Xenobiotics are chemical compounds that are not natural components of a living organism but are exposed to them. They undergo metabolic processes, including absorption, distribution, biotransformation, and excretion. Xenobiotics can enter insects through the cuticle or eggshell or through the oral route. Then, the detoxification process begins. The compounds undergo modifications and degradations, including oxidation‒reduction reactions that increase their solubility and facilitate their elimination from the organism. Subsequently, the modified xenobiotic is excreted first from cells and finally from the organism by different types of transporters23.Many insects that are crop pests are exposed to glycoalkaloids. The purpose of this study was to perform a quantitative analysis of GAs at specific time points in different tissues after their injection to assess how they are distributed and accumulated throughout the insect as well as how quickly they are metabolised and eliminated by the insect.Under natural conditions, GAs can enter the insect body with food. Most studies involve feeding insects plants containing GAs or the preparation of an artificial diet supplemented with GAs13,14,15,18,24. However, without knowledge about the exact concentrations of these compounds in insects, it is impossible to understand the precise mechanism of action of GA. Thus, in this research, we applied SOL and CHA by injection to deliver the exact amount of the compound to the larvae. We tested samples of haemolymph, gut, and samples obtained from the remaining part of the insect body, which mainly consisted of the fat body, Malpighian tubules, and cuticle. Insect haemolymph is composed of fluid plasma containing haemocytes, and it circulates around other tissues in the insect body. The fat body fills the body cavity surrounding the digestive tract. It is immersed in the haemolymph, which facilitates the exchange of metabolites. It is the main organ involved in intermediary metabolism in insects. Therefore, it is not surprising that the applied GAs were detected in the fat body and haemolymph (Figs. 2, 4). However, the results also indicate that the GAs were transported to the insect gut (Figs. 2, 4). They might be transported from the haemolymph directly or/and with Malpighian tubules. This may be one of the explanations for the GA loss in the haemolymph over time. The Malpighian tubules are long tubes that are connected to the gut between the midgut and hindgut. They build up the excretory system, which is responsible for maintaining homeostasis25.When a xenobiotic enters an insect, it may undergo different detoxification reactions. The type of process depends on the chemical nature of the compound. GAs are classified as glycosides because they are composed of carbohydrate chains, and the aglycone is connected by a glycosidic bond. Glycosides, in turn, are acetal compounds with the general formula R2C(OR′)210. Acetals are obtained during the nucleophilic addition of two alcohol molecules to an aldehyde or ketone in the presence of an acid catalyst26. This condensation reaction is called acetalisation. Acetals are stable to bases, reducing agents, and nucleophiles; however, they break down in an acidic environment26. SOL and CHA are produced in plants through the cholesterol pathway through the glycosylation of carbohydrates (carbonyl compounds) with solanidine (alcohol)7,8. Additionally, GAs are derived from alkaloids.The biotransformation of GAs involves the hydrolysis process, which leads to different products. Carbohydrate groups are susceptible to hydrolysis in acids as well as to hydrolysis catalysed by enzymes. First, the detachment of particular sugar molecules leads to the formation of β-compounds, followed by the formation of γ-derivatives. The aglycon part, called solanidine, remains when all sugar chains are removed from the SOL or CHA molecule (Fig. 8)7,8. Hydrolysis of the glycosidic bond results in the loss of GA activity10; thus, biotransformation is an ability of many organisms (to avoid toxicity) as well as of different plant species (to eliminate autotoxicity risk), although nitrogen-containing chains often show high resistance to transformation. Many bacterial species have the ability to metabolise GAs by detaching carbohydrate groups or oxidising hydroxyl groups27. Plants and phytopathogenic fungi contain glycosidases that hydrolyse GA molecules. However, it is not known whether mammalian glycosidases also have such properties7,8. Glycosidases have been identified in insects of various orders, such as Orthoptera, Hymenoptera, and Coleoptera28. These enzymes were also reported in adults of T. molitor29 as well as in larvae30. However, contrary to expectations, none of the GA hydrolysis products were detected in this study. One possible explanation is that glycosidases present in T. molitor larvae have high substrate specificity and do not react with GA compounds.Figure 8Hydrolysis products of GAs.Insects have developed complex protection systems for defence against different xenobiotics23. Some toxic molecules can be metabolised into easily excreted compounds and eliminated from the body by the excretory system. Other xenobiotics can be modified into safer chemicals to facilitate their accumulation in tissues25. The tested GAs did not cause lethal toxicity for 10 days after application (Fig. 7); thus, insects can use a variety of strategies to deal with xenobiotics. One of the physiological adaptations of the organism to prevent poisoning is rapid intestinal passage, which protects against the accumulation of toxins31. Usually, cytochrome P-450 is involved in detoxification processes. It catalyses the oxidation of different xenobiotics, such as phytochemicals and insecticides32. For example, nicotine (another alkaloid of Solanaceae plants) given in food to Manduca sexta larvae induces cytochrome P-450 in the midgut epithelium33. Nicotine present in the haemolymph is metabolised, and the product of its oxidation is actively transported to the Malpighian tubules via a nonspecific alkaloid pump and is subsequently excreted25,33. The active transport of alkaloids to urine has also been reported in the larvae of Rhodnius prolixus and Pieris brassicae34. However, G-strophanthin, a cardiac glycoside, is also actively transported in Zonocerus variegatus, while in Locusta migratoria, it moves passively into the Malpighian tubules35. The detoxification enzymes act in the fat body and Malpighian tubules; however, they are the most active in the insect midgut25,36. Some species, such as the butterfly Danaus plexippus, maintain oxidising conditions in the midgut to defend against plant-derived compounds37. In Spodoptera litura, many detoxification-related genes were upregulated after tomatine treatment. In addition to the P450 genes, glutathione S-transferases, ABC transport enzymes, UDP-glucosyltransferases, and carboxylesterases were also upregulated, mainly in the midgut and fat body36. The molecular mechanisms involved in the action of all these enzymes in the Spodoptera genus were described in a review38, while the regulation of their expression in insects was described in the study of Amezian et al.39. In addition to the oxidation system, xenobiotics that enter the insect body can be sequestered and stored in the cuticle, glands or haemolymph25,40. For example, Oncopeltus fasciatus (Hemiptera) can sequester g-strophanthin41. Thus, one possible mechanism for the detoxification of GAs is oxidation and/or sequestration.Various xenobiotics are removed from insects in different ways. The elimination path depends on the type of detoxification process that takes time. This is the first study to analyse changes in GA concentration in T. molitor tissues over time. The change in the applied SOL percentage at different time points is shown in Fig. 2. The oxidation/excretion processes did not occur during the first 30 min after application because most of the GA was detected in the samples. At the end of the experiment (24 h after injection), 73.9% of the applied SOL remained in the larvae. On the other hand, the CHA content was much lower than the SOL percentage after 0.5 h (87.9% of the applied CHA) and 24 h after injection (63.1%) (Fig. 4). The results indicate that CHA is eliminated immediately after injection, while there is a delay in SOL elimination. Moreover, GA excretion processes are relatively slow because 24 h is not enough to remove all GA from the larval organism.As expected, the highest percentage of applied GA among the tissues tested was in the FB sample (Figs. 2, 4), which mainly contained a fat body and Malpighian tubules, due to the function of these tissues described above. Furthermore, these results are consistent with other studies because the lipid droplets in the fat body of the T. molitor larvae, as well as the G. mellonella larvae treated with the extract of S. nigrum, solasonine, and solamargine showed decreased homogeneity and lysis of the content of lipid droplets15,18. Thus, GAs can alter fat body structure. Moreover, SOL, CHA, and tomatine affect lipid metabolism19. Despite GA delivery by injection through the cuticle, the compounds were also detected in the gut tissue. This indicates that GAs can be transferred to the gut, where they are involved in GA metabolism and/or elimination. This finding was also reported by Li et al.24, who studied GA accumulation in the potato tuber moth P. operculella. In this study, the concentrations of GAs applied to food-containing insects were analysed in the head, foregut, midgut, hindgut, cuticula, and faeces of larvae. In the insects fed potato leaves, SOL was detected in the faeces and midgut, while CHA was excreted in the faeces and accumulated in the hindgut, head, midgut, and cuticle (in order of decreasing GA content). In the insects fed 0.3% GAs containing an artificial diet (1 mL of CHA and 0.75 mL of SOL), SOL was found in the midgut and faeces, while CHA was detected in the midgut, hindgut, faeces, head, and cuticle (in order of decreasing GA content). None of these GAs were detected in the foregut. Unfortunately, neither the haemolymph nor the fat body was studied therein. The excretion of GAs in faeces might be the most effective method for their detoxification. These results are consistent with our suggestions that SOL and CHA are excreted by T. molitor mainly through the faeces and cuticle.The concentration of GAs in insects depends on the type of tissue (Figs. 3, 5) and are eliminated at different rates (Fig. 6). The concentration of GAs in the FB sample was relatively low and did not change with time (Figs. 3, 5), thus showing a low affinity for that tissue. Moreover, their concentration change rates were almost constant (Fig. 6A). In the haemolymph, the SOL and CHA concentrations decreased within 8 h after application (Figs. 3, 5), and the elimination rate tended to be the highest at the beginning of the experiment (Fig. 6B). In the gut, similar to that in the FB sample, the GA concentration was also quite low, and there were no changes in GA concentration with time (Figs. 3, 5). On the other hand, during the first tested period (0.5–1.5 h), SOL was eliminated at the fastest rate in the whole experiment and significantly faster than CHA (Fig. 6C). A possible explanation for this finding might be that GAs present in the haemolymph are transported to the gut (directly or/and with the Malpighian tubules), maintaining a constant, maximum level. This result can also be explained by the direct transfer of these compounds to the cuticle. Considering the whole insect, the tested GAs were eliminated from the larval body throughout the entire experiment (Fig. 6D). Thus, in addition to GA transport between tested tissues, SOL and CHA must be eliminated outside the body, for example, via faeces. These results corroborate the findings of24, who reported GA excretion in faeces as well as during moulting. It is possible that the amount of GA in the gut as well as in the fat body would decrease when it reached the saturated concentration in the haemolymph. The observed changes might also be attributed to the sequestration of some of these plant secondary metabolites in the insect body.In the present study, SOL and CHA, which are toxic to the larvae of T. molitor, were injected into beetles, and the percentage amounts of GAs were analysed in different tissues within 24 h at specific time points. Tested GAs were reported in the gut, haemolymph, and remaining tissues together (mainly the fat body and Malpighian tubules), with the highest percentage in the latter. The present study raises the possibility that SOL and CHA are not hydrolysed in the larvae of T. molitor by glycosidases because none of the hydrolysis products were detected in the tested samples. One possible mechanism for the detoxification of GAs is oxidation and/or sequestration. On the other hand, the GA concentration was the highest in the haemolymph. The SOL and CHA concentrations decreased in the haemolymph during the experiment, while they did not change in other tissues. Thus, they may be excreted by Malpighian tubules, with faeces or with cuticles during moulting. Moreover, GA excretion processes are relatively slow because 24 h is not enough to remove all the applied GAs from the larvae. Despite this, there were no lethal effects during the 10 days following GA administration. The rate of CHA elimination in the entire insect was the highest immediately after injection (0–0.5 h), while SOL was eliminated the fastest later (between 0.5–1.5 h). The presented results are significant because they facilitate the interpretation of the conducted research and future research related to the effects of GAs on insect metabolism. Further work is needed to explore the longer-term excretion of GAs in insects, as well as to evaluate the impact of the way in which insects are exposed to GAs on the detoxification of these compounds.
