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Introduction
The Asteraceae family has about 380 genera in Mexico, with more than 3000 species currently known (Ezeta-Miranda et al., 2020), among them the Artemisia genus is receiving increasing attention for the biological and chemical diversity of its components (Carvalho et al., 2011). Artemisia ludoviciana commonly known as "estafiate" is a widespread species in Mexico (Damian-Badillo et al., 2010) since pre-Hispanic times (Andrade-Cetto, 2009). The stem of the plant is used in oral infusion for the treatment of parasitic diseases, stomach upset, diarrhea, painful discomfort, gallbladder malfunction, and diabetes (Lopes-Lutz et al., 2008). Different metabolites of Artemisia essential oil such as α-pinene, camphene, 1,8-cineole, camphor, borneol, nonanal, linalool, carvacrol, and p-α-dimethylbenzyl alcohol (Anaya-Eugenio et al., 2014), act as antioxidants, anti-inflammatory, (Kim et al., 2008) antibacterial, antiallergic, anticancer, (Nageen et al., 2011) and immunosuppressive, (Nam et al., 2013). However, there is little literature on studies of the biological effects of A. ludovicina on insects, from the first report we found that Smith et al. (1983) evaluated the effects of feeding Hypoclora alba and Menaloplus sanguinipes produced with Artemisia ludovicina leaves with trichomes and without trichomes, and later Durden et al. (2008) evaluated the effects of feeding A. ludovicin against neonate larvae of Cydia pomonella. On the other hand, the biological effects of other Artemisia species have been reported, such as Hwang et al. (1985) who reported the repellent activity of A. vulgaris on Aedes aegypti mosquitoes, and Tripathi et al. (2001) studied the contact and fumigation toxicity of A. annua against Tribolium castaneum. Likewise, Maggi et al. (2005) investigated the feeding inhibition of A. annua against Epilachna paenulata and Spodoptera eridania.Liu et al. (2010) reported the insecticidal activity of A. capillaris and A. mongolica on Sitophilus zeamais.Creed et al. (2015) evaluated extractions of A. arborescens on Cydia pomonella infestations and recently Hu et al. (2019) investigated the toxic and repellent activity of A. brachyloba essential oil against the insect T. castaneum. This work aimed to evaluate the toxic effects of methanolic extracts on the first instar larvae of Spodoptera frugiperda.
Material and Methods
Plant material
Plant samples (A. ludoviciana) were purchased from a convenience store of the brand ''Infusionate'' (produced and distributed by Planta de Vida S.A. De C.V., Mexico). Three hundred g of leaves and stems previously washed in distilled water were processed and dehydrated in a forced draft stove at 30 °C. These were placed in 1 L of methanol and left at room temperature for 7 days. Then were filtered and the solvent was removed with a rotary evaporator. The dried material was placed in a glass container at 5 °C.
Glass column chromatography
A glass column 21 cm long and 1.5 cm thick packed with 31 g of 230-400 mesh silica gel with a particle size of 63 µm (Sigma-Aldrich) was used. A mobile phase of 100% benzene (C6H6) to 100% ethyl acetate (C4H8O2) was used to obtain 10 fractions. In each fraction, the solvents were removed by evaporation and stored refrigerated at 5 °C.
Bioassay of mortality and weight in larvae
1mg/mL aliquots of each mobile phase were prepared using methanol as solvent, the samples were vortexed and a 100 μL drop was placed on the surface of the diet (soybean meal, wheat germ, yeast, agar, salts, and vitamins) in 10 2-ounce plastic cups with 5 replicates per treatment, one cup was used as a negative control to which only methanol was added and allowed to evaporate at room temperature for 2 h. One neonate larva was then placed in each beaker with a plastic lid and stored in paper bags at 28°C for 7 days. After some time, the mortality and weight of larvae in treatment and control were recorded.
Gas Chromatography-Mass Spectrometry (GC-MS)
Methanol extracts were analyzed using a 6890-5975 GC-MS system (Agilent Technologies) equipped with an HP-5 MS fused silica capillary column (30 m x 0.25 mm film thickness of 0.25 µm). GC-MS detection uses an electron ionization system with an ionization energy of 70 eV. The carrier gas was helium at a constant flow rate of 1 mL/min. The injector and mass transfer line temperatures were set at 250 °C and 280 °C, respectively. The injection volume was 2 μL of solution (1:100) and was analyzed under the following column conditions: initial column temperature maintained at 40 °C for 1 min, then raised to 250 °C at a rate of 3 °C/min and maintained at 250 °C for 20 min.
Results and Discussion
The mortality of each mobile phase was evaluated, with the crude and 90:10 phases showing the highest mortality of approximately 30% (Table 1).
Table 1 Mortality of methanolic extracts of A. ludoviciana.
| Treatment | Mortality (%) ± Standard Error | Tukey |
|---|---|---|
| Negative | 3.33 ± 3.33 | ab |
| crude | 26.67 ± 3.33 | c |
| 90:10 | 33.33 ± 3.33 | c |
| 80:20 | 6.67 ± 3.33 | ab |
| 70:30 | 0.00 ± 0.00 | a |
| 60:40 | 3.33 ± 3.33 | ab |
| 50:50 | 6.67 ± 6.66 | ab |
| 40:60 | 3.33 ± 3.33 | ab |
| 30:70 | 0.00 ± 0.00 | ab |
| 20:80 | 10.00 ± 0.00 | a |
| 10:90 | 10.00 ± 0.00 | b |
| 0:100 | 8.72 ± 1.73 | b |
ANOVA. p ≤ 0.05. Tukey multiple comparisons. Equal letters have the same mean.
Regarding the effect of weight in neonate larvae, in the 90:10 phase it was reduced almost by half compared to the negative, but there was a very significant reduction in the crude treatment compared to the other treatments. 2
Table 2 Larval weights in milligrams of S. frugiperda larvae in different treatments.
| Treatment | Weight ± Standard Error | Tukey |
|---|---|---|
| Negative | 0.0191 ± 0.0091 | c |
| Crude | 0.0035 ± 0.0017 | a |
| 90:10 | 0.0072 ± 0.0025 | b |
ANOVA p ≤ 0.05. Tukey multiple comparisons. Equal letters have the same mean.
Concerning the chromatographic analysis, 5 metabolites related to biological activity in insects were found: limonene, thujone, camphor, borneol, and borneol acetate.1
One of the first studies of Artemisia spp. on Lepidoptera was carried out by Maggi et al. (2005), where the percentage of feeding inhibition by an ethanolic extract at a maximum dose of 1.5 mg/cm² (2.4 mg/mL) against Spodoptera eridania showed a feeding inhibition percentage of 87.1% and a larval weight loss different from the control and a mortality of 50% at the same concentration (statistical data not shown). In the same study, the metabolite artemisinin was analyzed, which showed inhibition at low doses of 60 to 75%, suggesting that this metabolite is phytotoxic. Durden et al. (2008) carried out a test of antifeedant effects with extracts of A. absinthium, A. arborescens, and A. ludovicina at a concentration of 10 mg/mL, where all extracts showed these effects against Cydia pomonella larvae. However, the specific toxicity of the metabolites was not analyzed. On the other hand, Karahroodi et al. (2009) evaluated extracts of A. dracunculus and A. absinthium on the lepidopteran Plodia interpunctella at a concentration of 2 µL of essential oil in 2 g of food, causing a repellent effect of 40 and 64%, respectively.
On the other hand, Khosravi et al. (2010) estimated the feeding deterrent effect of methanolic extracts of A. annua between a concentration of 0.625 to 5 % against Glyphodes pyloalis, showing a deterrence of 60 % to 90 %. Hasheminia et al. (2011) evaluated LC50 against Pieris rapae calculating a concentration of 9.38 % with methanolic extracts of A. annua and deterrence of 29 % at a concentration of 0.625 %. Durden et al. (2011) tested the same insects focusing on two metabolites found in A. annua extract, artemisinin, and 1,8-cineole, at a wider dose range. The feeding deterrent effect at the 1 mg/mL dose of the crude extract was 61.3 %, while for artemisinin and 1,8-cineole it was 28 and 8.8 %, respectively. Knaak et al. (2013) estimated the LC50 of Artemisia absinthium essential oil to be 2.09 µL, while the LD50 by topical application was 5.51 µL against Spodoptera frugiperda, but repellency on first instar larvae in this lepidopteran was not observed. Creed et al. (2015) evaluated the metabolite α-thujone at 1 mg/mL which did not show a deterrent effect on Cydia pomonella, crude extracts of A. ludoviciana, A. annua, and A. absinthium evaluated at 10 mg/mL also did not show a deterrent effect. While the deterrence study of the metabolite α-thujone of A. arborescens between 1 to 300 mg/mL caused 90% deterrence, so also, the crude extract evaluated between 1 to 10 mg/mL showed similar deterrence percentages in all cases.
Obtained results indicate mortality against S. frugiperda in the methanolic extract of A. ludoviciana, which was around 30 % at a dose of 1 mg/mL, was not statistically significant, while Knaak et al. (2013) reported mortality towards this lepidopteran using Artemisia absinthium essential oil.
Regarding the antifeedant effect, a 50% reduction in the weight of treated larvae was observed compared to the negative control. This effect was also visible on the surface of the food, as the larvae did not consume the same area as the control and the amount of excrement in the vessels decreased with the food. These antifeedant effects were noticeable at 1 µg/mL, which is a low concentration compared to the literature cited. It is worth mentioning that there is currently no information on the biological activity of estafiate against codling moths, but existing information on different Artemisia species against various lepidopteran insects suggests antifeeding and feeding deterrent effects. It is worth mentioning that this type of biological activity can be used for plant protection of agriculturally important crops and also to interrupt the life cycle of Spodoptera sp. larvae as a strategy in biorational control.
On the other hand, MC-GC analysis detected several components such as limonene, thujone, camphor, borneol, and borneol acetate related to biological effects on insects. R-limonene is reported to have activity on Diptera, Hymenoptera, and Lepidoptera, leading to several adverse nutritional and reproductive effects on Spodoptera frugiperda larvae (Oliveira et al., 2021; Johnston et al., 2022; Cruz et al., 2017). While α-thujone in combination with camphor has an insecticidal effect on Lepidoptera (Chen et al., 2021), the compound borneol is also reported to have effects on decreasing pupation and emergence against the same species (Magierowicz et al., 2020), and borneol acetate have toxicity towards stored grain insects (Feng et al., 2020).
Conclusions
The methanolic extract of A. ludoviciana contains biologically active compounds against various insect pests, although the insecticidal effect on Spodoptera frugiperda is low, it has an antifeedant effect on S. frugiperda larvae. Hence it can be used in biorational control for crop protection achieving the design of a more economical, simple, and friendly formulation with the environment and living beings.
