems, have been reported as the major metabolizing enzyme of benzoylphenyl urea chitin synthesis inhibitors (Xianchun et al., 2007;Elbert andNauen, 2000 andLi et al., 1998).
This study aims at clarifying the role of carboxylesterase metabolic enzymes in the in vivo degradation of chlorfluazuron and assessing the in vitro activity of the involved esterases in chlorfluazuron susceptible and resistant strains of the black cutworm.

Insects:
Chlorfluazuron resistant and susceptible strains, of the same origin, of the black cutworm, Agrotis ipsilon (Hfn.) were used in this study.The resistance ratio of the resistant strain was more than 100 folds that of the susceptible strain.Rearing technique adopted by Abdin, (1979) was carried out under incubation at a constant temperature 25°C and 70 % relative humidity.

Chemicals:
Chlorfluazuron and 14 C-labeled chlorfluazuron (654 MBq/nmol) which was uniformly labeled at the 2,5-difluorophenyl ring were used for larval treatment.Unlabeled proposed chlorfluazuron metabolites i.e. 2,6-difluorobenzoic acid and 2,6-difluorobenzamide along with the parent compound (chlorfluazuron) were separated by thin layer chromatography (TLC) to assess their RF values.Triphenyl phosphate (TPP) (95% pure) was used as a synergist.Silica gel 60 F 254 pre-coated plates were used for TLC and X-ray films were used for tracing radioactivity.Acetyl p-nitroanilide was used as a substrate for assaying the in vitro activity of carboxyamidase.

Synergism study
Chlorfluazuron was mixed with the same concentration of triphenyl phosphate (TPP) at the ratio of 1 : 1 (v/v).Castor oil leaves, Ricinus communis were dipped in this mixture or in chlorfluazuron alone, left to air-dry at room temperature then offered to the newly moulted 4 th instar larvae.

In vivo metabolism of 14 C-chlorfluazuron:
Early 4 th instar larvae were previously starved for 24 hours before experiment to ensure a whole consumption of the treated castor oil leaf.Two μ l of acetone solution containing 10 -3M of radiolabeled chlorfluazuron, or chlorfluazuron with TPP synergist at 1 : 5 ratio (w/w), was applied to a small castor oil leaf disc (3 mm X 3 mm).They were left to airdry and then put singly to each glass homogenizer.Larvae were then introduced singly to each glass homogenizer.Five larvae with three replicates were used for each treatment.Each leaf disc was totally consumed by each larva within four hours.The five larvae of each test were then collected in one of the glass homogenizers and incubated for 96 hours at 25 o C. The glass homogenizers were then rinsed three times with 1 ml of extraction solvent (acetonitrile : ethyl acetate : methanol : water, 1:1:1:1 v/v) and the rinsing solution was added to the glass homogenizer containing the larvae.The larvae were then homogenized in the same solution for one minute at 4 o C (El-Saidy et al., 1989).The products were extracted three successive times by addition of one ml of the extraction solvent then centrifuged at 15000 rpm for 5 minutes.The combined extracts were concentrated under vacuum at 35 o C using a rotary evaporator.
The extract was analyzed by TLC using a developing solvent system (ethyl acetate: toluene: acetic acid, 50:45:5, v/v).The plate was then exposed to X-ray film for one week to trace radioactivity.After that, the silica gel plate was cut to small pieces (5mm width), scratched and introduced to a scintillation glass vial containing 5 ml aqueous counting scintillant, ACS II ® then radioactivity was measured using a liquid scintillation spectrophotometer.Ultraviolet light was used to detect the authentic unlabeled compounds.

In vitro assay of carboxyamidase:
Fourth instar larvae of black cutworm were homogenized in 0.05M, pH 7.5 Tris-HCl buffer, centrifuged at 1000g and the supernatant of the homogenate was used as enzyme source.The assay of carboxyamidase with acetyl p-nitroanilide as a substrate was performed at 30 o C (Woods et al., 1979).

RESULTS
The synergistic effect of triphenyl phosphate (TPP), a carboxylesterase inhibitor, on chlorfluazuron was tested in chlorfluazuron resistant and susceptible strains of the black cutworm larvae and the results are shown in Table (1).TPP did not exert any remarkable synergistic action in the chlorfluazuron susceptible strain of the black cutworm.On the other hand, addition of TPP in the resistant insects has led to a clear synergistic effect on chlorfluazuron, evidenced by the sharp decline in the LC 50 value (from 445 to 48.11 ppm).
Table 2: In vivo metabolism of C 14 -chlorfluazuron 96 hours after larval treatment with and without the addition of TPP.
Addition of TPP did not have any remarkable effect on the amounts of major metabolites of chlorfluazuron in the susceptible insects (without TPP = 4.0 and +TPP = 3.1).On the other hand, addition of TPP in the chlorfluazuron resistant insects has led to a remarkable decrease in the amount of major metabolites (without TPP = 16.6% and + TPP = 3.9%, respectively).Carboxyamidase activity from the susceptible and resistant strains of the black cutworm was assayed in vitro using acetyl p-nitroanilide as a substrate.The results or this assay are shown in Table (3).
Table 3: In vitro, carboxyamidase activity in chlorfluazuron susceptible and resistant black cutworm. -

DISCUSSION
Triphenyl phosphate, TPP is known to be a typical carboxylesterase inhibitor (Ishaaya, 1992 andHaubruge et al., 2002).In the present study, addition of TPP has synergized chlorfluazuron (through inhibition of carboxylesterases) in the resistant insects but not in the susceptible ones.This suggests that carboxylesterases are involved in the mechanism of resistance to chlorfluazuron in the black cutworm.The same pattern of synergist was also reported in chlorfluazuron resistant and susceptible strain of the diamondback moth (Fahmy andMiyata, 1998 andWu QingJun, 1998).Usmani et al. (2001) have detected S,S,S-tri-n-butyl phosphorotrithioate (DEF)-sensitive enzymes that hydrolyzed trans-cypermethrin in adults and larvae of the black cutworm.DEF is also known to be an esterase inhibitor.
In order to confirm the role of carboxylesterases as a suspected mechanism of chlorfluazuron resistance in this insect pest, In vivo metabolism of 14 C-chlorfluazuron was investigated.2,6-difluorobenzoic acid and 2,6-difluorobenzamide were the major metabolites recovered and detected 96 hours after treating the larvae with 14 C chlorfluazuron (Table 2).The amounts of these metabolites were four times higher in the resistant insects than Role of Carboxyamidase in the in vivo metabolism of Chlorfluazuron in the Black Cutworm 69 susceptible ones.Detected metabolites labeled at the 2,6 positions suggest that cleavage of chlorfluazuron molecule took place at the C-NH-C urea bonds and this strongly supports the hypothesis of involvement of a TPP-sensitive enzyme system (presumably carboxyamidase) in the metabolic breakdown of chlorfluazuron in resistant insects.
Again, addition of TPP did not affect the pattern of chlorfluazuron metabolism in the susceptible strain, but it led to a significant decrease in the amount of major metabolites in the resistant insects due to the fact that TPP inhibits the carboxyamidase enzymes responsible for chlorfluazuron degradation.
The responsibility of carboxyamidase enzyme for chlorfluazuron degradation was further confirmed by the in vitro assay of carboxyamidase activity in chlorfluazuron resistant and susceptible insects using p-nitroanilide as a substrate.Results clearly show that carboxyamidase activity was about four times higher in the resistant insects than the susceptible ones which confirm the same hypothesis.
Only little information is available in the literature on the metabolic breakdown of chlorfluazuron in insect bodies.In most studies on resistance mechanisms, esterases have been confirmed to be involved in resistance by the addition of synergists (esterase inhibitors) or through the in vitro assaying of esterase activity in resistant and susceptible insects (El-Saidy et al., 1998;Haubruge et al., 2002 andXianchun et al., 2007).In this study the in vivo degradation pattern of 14 C-chlorfluazuron elucidates the cleavage of chlorfluazuron molecule, metabolites resulted, amount of unchanged parent compound and the involvement of carboxyamidases.
However, it is still necessary to investigate the pattern of metabolite distribution in both larval body and the excreta separately, since the amount of unchanged chlorfluazuron might then show difference due to the speed by which chlorfluazuron reaches its target site inside insect body or excreted with excreta.
Fig.1 Cleavage of chlorfluazuron molecule at the urea bond by carboxyamidase enzyme.