Phenolic secondary metabolites from Acorus calamus (Acorales: Acoraceae) rhizomes: the feeding deterrents for Spodoptera litura (Lepidoptera: Noctuidae).

Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is one of the most destructive pests of various crops cultivated in Thailand. Spodoptera litura larvae, at early stages, attack the leaves and feed on every part of infested crops in later stages. Acorus calamus essential oil contains toxic asarones, which are generalistic cytotoxic compounds. However, the present study is the first attempt to look at safer metabolites from the rhizomes that could deter insect feeding. The objective was to use such compounds as safer residues on crops that would prevent the feeding of herbivorous lepidopterans. Accordingly, phenolic metabolites were isolated and evaluated to establish the feeding deterrence against polyphagous S. litura larvae. Methanol extract of A. calamus, chrysin, and 4-hydroxy acetophenone compounds were the most effective feeding deterrents with FD50 of 87.18, 10.33, and 70.77 µg/cm2, respectively, after 4 h of feeding on treated kale leaves in a no-choice leaf disc assay. Chrysin also reduced carboxylesterase activities (1.37-fold), whereas A. calamus methanol extract reduced glutathione-S-transferase activities (1.44-fold). Some larvae were also seen dead if they consumed the treated kale leaves. Feeding deterrent activity in the methanol extract of A. calamus was due to chrysin and 4-hydroxy acetophenone. The large-scale utilization of such compounds could help develop feeding deterrent strategies in the integrated pest management of lepidopterans.

Insecticidal and growth inhibitory effects of some thymol derivatives on the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) and their impact on detoxification enzymes.

BACKGROUND: Thymol is a known natural product with insecticidal activity against several insect species. A recent study on structural modifications of thymol to thymyl esters and their efficacy against Spodoptera litura suggested that such an approach could develop generalized novel insecticides/insect growth inhibitors and requires further studies to establish the efficacy against lepidopterans. RESULTS: Thymol and structurally modified eight esters were evaluated against beet armyworm, Spodoptera exigua using the topical application. Thymyl butanoate was the most toxic compound with a median lethal dose (LD50 ) of 2.33 and 1.62 µg/larva after 24 and 48 h posttreatment, respectively. All thymyl esters were potentially better than the parent compound thymol, except thymyl dibromoacetate, in their efficacy against Spodoptera exigua. Essentially, there were three levels of activity vis-à-vis the compounds used, that is, with the LD50 range of 1.5 to 5.0, 7.0 to 15.0, and > 20 µg/larva, respectively. Ovicidal activity and reduction in larval growth were also determined by treating third instars at sub-lethal doses, that is, LD50 doses of second instars. Thymyl butanoate treated larvae inhibited glutathione S-transferase, carboxylesterase, and acetylcholinesterase activities, whereas the other thymyl esters induced these enzymes. CONCLUSION: Thymyl butanoate exhibited higher toxicity against Spodoptera exigua and is the first to report about > 15.5× more toxicity than thymol and > 6.5× than thymyl cinnamate, which suggests that the efficacy was species-specific versus the chemical structural variation of the esters. © 2021 Society of Chemical Industry.

The Study of Isolated Alkane Compounds and Crude Extracts From Sphagneticola trilobata (Asterales: Asteraceae) as a Candidate Botanical Insecticide for Lepidopteran Larvae.

The antifeedant and contact toxicity of Sphagneticola trilobata L. (Asterales: Asteraceae) extracts and isolated alkane compounds were investigated. Leaves of S. trilobata were sequentially extracted with hexane, dichloromethane, ethyl acetate, and methanol. Each extract and the compounds isolated were evaluated against the third instars of Spodoptera litura (F.) (Lepidoptera: Noctuidae), Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), and Plutella xylostella L. (Lepidoptera: Yponomeutidae). Ethyl acetate extract and isolated alkanes were feeding deterrents as well as contact toxins against all the three species evaluated (FI50 ~ 0.27-2.34 mg/ml; LD50 ~ 0.88-4.2 µg/larvae for ethyl acetate extract, and FI50 ~ 0.06-4.35 mg/ml; LD50 ~ 0.72-3.54 Ethyl acetate extract for isolated alkane). Impact on detoxifying enzymes was variable. The ethyl acetate crude extract reduced carboxylesterase activity in S. litura and P. xylostella while in S. exigua the enzyme was induced. In contrast, glutathione-S-transferase activity was induced in S. exigua but no significant difference in P. xylostella and S.litura was observed. Our results suggest that the S. trilobata extracts have multiple biological activities that contribute to the toxicity in lepidopterans. Variable enzyme responses to the products evaluated in different lepidopteran species also confirm that some species-specific inductions do occur, suggesting the possibility of resistance development in the future, which cannot be summarily ignored. However, for this detailed biochemical studies are required. Multiple bioefficacies of S. trilobata makes it a potential botanical for further exploitation on larger scale so that field potential can be established in any integrated pest management (IPM) system.

EFFECTS OF AZADIRACHTIN ON CUTICULAR PROTEINS OF SPODOPTERA LITURA (LEPIDOPTERA: NOCTUIDAE) VIS-A-VIS THE MODES OF APPLICATION.

Azadirachtin is a known botanical insecticide with multiple modes of action. Whether these effects have any relation with the modes of application, specifically during ecdysis process, has been the objective of the present study and accordingly the impairment, if any, among cuticular proteins of Spodoptera litura (Fab.) was determined. Azadirachtin was applied topically, via injection or oral administration. Azadirachtin administered via injection and topical applications severely impaired the ecdysis by 86.67 and 80.0%, respectively. However, this impairment via oral administration was significantly lower (73.33%). Using SDS-PAGE, the cuticular proteins were determined for treated insects under all the three modes of application. In all cases 6 protein bands (MW 9-34 kDa) were identified using markers as standard. In all treatments 3 induced proteins (MW. ~16, 20 and 23 kDa) and 1 reduced protein (~19 kDa) were observed. In case of the topical treatment a different induced protein of ~18 kDa was identified. The change in cuticular proteins, their possible role in ecdysis impairment vis-a-vis the mode of application of azadirachtin is being correlated. This will help in understanding the mode-of-action at cuticular level and also will allow developing a suitable application strategy under field conditions in insect pest management.

Acute, sublethal and combination effects of azadirachtin and Bacillus thuringiensis toxins on Helicoverpa armigera (Lepidoptera: Noctuidae) larvae.

The efficacy of neem (1500 ppm azadirachtin (AI)), Delfin WG, a biological insecticide based on selected strain of Bacillus thuringiensis Berliner (Bt) subspecies kurstaki, and Cry1Ac protein, either individually or in combination, were examined against first to fourth instar Helicoverpa armigera (Hübner) larvae. Using an oral administration method, various growth inhibitory concentrations (EC) and lethal concentrations (LC) were determined for each bioagent. Combinations of sublethal concentrations of Bt spray formulation with azadirachtin at EC50 or EC95 levels not only enhanced the toxicity, but also reduced the duration of action when used in a mixture. The LC20 and LC50 values for Cry1Ac toxin were 0.06 and 0.22 microg ml-1, respectively. Bt-azadirachtin combinations of LC50+EC20 and LC50+EC50 result in 100% mortality. The mortality also was significant in LC20+EC20 and LC20+EC50 mixtures. These studies imply that the combined action is not synergistic but complimentary, with azadirachtin particularly facilitating the action of Bt. The Bt spray-azadirachtin combination is more economical than combinations that involve isolating the toxic protein, as the Bt spray formulations can be combined in a spray mixture with neem. These combinations may be useful for controlling bollworm populations that have acquired resistance to Bt as they may not survive the effect of mixture. Azadirachtin may be useful as a means of reducing the endotoxin concentrations in a mixture, to promote increased economic savings and further reduce the probability of resistance development to either insect control agent.

Bioefficacy and mode-of-action of some limonoids of salannin group from Azadirachta indica A. Juss and their role in a multicomponent system against lepidopteran larvae.

Biological activities of the salannin type of limonoids isolated from Azadirachta indica A. Juss were assessed using the gram pod borer Helicoverpa armigera (Hubner) and the tobacco armyworm Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Inhibition of larval growth was concomitant with reduced feeding by neonate and third instar larvae. All three compounds exhibited strong antifeedant activity in a choice leaf disc bioassay with 2.0, 2.3 and 2.8 microg/cm(2) of 3-O-acetyl salannol, salannol and salannin, respectively deterring feeding by 50% in S. litura larvae. In nutritional assays, all three compounds reduced growth and consumption when fed to larvae without any effect on efficiency of conversion of ingested food (ECI), suggesting antifeedant activity alone. No toxicity was observed nor was there any significant affect on nutritional indices following topical application, further suggesting specific action as feeding deterrents. When relative growth rates were plotted against relative consumption rates, growth efficiency of the H. armigera fed diet containing 3-O-acetyl salannol, salannol or salannin did not differ from that of starved control larvae (used as calibration curve), further confirming the specific antifeedant action of salannin type of limonoids. Where the three compounds were co-administered, no enhancement in activity was observed. Non-azadirachtin limonoids having structural similarities and explicitly similar modes of action, like feeding deterrence in the present case, have no potentiating effect in any combination.

Biological activity of volatile di-n-propyl disulfide from seeds of neem, Azadirachta indica (Meliaceae), to two species of stored grain pests, Sitophilus oryzae (L.) and Tribolium castaneum (Herbst).

Head space volatiles, including 73% di-n-propyl disulfide, were collected from freshly crushed neem seeds. This compound along with previously reported diallyl disulfide (di-2-propenyl disulfide) were toxic when applied topically or as a fumigant to Tribolium castaneum adults and 8-, 12-, and 16-d-old larvae, and Sitophilus oryzae adults. Di-n-propyl disulfide significantly decreased the growth rate and dietary utilization with moderate inhibition of food consumption in both insects. The total coefficient of deterrence for this compound ranged between 68.5 and 178.6, which suggests that it has medium to very good deterrent activity vis-à-vis the treatment concentration and instar. Di-n-propyl disulfide and diallyl disulfide presented a similar effect on efficiency of conversion of ingested food, which is reduced 3-fold; this implies that both compounds are physiological toxicants. Present studies clearly demonstrate that di-n-propyl disulfide could be a potent toxicant, fumigant, and feeding deterrent for stored grain pests, if a suitable formulation and application procedure are developed.

Antifeedant effects of the limonoids from Entandrophragma candolei (Meliaceae) on the gram pod borer, Helicoverpa armigera (Lepidoptera: Noctuidae).

The biological activity of the limonoids prieurianin and epoxyprieurianin isolated from Entandrophragma candolei (Harms) (Meliaceae) and their respective acetates was assessed using the gram pod borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). The compounds exhibited strong antifeedant activity in a diet choice bioassay with epoxyprieurianin acetate being most effective with 48.3 ppm deterring feeding by 50% (DI50) and prieurianin the least effective (DI50 = 91.4 ppm). The effect on growth of larvae was concomitant with the reduced feeding by neonate and third instar larvae. In nutritional assays, all the compounds reduced growth and consumption when fed to larvae without any effect on efficiency of conversion of ingested food (ECI), suggesting antifeedant activity alone. No toxicity was observed nor was there any significant affect on nutritional indices following topical application, further suggesting that prieurianin-type limonoids act specifically as feeding deterrents.

6beta-hydroxygedunin from Azadirachta indica. Its potentiation effects with some non-azadirachtin limonoids in neem against lepidopteran larvae.

The biological activity of 6beta-hydroxygedunin isolated from Azadirachta indica A. Juss. was assessed using the gram pod borer, Helicoverpa armigera (Hubner), and Asian armyworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), alone and in combination with other limonoids, gedunin, salannin, nimbinene, and azadirachtin. The compound exhibited growth inhibitory activity in artificial diet bioassays, with 24.2 and 21.5 ppm, respectively, inhibiting growth by 50%. This efficacy was higher in comparison to gedunin (EC(50) = 50.8 and 40.4 ppm), salannin (EC(50) = 74.5 and 72.0 ppm), and nimbinene (EC(50) = 391.4 and 404.5 ppm). Azadirachtin, however, remained the most active neem allelochemical against both insect species. Nutritional assays clearly demonstrated that, though relative consumption and growth rates of fourth instar larvae were reduced, gedunin-type compounds induced physiological toxicity, evident by reduced efficiency of conversion of ingested food (ECI) in feeding experiments. Salannin and nimbinene, on the contrary, induced concentration-dependent feeding deterrence only. In feeding experiments, combinations of the compounds revealed that when azadirachtin was present in a mixture, EC(50) values did not deviate from the individual efficacy of azadirachtin (0.26 and 0.21 ppm, respectively) against H. armigera and S. litura larvae. However, a combination without azadirachtin did show a potentiation effect with potent EC(50) values among structurally different molecules, i.e., when salannin or nimbinene was combined with 6beta-hydroxygedunin or gedunin rather than structurally similar salannin + nimbinene or 6beta-hydroxygedunin + gedunin. Obviously, azadirachtin being the most active compound in neem is not synergized or influenced by any other limonoid, but other non-azadirachtin limonoids were more potent in specific combinations vis-à-vis the structural chemistry of the compound. It is obvious from the present study that potentiation among non-azadirachtin limonoids having explicitly two different modes of action, such as feeding deterrence and physiological toxicity, may be playing a significant role in the potentiation effect.