In recent years, the knowledge about the potential alternatives for synthetic pesticides, such as plant derivatives, has significantly increased [1
]. Natural products are already in use on the markets worldwide, for example, in organic agriculture [4
]. Some of the active compounds from extracts have been changed structurally to obtain more persistent substances, such as the neem tree (Azadirachta indica
) extract, which has progressively become increasingly popular [5
], or pyrethrins obtained from Chrysanthemum cinerariifolium
, which in the 1970s became a source for the third class of synthetic pyrethroids [6
]. One of the most difficult aspects of crop protection is the application of substances to stored commodities. This step requires easily degradable compounds that are relatively nontoxic to mammals. From an economic point of view, substances that are used in crop protection should be inexpensive and their emission should not lead to their environmental accumulation or to toxic effects on nontarget organisms. Natural substances that are produced by plants to deter herbivores meet the criteria outlined above. They not only are effective in the control of pest insect populations, causing up to 100% mortality at concentrations as low as 0.5 mg/cm3
after 24 h of fumigation [7
], but also exhibit selective action against various species [8
]. The effectiveness of plant derivatives may strongly depend on the dosage, method of extraction and solvent [10
], as well as the application method [13
]. Given that there are an enormous number of possible plant extracts containing active ingredients and that there are many solvents that can be used, there is a great need to study the possible combinations to obtain the most effective substances for specific species. The majority of research has focused on direct toxic effects, which can be used to limit pest populations [1
]. However, understanding the mode of action of plant derived substances can be useful in planning strategies for pest control. The nature of the effects on both the target and nontarget tissues can be tested by exposure of the target species to low doses of the plant-derived substances. Although the effects may be subtle and even statistically non-significant, some discrete malformations and malfunctions may be observed before the massive toxic effects appear when low concentrations of these substances are used [14
]. Furthermore, the sublethal doses and concentrations can reveal the first effects and the mode of action at the level of organs, tissues, or even cells.
In this study, we tested the extract that was obtained from the unripe fruits of Solanum nigrum
, a plant commonly distributed in Europe, which is known to produce glycoalkaloids (GAs). Previous studies have demonstrated that the extract caused larvicidal effects in mosquitoes (Diptera), such as Culex vishnui
], Culex quinquefasciatus
], Culex pipiens
, Aedes caspius
], Anopheles culicifacies
, Aedes aegypti
], and Anopheles stephensi
]. Toxic effects have also been found in the fruitfly (Drosophila melanogaster
: Diptera) [22
] and the Colorado potato beetle (Leptinotarsa decemlineata
: Coleoptera) [23
]. S. nigrum
extract, in addition to its toxic effects, was reported to have promising anticancer [24
] and antimicrobial properties [25
]. The tested extract contains 10 GAs, but two, solasonine and solamargine, are present in greatest amounts [22
]. Studies have shown not only the toxic influence of glycoalkaloids on animal health [26
], but also the beneficial effects, such as anticancer properties [27
]. Since alkaloids have been reported as promising tools for pest management (for review see: [1
]), we decided to examine the extract from S. nigrum
fruits as well as pure solasonine and solamargine and to compare their effects on a model organism in ecotoxicological studies and a pest of stored products—the yellow mealworm beetle T. molitor
We addressed the following research questions:
Do the extract and the pure GAs cause lethal toxic effects and disturb the development of T. molitor larvae?
Do the tested substances cause malformations of the cells in the exposed tissues?
Do the tested substances affect the biochemical parameters of the exposed tissues?
Do the tested substances affect the physiological parameters of T. molitor larvae?
Do the effects of the extract differ from the effects of pure GAs, and (if yes) what aspects of the toxicity may be caused by solasonine, solamargine or other compounds of the extract?
To answer these questions, we conducted some observational studies and tests of various levels of biological organization. This study included an analysis of the general toxic activity of the S. nigrum
extract given in the food on the growth of T. molitor
larvae. Since we had already observed some ultrastructural changes in response to exposure to Solanaceae
plant extracts [2
], we decided to test the ultrastructure of the midgut and fat body, which are important tissues for the ingestion and distribution of toxic agents within insect bodies. The midgut was directly exposed to the agents present in the ingested feed. To complement the changes that were observed with electron microscopy, biochemical assays of parameters, such as the content of lipids, glycogen, and proteins in the fat body were conducted. Next, further studies included the analysis of the influence of the extract and pure glycoalkaloids on the visceral muscles and myocardium contractile activity under in vitro conditions, to check their utility as possible factors affecting muscle activity. The modulation of muscle contractility of organs, such as the heart, hindgut, or oviduct may result in impaired development, food intake, and reproduction. Hence, the above mentioned parameters may be crucial for better understanding the toxic mode of action of the tested alkaloids, and they may also contribute to the more efficient application of plant derived substances in plant protection. Consequently, this may lead to the decreased use of both synthetic and natural substances in plant protection, with the benefits of limiting treatment of crops and food products and reducing environmental pollution.
In the present study, an expanded description of the effects that are caused by the extract of S. nigrum and pure GAs on T. molitor physiology was conducted. The tested substances do not appear to have acute toxic effects on the T. molitor larvae. In addition, the results differed between the extracts and pure GAs. The results showed that the exposure of the larvae to the tested substances caused slight changes in the body mass, ultrastructure of the midgut and fat body, and biochemical parameters of the fat body. However, it must be noted that these results were obtained over the relatively short observation periods that were used for the experiments. Hence, the observed effects often showed a trend, but the changes were not always statistically significant.
At the subcellular level, one can observe the very early effects, which appeared only in some cells. Therefore, the observed malfunctions and malformations can be used as bioindicators of stress caused by toxic substances. Perhaps, extended exposure or increased dosage would have given more significant effects. However, the aim of the study was to examine the direct, immediate effects and the mechanisms of the toxicity of S. nigrum
extract and pure GAs. Furthermore, sublethal effects are often very important, they can limit crop or stored food loss [2
] or affect the cost-to-benefit ratio similar to that obtained with synthetic insecticides [33
The weight of the larvae did not change significantly but some tendencies were observed. It appears that the plant-derived substances may cause bimodal effects. For example, exposure to the low concentrations of solasonine slightly decreased the larval gain in body mass, while higher concentrations did not cause such an effect. On the other hand, only the highest concentration of the extract decreased the body mass, and the lower concentrations increased the body mass. Alkaloids in high concentrations may deter herbivores from feeding, as usually they are present in unripe fruit. Glycoalkaloids, such as solamargine and solasonine, have been tested by Weissenberg et al. [8
] on Tribolium castaneum
, belonging to the same family (Tenebrionidae) as T. molitor
. The results showed that these compounds acted as growth inhibitors in larvae, but the experiment lasted 15 days. This finding also proves that GAs may be useful in plant protection in sublethal doses, thus limiting pest feeding. The results indicate that the plant must produce other substances that are responsible for the limitation of feeding and the growth of herbivores.
The effects on muscles differed between the tested substances. The application of the extract altered the contraction of heart, oviduct, and hindgut musculature, whereas the effects of solasonine and solamargine were much weaker, with solasonine decreasing the oviduct activity. These data suggest that neither solasonine or solamargine was responsible for the observed effects after application of the extract and indicate that plant extracts must contain other active substances in addition to the main GAs, perhaps alkaloids present in a lower concentrations, which affect muscle physiology. Both the glycoalkaloids may influence each other or interact with other ingredients of the extract. A stronger reaction of the insects to extracts than to single compounds has been described previously [12
]. Moreover, the opposite effect was observed after the application of the extract to the heart, where the contraction frequency decreased, as compared to that on the oviduct, where the frequency increased. This finding suggests that the extract has a different mode of action on the two muscle tissues, and its components may interact with different receptors. The obtained results are similar to those that were obtained by Ventrella et al. [34
], where black nightshade extract was used on the heart of Zophobas atratus
(Tenebrionidae), and at a concentration of 0.5 mM, it caused a reversible negative chronotropic effect.
The microscopic observations revealed the differences between the effects caused in both of the tissues. Perhaps the differences were caused by a longer time of exposure of the fat body than of the midgut. Columnar cells transport ingested substances to other tissues, and trophocytes store substances and play crucial roles in detoxification. Therefore, these cells can reveal more drastic effects. A similar phenomenon was observed in the case of exposure to other toxic substances, such as boric acid [35
] and tomato or potato leaf extract [36
]. Although the intensity of the ultrastructural malformations differed between the tested organs, species, and substances, some of those effects were similar: swollen nuclear envelopes and endoplasmic reticulum, cytoplasmic vacuolization, or swollen mitochondria have been reported for many toxic substances, including synthetic pesticides [16
] or plant-derived substances [15
]. These ultrastructural changes may be due to the increased production of reactive oxygen species, which has been reported for various toxic substances [35
]. It is noteworthy that the biochemical parameters of the fat body depend on the biochemical parameters of the hemolymph, which transfers compounds to and from the fat body. Therefore, in the near future, we plan to further observe of the hemolymph biochemical parameters, such as sugar and lipid levels, and to correlate the obtained results with those of the fat body biochemistry and ultrastructure. To compare the effects that are caused by the S. nigrum
extract and its pure GAs, both of the substances were given to the larvae and changes in the ultrastructure of the midgut and fat body cells were observed. While the extract caused a disruption of the nuclear membranes and cytoplasm vacuolization in the midgut, solasonine, and solamargine did not show any visible effects on these cells. This suggests that other glycoalkaloids present in the extract may be responsible for the observed effect or that solasonine and solamargine act synergistically in the extract or that the other compounds found in the extract play crucial roles in the process of membrane lysis by GAs.
Ultrastructural studies showed changes in the chromatin condensation that could lead to the altered expression of genes. The results suggest that solamargine is mostly responsible for chromatin condensation. For both tested concentrations of solamargine, the correlations with the effects on chromatin condensation were the strongest observed in this study. On the other hand, bimodal effects were observed in the case of solasonine in the fat body cells. Therefore, the correlation coefficient cannot be treated here for the whole range, as it measures linear relationships. The condensation of chromatin was also reported for cancer cells that were exposed to solamargine [43
]. Solasonine and solamargine are both glycosides of solasodine (aglycone, the true alkaloid). They are characterized by sugar moieties and aglycone moieties. The electrophilic behavior of these compounds is regulated by the polarity of the sugar moieties and by the presence of oxygen and nitrogen ion pairs in the aglycone moiety, which does not contain aromatic rings. These features are responsible for GA-induced membrane disruption and interactions with nucleic acids, which result in DNA malformations [44
]. Again, the weak effects that were observed in this study probably reflect the relatively low doses of applied substances that affected the nuclei in both the tested tissues. However, the teratogenic activity of GAs has been proven [46
] and it may be due to the disruptive effect of GAs on membranes and nucleic acids.
The results of the biochemical studies showed that the level of glycogen decreased significantly after the application of the extract at only one concentration 0.1% (Figure 15
). Glycogen is the first source of glucose during periods of starvation or detoxification; hence, one can suppose that the extract might have had a significant effect on the larvae. However, the weight gain of the larvae treated with this extract concentration was the highest among the used concentrations, which did not confirm the theory of starvation. Rather, glycogen, as an energy source, might have been used for detoxification and the vitality of the insects was not decreased. A slight decrease in the glycogen content of the fat body was also observed after application of the extract at a concentration of 10%, and the percentage of the larval weight gained seemed to decline. Perhaps a longer exposure would demonstrate whether this effect could be intensified. Such an effect suggests that S. nigrum
produces substances that can be used as deterrents. Satake et al. [49
] observed a decrease in the fat body glycogen content after starvation of the larvae of Bombyx mori
, and that can be an early bioindicator of the inhibition of food intake. Another place, where carbohydrates are stored by insects and are transported to or from the fat body, is the hemolymph, which was not studied in our research. Previous studies claimed that the hemolymph glucose level can be an indicator of fat body carbohydrate metabolism [50
]. The correlation between these factors results from the inhibition of glycolysis in the fat body by the key enzyme regulating this process present in the hemolymph—fructose-2,6-biphosphate. Solamargine caused an increase in the glycogen content, as compared to that of the control. The difference was significant after application of the concentrations of 7.23 × 10−7
M and 7.23 × 10−5
M. The increase in the glycogen amount may be connected with the increase in the food intake by the larvae, which corresponds with the percentage body mass gain after solamargine application. The results showed the highest body mass gain in the case of solamargine. However, in general, the content of glycogen in the fat body was higher after the application of both GAs than after the extract application. A significant difference was obtained between the extract 0.1% concentration and the equivalent concentration of solamargine, which suggests different modes of action in the extract.
extract significantly decreased the lipid content in the fat body (Figure 16
) after application of the 1% concentration. This result does not correspond with the gain in body mass, which increased when compared to that of the control. One supposes that the reason for this discrepancy may be, again, not the observed level of lipids in the hemolymph. Lipids from the fat body could have been moved to the hemolymph, which may have caused the decrease in the lipid levels of the fat body, without changing the body mass of the insects. Lipid mobilization is activated during starvation stress [50
], which supports the theory that food intake is inhibited by alkaloids. Significant differences were present not only between the level of lipids in the control and extract treatment groups, but also between the extract and solamargine treatment groups. These data show, similar to the case of glycogen, that in the extract, other substances can be present that modulate its mode of action and play a crucial role in the toxic action of the major GAs. Significant differences in the lipid content were obtained between both the GAs at all the tested concentrations. These results show different influences of both the GAs on the lipid metabolism in the fat body.
No significant changes in the protein content were observed after the application of the tested substances. Only slight changes in the amount of protein were present when compared to that of the control. However, the quantity of protein was measured, but their profiles were not assessed. We do not know if some proteins are specifically produced, which would not necessarily influence the general protein content of the fat body, but might greatly alter its metabolism. This is a very interesting question that we will address in future studies. Furthermore, the observed changes in the chromatin density of the fat body, as well as its other ultrastructural alterations, suggest that the metabolism of the fat body may change dramatically during exposure to the GAs and their detoxification.
The difference between the effects that were observed with the transmission electron microscope and the biochemical analyses is also of interest. The altered homogeneity of the lipid droplets, cytoplasm density, and therefore glycogen content, as well as the stored protein disintegration observed by microscopy, did not strongly correspond with the results of the biochemical analyses. The main reason for this difference is that the biochemical analyses were conducted on dry tissue. In addition, it is very likely that the ultrastructural changes occurred before they could be analyzed biochemically and before significant lethality appeared. These results suggest that the use of transmission electron microscopy as a tool for early changes in detection, especially when employing short tests periods is highly justifiable. Furthermore, this implies that ultrastructural malformations may be used as environmental and functional bioindicators of exposure to the low concentrations of stressors, or they may represent the early stage of exposure.