Control of the crop pest African cotton leafworm, Spodoptera littoralis (Boisduval), by chemical insecticides has led to serious resistance problems. Ajuga plants contain phytoecdysteroids (arthropod steroid hormone analogs regulating metamorphosis) and clerodanes (diterpenoids exhibiting antifeedant activity). We analyzed these compounds in leaf extracts of the Israeli Ajuga iva L. by liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) and thin-layer chromatography (TLC), and their efficiency at reducing S.littoralis fitness. First and third instars of S. littoralis were fed castor bean leaves (Ricinus communis) smeared with an aqueous suspension of dried methanolic crude extract of A. iva phytoecdysteroids and clerodanes. Mortality, larval weight gain, relative growth rate and survival were compared to feeding on control leaves. We used ‘4’,6-diamidino-2-phenylindole (DAPI, a fluorescent stain) and phalloidin staining to localize A. iva crude leaf extract activity in the insect gut. Ajuga iva crude leaf extract (50, 100 and 250 µg/µL) significantly increased mortality of first-instar S. littoralis (36%, 70%, and 87%, respectively) compared to controls (6%). Third-instar larval weight gain decreased significantly (by 52%, 44% and 30%, respectively), as did relative growth rate (−0.05 g/g per day compared to the relevant controls), ultimately resulting in few survivors. Crude leaf extract (250 µg/µL) reduced gut size, with relocation of nuclei and abnormal actin-filament organization. Ajug iva extract has potential for alternative, environmentally safe insect-pest control.

Control of the crop pest African cotton leafworm Spodoptera littoralis (Boisduval) by chemical insecticides has led to serious resistance problems. Ajuga plants contain phytoecdysteroids (arthropod steroid hormone analogs regulating metamorphoses) and clerodanes (diterpenoids exhibiting antifeedant activity). We analyzed these compounds in leaf extracts of the Israeli Ajuga iva L. by LC-TOF-MS and TLC, and their efficiency at reducing S. littoralis fitness. First and third instars of S. littoralis larvae were fed on castor bean leaves smeared with an aqueous suspension of dried methanolic crude extract of A. iva phytoecdysteroids and clerodanes. Mortality, larval weight gain, relative growth rate and survival were compared to feeding on control leaves. We used DAPI and phalloidin staining to localize A. iva crude leaf extract activity in the insect gut. A. iva crude leaf extract (50, 100 and 250 µg/µL) significantly increased mortality of first-instar S. littoralis larvae (36%, 70% and 87%, respectively) compared to controls (6%). Third-instar larval weight gain decreased significantly (by 52%, 44% and 30%, respectively), as did relative growth rate (–0.05 g/g per day compared to the relevant controls), ultimately resulting in few survivors. Crude leaf extract (250 µg/µL) reduced gut size, with relocation of nuclei and abnormal actin-filament organization. A. iva extract has potential for alternative, environmentally safe insect-pest control.

20Insects cause severe damage to numerous crops and their control relies on pesticides. 21Green control is becoming increasingly popular due to concerns about the negative 22 impacts of pesticides on the environment. Phytoecdysteroids are found in Ajuga plants 23and affect a wide range of insects at very low concentrations. Here we demonstrate that 24 crude extract from Ajuga iva alters the development of Spodoptera littoralis. 25Phytoecdysteroids may therefore be beneficial in IPM programs. 26 27 28 29 Abstract 30The African cotton leafworm Spodoptera littoralis, a major crop pest worldwide, is 31 controlled by chemical insecticides, leading to serious resistance problems. Ajuga plants 32contain phytoecdysteroids (analogs of arthropod steroid hormones that regulate 33 metamorphoses) and clerodanes (diterpenoids exhibiting antifeedant activity). We 34 analyzed phytoecdysteroids and clerodanes in leaf extracts of the Israeli Ajuga iva by LC-35 TOF-MS and TLC, and their efficiency at reducing S. littoralis fitness. Castor bean leaves 36were smeared with an aqueous suspension of dried methanolic crude extract of 37 phytoecdysteroid and clerodanes from A. iva leaves (50, 100 and 250 µg/µl). First and 38 third instars of S. littoralis larvae were fed with 1 treated leaf for 3 and 4 days, respectively. 39Mortality, larval weight gain, relative growth rate and survival were compared to feeding 40 on control leaves. To evaluate and localize A. iva crude leaf extract activity in the insect 41 gut, we used DAPI and phalloidin staining. Crude extract of A. iva leaves (50, 100 and 250 42 µg/µl) significantly increased mortality of first instar S. littoralis larvae (36%, 70% and 87%, 43 respectively) compared to controls (6%). Third instar larval weight gain decreased 44 significantly (by 52%, 44% and 30%, respectively), as did relative growth rate (-0.05 g/g 45 day, compared to the relevant controls). S. littoralis larvae were further affected at later 46 stages, with few survivors. Insect-gut staining showed that 250 µg/µl crude leaf extract 47 reduces gut size, with relocation of nuclei and abnormal actin-filament organization. Our 48 results demonstrate the potential of A. iva extract for alternative, environmentally safe 49 insect-pest control. 50 51