Insecticidal Effects of Organotin(IV) Compounds on Plutella Xylostella (L.) Larvae. II. Inhibitory Potencies Against Acetylcholinesterase and Evidence for Synergism in Tests With Bacillus Thuringiensis(BER.) and Malathion

Features of pesticide synergism and acetylcholinesterase (AChE) inhibition (in vitro) were studied using a selected range of organotin compounds against the early 4th instar larvae of a highly resistant strain of the diamondback moth (DBM), Plutella xylostella, a major universal pest of cruciferous vegetables. Fourteen triorganotin compounds were evaluated for their ability to enhance the toxicity of the microbial insecticide, Bacillus thuringiensis (BT) and of the commercial insecticide, Malathion to Plutella xylostella larvae. Supplemental synergism was observed with triphenyl- and tricyclopentyltin hydroxides in combinations with Bacillus thuringiensis. Increased synergism was observed with an increase in the number of cyclopentyl groups on tin in the mixed series, Cypn Ph3-n SnX, where X = OH, and 1-(1,2,4-triazolyl). The combination of (p-chlorophenyl)diphenyltin N,N-dimethyldithiocarbamate at LD10 and LD25 concentrations with sublethal concentrations of Malathion as well as of tricyclohexyltin methanesulphonate at the 0.01% (w/v) concentration with Malathion exerted strong synergistic effects (supplemental synergism) with toxicity index (T.I) values of 7.2, 19.8 and 10.1, respectively. Studies on the in vitro inhibition of acetylcholinesterase prepared from the DBM larvae showed that while most of the triorganotin Compounds tested were without effect on the enzyme, compounds containing the thiocarbamylacetate or the dithiocarbamylacetate moieties demonstrated appreciable levels of inhibition, being comparable in efficacy to commercial grades of Malathion and Methomyl.


INTRODUCTION
Insecticide synergists have considerable practical importance in the control of arthropod pests by "making more efficient the economical use of insecticides, thereby increasing the spectrum of activity of an insecticide and restoring its activity in a resistant strain". The enhancement of the toxic effects of one compound by another or synergism, was first reported in pharmacological studies by Macht. 2 On the other hand, if the combination turns out to be antagonistic, it will result in reduced mortality of the test organism.
Author to whom correspondence should be addressed study. The R-strain was obtained locally from Kea Farm, Cameron Highlands and the larvae were reared in muslin-mesh cages of size 30x30x30 cm, at 28 + 2C with a constant relative humidity of 90 +/-6% and a photoperiod of 12:12 (L:D) without exposure to any insecticide. The adults were fed with drops of Holloway medium 4 on cellophane and the larvae were fed on fresh Brassica chinensis leaves. For the in vitro inhibition studies on ACHE, early fourth instar DBM larvae of the susceptible strain obtained from National Chung-Hsiang University, Taichung (Taiwan) were also included.

Insecticides
A total of eighteen organotin compounds, two conventional insecticides [Malathion (Gold CoinR,84% a.i.) and Methomyl (LannateR, 90% a.i.)] and one microbial biocide, Bacillus thuringiensis Berliner (BT) were used in the toxicological studies. The organotin compounds used in this study were synthesised according to established procedures 26 and were of analytical grade purity. The standard insecticides used were of technical grade quality.

Topical bioassay
Early 4th instar larvae in batches of ten, of average weight 28 +/-3 mg, were lightly anaesthesized with CO 2 and treated topically on the dorsal surface by using a Drummond microcap applicator with 1.0 pLof test solution. Treatments were carried out at six concentrations for each test compound, while the controls were treated with solvent (acetone) alone. For a complete test, 3 batches of ten larvae each were treated with each of the 6 doses of a given test compound. The treated larvae were then transferred onto fresh Brassica leaves and kept at 28 +/-C in plastic finger bowls provided with ventilated covers.
Mortality was assessed at 24h and 48h after the topical applications. Larvae that failed to respond to gentle mechanical stimulation were considered dead. The bioassay data were analysed by the Probit Method of Finney 27 to obtain the lethal dosage index values, LDso.
Determination of BT toxicity An aqueous suspension of ThuricideR, a F-Zuelling product containing Bacillus thuringiensis Berliner (BT) (1600 IU/mg) as biotic insecticide, together with a wetting agent (Teepol) was applied to both sides of the Brassica test leaf.
For each dosage of ThuricideR, the effects on the mortality of ten 4th instar larvae were assessed in triplicate and the mortality data were counted 48h after larval feeding on the treated leaf.

Determination of synergistic effects
In this study two methods for evaluating synergistic effects were employed. In the first method (Method A), a constant sublethal concentration [LDlo, LD25 or 0.01% (w/v)] of the triorganotin compound was applied topically on early 4th instar larvae, pre-starved for 6 hours, and the larvae were then released onto the BTor Malathion-treated leaves. The Malathion solutions were prepared from the commercial sample of Malathion diluted in distilled water containing a surfactant (" Teepol') to ensure complete wetting of the leaf surface. Four concentrations of BT and Malathion (LDlo, LD25, LD4o and LD, o) were used; for each concentration 100 pL of solution was applied to the leaf surface. Following air-drying, the leaves were kept with their petioles wrapped in moist cotton wool in plastic finger bowls provided with ventilated covers and at the temperature of 28 +_ 1C. Ten treated larvae were then released onto each leaf. Three replicates were performed for each BT and Malathion concentration.
In the second method (Method B), which more accurately reflects field exposure than the topical application method, both BT or Malathion and the organotin [fixed at a given topical dosage concentration of LDlo, LD25 or 0.01% (w/v)] were applied as a mixture on the test leaf, prior to the release of the pre-starved (6 h) larvae on its surface. For both bioassays the data procured at the end of 48h were corrected for control mortality using Abbott's formula,28 and analysed by Finney's probit method 27 to obtain the LDso (or LCso) value of the combined BT or Malathion triorganotin application. The toxicity index 2 (T.I) as defined by the formula below, was calculated.
Toxicity Index (T.I.) LDso (BT or Malathion) LDso (BT or Malathion + Organotin) Values of T.I. greater than unity are indicative of synergism, and values less than unity of antagonism.
In vitro assay of AChE activity and its inhibition by a selected range of triorganotin compounds The activity of acetylcholinesterase was measured by the colorimetric method of EIIman 3 using acetylthiocholine iodide (ASChl) as the substrate and 5,5 dithiobis(2-nitrobenzoic acid) (DTNB), as the reagent. The enzyme activity was measured by following the increase in absorbance at 412 nm arising from the formation of the thionitrobenzoate anion in the enzyme catalysed reaction.
Twenty larvae were homogenized in 30 mL of 0.1 M phosphate buffer (pH 7.0) using a glass homogenizer in an ice-bath. Homogenates were centrifuged for 20 min at 1,500 g. The supernatant was used as the enzyme source.
The reaction mixture consisted of mL enzyme solution, 1.8 mL of 0.1 M phosphate buffer, pH 7.0, 0.1 mL of staining solution (DTNB) and 0.1 mL of substrate (ASChl). The final concentration of substrate and DTNB in the reaction mixture were 0.5 mM and 0.3 mM, respectively. The incubation period was 6 min. Optical density was read on a Bausch & Lomb spectrophotometer at 41 2 nm against a blank provided by incubating an equivalent volume of the same mixture but omitting the enzyme.
Inhibition studies on the enzyme were performed using 28 triorganotin compounds along with the two commercial insecticides, Malathion and Methomyl, for comparison purposes. A series of six concentrations of the various compounds (10 .3 10 .8 M were tested. Three replicates were performed for each assay. From the optical density values, the percentage inhibition was evaluated using the formula: Abs. of control Abs. of test solution % Inhibition x 100 Abs. of control The results obtained were subjected to simple linear regression analysis using the statistical programme (Statsgraphic) to obtain 50% inhibition values (15o). Insecticidal Effects of Organotin (IV) Compounds on Plutella Xylostella Vol. 1, No. 1, 1993 (L.) Larvae. IIInhibitory Potencies Against ,4cetylchlolinesterase and Evidence for Synergism in Tests with Bacillus Thuringiensis (BER.) and

Malathion
Synergistic combinations require only sub-lethal doses of both chemical and biotic insecticides. Hence synergism has become a favourite topic of research since synergistic combinations not only offer many possibilities in the more potent use of available insecticides, but also, as a result of reduced amounts of chemical insecticides used, check rapid resistance development in the pest. Benz a classified synergism on the basis of mortality data into five types, viz. independent, subadditive, supplemental, potentiative and coalitive action synergism.
The diamondback moth, Plutella xylostella (L.), represents one of the most notorious cases of control failure caused by insecticide resistance. The high level of resistance of the larvae of this pest to all major groups of chemical insecticides4e, i.e. chlorinated hydrocarbons, organophosphates, carbamates, pyrethroids and benzoylphenylureas, as well as microbial insecticides a, such as the bacterium, Bacillus thuringiensis (Ber.), has prompted studies on other control measures such as the use of a synergist to regulate the moth population.
Piperonyl butoxide is a well established synergist which has been used in conjunction with a range of insectides in the control of a number of lepidopterous larvae, but is conspicously ineffective against the DBM larvae. 1 Indeed, no effective synergist has been found todate to overcome insecticide resistance in DBM. TM Applications of insecticides on a rotational basis or their use as mixtures are other possible approaches for overcoming the resistance problem. 14 Resistance mechanisms in DBM appear to be complex, and relate to several factors such as decreased penetration of insecticides, 15 enhanced detoxification by esterases 6 and glutathione S-transferase, 7's and reduced sensitivity of ACHE. 5'7'19 It was therefore of considerable interest to examine a selected range of triorganotin compounds for their potency as synergists when combined with the microbial biocide Bacillus thuringiensis and the commercial insecticide Malathion towards the DBM larvae. Fourteen triorganotin compounds were chosen for this purpose based on their acute toxicity data and antifeedant effects. 2 This paper also includes a study on the effects of organotins on the enzyme acetylcholinesterase (ACHE), which is known to be the target molecule in insects for the exertion of lethal effects by the organophosphate and carbamate group of insecticides. 2'22 One way in which insect populations become resistant to these insecticides is by evolution of an altered enzyme. 2a'2 Whereas organophosphates and carbamates have been extensively studied as potent AChE inhibitors, there are no reports todate of a systematic screening of triorganotin compounds substituted with organophosphate or carbamate moieties in them as potential inhibitors against this enzyme. This has prompted the additional investigation reported herein of the in vitro inhibition of the above enzyme, isolated from resistant strains of Plutella xylostella larvae, by a limited range of triorganotin compounds, selected in most cases, on the basis of their high acute larval toxicity. Included among the compounds are several which have previously been shown to be effective inhibitors of glutathione -Stransferase, 2s a major detoxifying enzyme present in relatively high levels in resistant strains of DBM larvae. 18

Insects
Early fourth instar larvae of a highly resistant DBM strain were used throughout this

Synergistic effects in tests with BT
The LDso values for a selected range of triorganotin compounds tested against early 4th instar DBM larvae (R-strain) are given in Table I CYP3SnCI (5) CypSnCI'Ph3PO (6) Cyp2PhSnNCH:NCH:N (7) 2.75 1.11 +/- The organotins were topically applied on pre-starved larvae at the pre-determined LDs dosage level, and the larvae were then released onto BT-treated test leaves.  (Table III) CypaSnCI (5) CypaSnCI'Ph3PO (6 In Method B, the organotin (at either the LDo or LD25 topical dosage level), although of a concentration not sufficient to cause larval mortality on the leaf, however, proved highly active in combinations with BT, leading to mortalities higher than expected on the basis of the sublethal concentrations of BT used. For tricyclopentyltin hydroxide, the toxicity index is about 3 at the oLfD7 and 0.01%(w/v) dosage levels and 2 at the LDlo level, whereas a higher toxicity index (Table V) is obtained when the test is performed by Method A. However, in practical applications in the field where the larvae are found on the leaves, the use of tricyclopentyltin BT mixtures at the LD_ s level may be anticipated to yield a better performance than that indicated by the data in MZethod B.
The percentage mortality data assessed by Method A are tabulated in Table VIII. For all the organotin compounds mortality values obtained were less than the algebraic sum of the single effects of Malathion and the specific organotin, but the values were, however, greater than the computed mortalities based on independent synergism (vide supra). This result is characteristic of subadditive synergism, and is corroborated also by the data in  The organotins were topically applied on prestarved larvae at the predetermined LDs, LDlo and 0.01%(w/v) dosage levels and the larvae were then released onto Malathion-treated leaves. LC4o or LCso concentrations with this compound at the LD2 concentration level led to larval mortalities on the leaf of 66.7, 73.3, 76.7 and 80.0 percent, respectively. Inasmuch as the mortality values are significantly higher than for the other cases, it is conceivable that supplemental rather than subadditive synergism might be operative with compound (14).
One possible reason for the increased efficacy of compound (1 4) is that it contains the dithiocarbamato ligand fragment, which latter is known to be insecticidally active in its own right. 31 Computations of Toxicity Index (T.I.) values (Table X)  It is to be noted that no larval mortality occurred at the end of 24h in the presence of organotin alone when applied on leaf at LD2s topical dosage concentration, whereas with Malathion, mortality was evident even at the end of 24h. Thus, in studies using Method B mortality was recorded at the end of 48h. The possibility that larval mortality recorded at the end of 48h could also be induced, at least partially, by starvation as a result of antifeedant effects exerted by the organotin compound under test cannot, of course, be excluded. The above result along with evidence for synergism with Bacillus thuringiensis dr Malathion presented herein suggests that the same series of compounds may also prove to be effective in combination with other classes of chemical or microbial insecticides. Further work in this direction should prove rewarding, including especially a biochemical study enquiring into the mode of synergistic action.

In vitro inhibition studies on AChE
The cholinesterases are a group of enzymes of which acetylcholinesterase (ACHE) is the most significant because of its role in the regulation of nervous impulses across neuron/neuron and neuron/target tissue synapses. In such a system, an action potential arriving at the distal end of an axon causes the release of the neurotransmitter acetylcholine which diffuses across the synapse to activate the cholinergic receptors of another neuron or the target tissue. As the enzyme AChE hydrolyzes acetylcholine, synaptic transmission ceases and the nerve membranes return to their resting potential and are prepared for the next stimulus. 32 AChE has important toxicological significance because it is readily inhibited by phosphate and carbamate esters that are commonly, used as insecticides. The inhibition causes nervous transmission to continue unchecked and results in a neuromuscular malfunction which can be lethal. 21'22 The potential of triorganotin compounds to inhibit acetylcholinesterase has been sparsely explored in the literature, the best documented work being that of Blum and Bower 33 who reported that triethyltin hydroxide was capable of causing rapid paralysis of house flies (Musca domestica) (L.) upon topical application. Triethyltin carboxylate esters, however, failed to inhibit cholinesterase but proved effective in blocking conduction in the isolated central nerve cord of the American cockroach, Periplaneta americana (L.). 33 In the present study, some twenty five selected triorganotin compounds were screened for their anticholinesterase activity using in vitro preparations of the enzyme derived from resistant and susceptible strains of DBM larvae. The results are presented in Tables XI and XII, where the 150 dose, expressed in moles L1, is the concentration of the test compounds that reduces the activity of the AChE to half the control level. In the majority of cases, the compounds are totally ineffective against the enzyme, with estimated 50 values well exceeding 10 6 moles L Noteworthy exceptions were provided by the compounds containing the thiocarbamyl and dithiocarbamyl moiety in the ester residue. Triphenyltin N,N-dimethylthiocarbamyl acetate, Ph3SnOC(O)CH2SC(O)NMe-'z is seen to be 103-fold more active than the corresponding dithiocarbamylacetate, PhzSnOC(O)CH2SC(S)NMe , but suprisingly a dramatic drop (over 106-fold) in activity attends the replacement of a tinbound phenyl group in PhoSnOC(O)CH SC(S)NMe 2 by a p-chlorophenyl group (Table XI). 2 This trend is not reflected =n the results from tests conducted on the enzyme derived from the suceptible strain of the DBM larvae, where a 10-fold increase in activity is noted for (p-CIC6H4)Ph2SnOC(O)CHSC(S)NMe^o ver the triphenyltin analogue (Table XII). This probably hints of an altered structure for A(hE in the resistant strain, but more work is necessary to establish this point. (L.) Larvae. 11Inhibitory Potencies Against Acetylchlolinesterase and Vol. 1, No. 1, 1993 Evidence for Synergism in Tests with Bacillus Thuringiensis (BER.) and Among the monothiocarbamates, no significant differences in act=vity are encountered upon changing the organic groups bound to nitrogen.

Malathion
A focussed study on the inhibition of AChE derived from the resistant strain of the DBM larvae was attempted using a range of triphenylstannylthiocarbamyl acetates at the 104M concentration. The percentage inhibition data (Table XIII)  The lowered activity of (p-CICH)Ph2SnOC(O)CH2SC(O)NHPh relative to the triphenyltin analogue is again apparent from the percentage inhibition data (1 5.81% vs 31.12%), although the difference is not as marked as that encountered with the dithiocarbamylacerates.
Returning to Table XI, it is apparent that the following organotin compounds have an inhibitory potency comparable to that of commercial grades of Malathion (15o 5