In this study, by A. adenophora aqueous extracts acting on the slug, the extracts were found to have insecticidal activity against slugs of different life cycles (stages). The insecticidal activities exhibited by the extract can be explained by the presence of flavonoids, saponins and tannins, which are secondary metabolites in the extract.
A. adenophora extract had an effect on the hatching rate of L. maximus, indicating that the molecular structure of the active ingredient of the plant extract is simple to make it easier to penetrate into the membrane pores involving embryos. However, according to previous studies, the high molecular weight of the egg membrane hinders the passage of the drug [26]. Since the mortality rate of eggs exposed to the extract for 48 h was higher than that of 24 h, indicating that a longer exposure time will have a greater effect on the hatching rate of slug eggs.
Temperature may be a factor affecting egg hatching rate and slug growth and development [27]. The high or low temperature will reduce the vitality of molluscs, which the phenomenon of drowsiness occurs when the environmental conditions are inappropriate. In addition, The high temperature increased the oxygen consumption of molluscs, thus accelerating the rate of energy consumption in vivo [28]. The temperature in this study was controlled at 21–24°C.
Souza et al confirmed that newly hatched snails were more sensitive to the shell of the cashew nut extracts [29]. Gohar et al reported the molluscicidal activity of Callistemon viminalis (Sol. ex Gaertner) G.Don ex Loudon against newly hatched Biomphalaria alexandrina [30]. However, in this study, the adult L. maximus exposed for 24 h and 48 h showed a signifcant reduction in food intake, movement slowing, darker body surface color and somnolence after 45 days, which led to some differences in growth and development and sexual maturity between the control group. The larvaes of L. maximus were less sensitive to extracts than adults, which may be due to the fact that pedal glands of larvaes secrete more mucus than that of adults [31]. Slugs respond to exogenous toxic substances by reducing their exposure to exogenous substances through mucus secreted by foot glands [32, 33].
Under stress, superoxide anion radicals, hydroxyl radicals, hydrogen peroxide and other reactive oxygen species accumulate in insects, which will cause damage to organisms. However, there are protective enzymes composed of SOD, POD and CAT in insects, which can scavenge excess free radicals. The three enzymes coordinate with each other to maintain the dynamic balance of free radical metabolism in the body, so as to protect the insect from injury or reduce injury [34]. Studies have shown that Nilaparvata lugens and Sogatella furcifera significantly increased the activities of SOD, POD and CAT in rice plants infected with rice black-streaked dwarf disease or southern rice black-streaked dwarf disease [35]. The results showed that the semi-lethal concentration (LC50) of A. adenophora extract showed an inhibitory effect on SOD activity in slug, an inhibition-activation-inhibition effect on POD activity, and an activation-inhibition effect on CAT activity.
CYP450, GSTs and AchE play an important role in decomposing exogenous toxicants and maintaining normal physiological metabolism [36]. Among them, GST can catalyze the sulfhydryl coupling between the electrophilic groups of toxic substances and reduced glutathione, and increase its hydrophobicity so that it can be easily excreted from the body [37]. It was found that the AchE activity of the 2nd instar larvae of Lymantria dispar was significantly inhibited by methylvitamin salt, and the GSTs activity was activated after 24 hours of treatment [38]. Carbendazim can inhibit the whole AchE of earthworm (Earthworm) and activate the whole GSTs. It can induce the activation of CYP450 at low concentration and inhibit it at high concentration [39]. Under the stress of sublethal concentration of tetrameric acetaldehyde, the activity of AchE increased at first and then decreased in the gill and abdominal foot of Pomacea canaliculata, while the activity of liver and intestine decreased at first and then increased [40]. The results showed that after treated with sublethal concentration of A. adenophora (LC50), the activities of CYP450 and GSTs in slugs were inhibited in varying degrees, but the activities of AchE were increased. Because the content of flavonoids in the extract was the highest, acetylcholinesterase was released in slugs, resulting in the cause of dehydration and death.
In this study, in the LC50 extract of A. adenophora, the content of flavonoids was the most, with an average content of 4.87%, followed by saponins and tannins, 1.85% and 1.35%, respectively. It can be inferred that flavonoids played a major role in the extract, but it can not be denied that the compounds with less content did not have an effect. If the flavonoids in Ageratina adenophora extract can be separated and extracted separately, whether it will have a more significant killing effect on slugs remains to be further confirmed in follow-up studies.
Flavonoids may play a role by inhibiting the detoxification system of snails. Some studies have found that the CYP450 enzyme of the land snail Cantareus aspersus changed after contact with tobacco leaves [41, 42], in this study, the activity of CYP450 enzyme decreased after L. maximus contact with Ageratina adenophora extract. This enzyme is part of a family of proteins that play a role in detoxification and can degrade various foreign substances [43]. In addition, some studies have shown that flavonoids can activate AchE [44], release a large amount of acetylcholinesterase in slugs, destroy the special mucus produced by slugs, and lead to rapid dehydration of slugs, destruction of body surface cells, and death of a large amount of body fluids [45]. In previous studies, it is concluded that saponins have hemolytic toxicity, which can destroy the cell membrane of red blood cells and cause cytoplasmic extravasation, leading to the disintegration of red blood cells [46, 47]. It can be inferred that the killing effect of the aqueous extract of R. roxburghii on L. maximus may be related to the hemolytic type of saponins. Tannin is a kind of polyphenol which contains several hydroxyl groups, which can complex with protein and cause precipitation, thus inactivating it [48, 49].
Slugs exposed to toxic substances or gases cause physiological stress, resulting in reduced carbohydrate and glycogen reserves [50], which begins to deplete the body's protein as a supplementary energy source [51]. Studies have shown that the hemolymph protein and uric acid levels of molluscs exposed to molluscs increase, indicating that the protein as an energy source is degraded [50]. However, the protein may precipitate after complexation and cannot be used as an energy substance [52], resulting in energy imbalance and death in the slug.
At present, chemical agents are still mainly used in the control of molluscs in agriculture in China ([3]. The disadvantages of chemical control in agriculture are becoming more and more obvious. in order to control this species more effectively, it is important that plant extracts are effective in all stages of life of the target organism. In addition, in order to be better applied to agriculture, factory production, preparation and treatment of extracts, selectivity at low concentrations, and harm to humans, the environment and crops need to be considered. more comprehensive studies on plant secondary metabolites in terrestrial slug species need to be strengthened.