Biochemical response of Mediterranean flour moth, Ephestia kuehniella Zeller (Lep.: Pyralidae) to the toxicity of trans-anethole

Essential oils are a variety of compounds, and their monoterpenoids, as the main constituents may impair insects' physiological and behavioral functions and could thus be applied in pest management. In addition to causing damage to stored products, the Mediterranean flour moth, Ephestia kuehniella Zeller, 1879 (Lep.: Pyralidae), is actually an easy model to grow in the laboratory and is one of the suitable insects to determine toxicity and in vivo interactions of xenobiotics with physiological systems of insects. In the present study, in order to better understand the mechanism of action of trans-anethole as one of the most important secondary metabolites of the Apiaceae family, the oral toxicity of this compound was evaluated on the activities of digestive and detoxifying enzymes, and metabolites involving in intermediate metabolism of the fourth instar of E. kuehniella.

Fourth instar larvae of E. kuehniella were randomly selected and separately exposed to 1 g of the artificial diet containing 1, 2, 4, 8, and 16 μL/g of trans-anethole while the control larvae fed on the diet containing acetone. The bioassay test was performed in 3 replicates with 10 larvae per replication. Mortality was determined after 24 h and LC50 value was calculated by POLO-plus software. Ephestia kuehniella larvae were exposed to the LC50 value of trans-anethole to determine its effects on the enzymatic and non-enzymatic components. After 24 of exposure, the larvae hemolymph, midgut, and fat bodies were extracted. Hemolymph samples were immediately centrifuged at 15000 × g at 4°C for 20 min, while samples of midgut and fat bodies were initially homogenized by a glass pestle and then centrifuged under the same conditions. The hemolymph samples were used to assay the detoxifying enzymes and enzymes of intermediary metabolism, while the midgut samples were used to assess the digestive enzymes. Fat body samples and head capsules were also used for energy reserves.

The LC50 value of trans-anethole against the larvae was 7.03 μL g-1. Significant decrease in the digestive enzyme activities (α-amylase, α and β-glucosidase, lipase) and specific proteases (trypsin, chymotrypsin, elastase, amino and carboxy peptidase) were observed. In contrast, the activity of the detoxifying enzymes (esterases, and glutathione S- transferase) increased in the treated insects. The activities of amino transferases (alanine, aspartate, and gamma-glutamyl) significantly increased in the treated larvae by trans-anethole. Significant decreases in lactate dehydrogenase and phosphatase (acid and alkaline) were observed after treatment. Moreover, the amount of storage macromolecules (total protein, glycogen, and triglyceride) in the treated insects was significantly decreased compared to the control.

Our research revealed that trans-anethole is a toxic compound to E. kuehniella by reducing the survival and digestive efficiency of the larvae. Moreover, trans-anethole significantly enhanced the detoxifying enzymes activities. When the larvae were exposed to LC50 of trans-anethole, the digestive activity was reduced due to the cytotoxic effects of trans-anethole on epithelial cells of the larval alimentary canal. Our results demonstrated that trans-anethole might have a promising potential to develop as a safe compound to suppress E. kuehniella population. For the practical use of trans-anethole to control E. kuehniella, it is required to determine the accurate mode of action, and using appropriate formulations to increase its efficacy in long-term applications.