Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae) (fall armyworm) is a pest native to the Americas that affects a variety of crops. Its control is based on chemical insecticides. However, this practice has been associated with changes in the susceptibility of pests to various insecticides. The use of plant products represents an eco-friendly alternative. The objective of this work was to evaluate the larvicidal activity of the chloroform extract of Senna crotalarioides (Kunth) H.S. Irwin & Barneby (Fabaceae) to control S. frugiperda. The chloroform extract of S. crotalarioides caused significant larval mortality, and reduced pupal weight and adult emergence. The analysis by gas chromatography coupled with mass spectrometry (GC-MS) revealed the presence of 22 compounds in the chloroform extract of S. crotalarioides leaves, with the straight-chain aliphatic fatty alcohol 1-octacosanol as the main component. This study revealed that the leaves of S. crotalarioides synthesize long chain alcohols, which increased the mortality of S. frugiperda in its larval stage, including the pupal stage. The extract also caused a decrease in the S. frugiperda pupal weight. The potential use of the chloroform extract obtained from S. crotalarioides and its principal chemical constituent is proposed as a promising alternative to control S. frugiperda.
The genus Spodoptera (Lepidoptera: Noctuidae) includes some of the most important insect pests that cause significant yield reductions and economic losses in the American and African continents (Aragón et al. 2011; Igyuve et al. 2018). Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae), commonly known as “gusano cogollero del maíz” (Spanish), fall armyworm, corn leafworm, and southern grass-worm, is a highly polyphagous pest that affects more than 180 crops, among which the following stand out for their importance in Western Hemisphere countries: Arachis hypogaea L. (peanut) (Fabaceae), Glycine max L. Merrill (soybean) (Fabaceae), Gossypium hirsutum L. (upland cotton) (Malvaceae), Linum usitatissimum L. (linseed) (Linaceae), Medicago sativa L. (alfalfa) (Fabaceae), Oryza sativa L. (Asian rice) (Poaceae), Phaseolus vulgaris L. (common bean) (Fabaceae), Saccharum officinarum L. (sugar cane) (Poaceae), Solanum lycopersicon L. (tomato) (Solanaceae), Solanum tuberosum L. (potato) (Solanaceae), Sorghum bicolor L. Moench (sorghum) (Poaceae), and Zea mays L. (maize) (Poaceae) (Hernández-Mendoza et al. 2008; Casmuz et al. 2010). In the case of maize, the larvae of S. frugiperda cause damage at all growth stages, including senescence (Rodríguez-del-Bosque et al. 2011). The presence of this indigenous insect in the Americas also has been reported in African cornfields (Goergen et al. 2016). Many yr ago, the control of Spodoptera species had been based on the use of conventional synthetic insecticides (approximately 3,000 tons of active ingredient per yr) (Blanco et al. 2014). However, the intense and non-rational use of these products has been associated with a strong selection pressure on insects, genetic variability (Pérez-Zubiri et al. 2016), and the development of insecticide resistance (León-García et al. 2012). This phenomenon limits the success of pest control in many countries. In addition, there is evidence of human intoxication due to exposure to the insecticides used in the management of Spodoptera pests (Barrientos-Gutiérrez et al. 2013). Alternative strategies have been proposed to control S. frugiperda, including the use of genetically modified crops (Aguirre et al. 2016), natural enemies (Nuñez-Valdez et al. 2008; Ordóñez-García et al. 2015), semiochemicals, and other natural product-based approaches (Guerrero et al. 2014).
The genus Senna Mill. (Fabaceae: Caesalpinioideae) comprises more than 350 species ( http://www.theplantlist.org) of herbs, shrubs, woody climbers, and tree species, unarmed or armed, distributed in a wide range of zones, with different climates and latitudes (Marazzi et al. 2006). Some species of the genus Senna are used as foods, ornamental plants, or with medicinal purposes (Mazzio & Soliman 2010), while others have become invasive species (Richardson & Rejmánek 2011; Singhurst et al. 2013), or are considered as noxious woody weed (Parolin 2005). Previous phytochemical studies have allowed the identification of a variety of compounds found in Senna spp., including alkaloids (Moo-Puc et al. 2007), anthraquinones (Branco et al. 2011), cardiac glycosides (Essiett & Bassey 2013), glucosides (sennosides) (Monkheang et al. 2011), naphthopyrones (Graham et al. 2004), phenols (Viegas Junior et al. 2013), saponins (Oluwole et al. 2016), triterpenes, (Luximon-Ramma et al. 2002), among others. The metabolic content of Senna species has been associated with a variety of biological properties, including insecticidal activity (Yagi et al. 2013; de Souza Tavares et al. 2014; Vasudev et al. 2015). Senna crotalarioides (Cassia crotalarioides Kunth [Fabaceae]) is an arborous species distributed in various states of Mexico (Estrada et al. 2004). Considering the wide range of secondary metabolites in the genus Senna and their insecticidal activity, the objective of this study was to evaluate the insectistatic and insecticidal activities of the chloroform extract of S. crotalarioides on the polyphagous lepidopteran S. frugiperda.
Materials and Methods
All reagents used in this study were of analytical grade and commercially available. Agar, brewer's yeast, L-ascorbic acid, and neomycin sulfate were purchased from Fisher Scientific (Thermo-Fisher Scientific, Waltham, Massachusetts, USA); acetone, ethanol, formaldehyde, methyl p-hydroxybenzoate, 1-octacosanol were from Sigma-Aldrich (St. Louis, Missouri, USA).
The aerial parts (leaves, stems, buds, pods, and seeds) of S. crotalarioides were collected in Sep 2017, from 10 random specimens in the locality of Comadres (22.616666°N, 100.4000000°W, 1640 masl), a municipality of Guadalcazar (state of San Luis Potosí, Mexico). The specimens of the plant were authenticated, based on macro- and microscopic features of the plant: texture, shape, apex and leaf margin, pods, seeds, stems, and floral structure, by the Biólogo José García-Pérez, Instituto de Investigación en Zonas Desérticas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, state of San Luis Potosí, Mexico. A voucher herbarium is preserved in the collection of the Isidro Palacios Plant Herbarium at the Universidad Autónoma de San Luis Potosí with the code number SPLM43012. The leaves were separated and dried in the shade at 27 ± 2 °C, for 15 d. Subsequently, the dry plant material was milled in a Thomas Model 4 Wiley™ mill to 1.0 mm particle size (Thomas Scientific, Swedesboro, New Jersey, USA). The ground material was placed in a 1 L flask with 500 mL of chloroform, and extracted under reflux conditions at 50 °C. Then, the supernatant was filtered under vacuum through a Büchner funnel (Corning Inc., Corning, New York, USA), and the solvent evaporated until dry under reduced pressure using a BUCHI R-210 rotary evaporator (Büchi, Flawil, Switzerland) to obtain the crude extract.
SPODOPTERA FRUGIPERDA TEST INSECTS AND DIET
The experiments were carried out in the Laboratorio de Compuestos Naturales Insecticidas, Universidad Autónoma de Querétaro, Querétaro, state of Querétaro, Mexico. Larvae of S. frugiperda obtained from the University of Querétaro were used throughout the study. The insects had been reared in the laboratory since 2012. Periodically, the population is replaced to avoid inbreeding. The artificial diet used to establish a laboratory population consisted of a mixture prepared with 30 g common bean grains and 90 g maize grains (ground in a Thomas Model 4 Wiley™ mill to 1.0 mm particle size), 20 g brewer's yeast, 10 g vitamin mix Lepidoptera # 722 (calcium pantothenate, crystalline biotin, folic acid, niacin, pyridoxine HCl, riboflavin, sucrose, thiamine HCl, vitamin B12, 1% mannitol) (Bio Serv, Flemington, New Jersey, USA), 17 mL of a 10% w/v ethanol solution of L-ascorbic acid, 2.5 mL formaldehyde, 1.7 g methyl p-hydroxybenzoate, and 0.6 g neomycin sulfate, mixed in 800 mL of boiled agar solution (12.5 g per L). Second instar S. frugiperda were reared according to the proposed methodology (Capataz-Tafur et al. 2007; Quintana-López et al. 2016) with minimal modifications. Under a laminar flow hood, in 25 mL disposable plastic containers with a lid (Envases Primo Cuevas, Ecatepec de Morelos, state of Mexico, Mexico), 1 larva of the second instar of S. frugiperda was deposited, the larva was fed with artificial diet (2–3 g cube), and replaced by a new one each wk until the larva completed its pupal stage. Larvae were maintained at 27 ± 2 °C, 70 ± 5% relative humidity (RH), and a 14:10 h (L:D) photoperiod, in a climatic chamber with a timer, and revised every third d. When molted to pupae after 24 h, the insects were collected and transferred to another plastic container. The plastic containers were closed with the lid to avoid contamination, and to prevent larvae from escaping until adult emergence. Twenty pupae were placed in each container.
Insect culturing and bioassays were run in the same experimental conditions, in a room kept at 27 ± 2 °C, 70 ± 5% RH, and a 14:10 h (L:D) photoperiod. Preliminary screening was performed by testing 5 logarithmic concentrations ranging from 0.5 to 5,000 ppm. For the final bioassay, the concentrations evaluated were 5,000, 4,000, 2,000, 1,000, and 500 ppm. Polyvinylpyrrolidone was used as co-solvent of distilled water to prepare all chloroform extracts of S. crotalarioides. The extracts were mixed with the larval diet ingredients during preparation. The extract was added when the temperature of the agar solution cooled to hand hot temperature (about 45 °C). Control larval diet was prepared by adding the same volume of distilled water and polyvinylpyrrolidone to the artificial diet. A 2 to 3 g cube of artificial larval diet was placed in each container. The artificial diet was replaced by a new one each wk. Bioassays were carried out using 20 second instars for each concentration and for the control, divided into 5 experimental units with 4 larvae each, selected randomly, distributed in 20 plastic containers with a larva each. The containers were covered with plastic lids and stacked close to each other. The larvae were maintained inside the same plastic containers at 27 ± 2 °C, 70 ± 5% RH, and a 14:10 h (L:D) photoperiod until reaching the pupal stage. The pupae were weighed 24 h after pupation (mg), and then each pupa was moved to another plastic container (3 × 3 × 3.8 cm) to allow the development of adults. The insecticidal and insectistatic parameters evaluated were mortality (%) of larval and pupal stages and cumulated, and duration from larva to adult (d). The median lethal concentration (LC50) of the larval population of S. frugiperda was calculated by using data from the total larval period.
GAS CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS
Samples of the S. crotalarioides extract were dissolved in distilled water. The gas chromatography-mass spectrometry analysis was performed using an Agilent 5973 inert Gas Chromatograph/Mass Spectrometer (Agilent Technologies Inc., Santa Clara, California, USA) equipped with an Agilent HP-5MS fused-silica capillary column (length 30 m; inner diameter 0.250 mm; film thickness 0.25 µm), coated with 5% phenyl-methylsiloxane, at 250 °C. Pure helium was used as a carrier gas with a flow rate of 1 mL per min. Split ratio was 2:1. The column temperature was initially 50 °C (for 3 min) and was gradually increased to 240 °C, at 3 °C min–1; this temperature was held for 2 min. The injector temperature was 250 °C and 1 L of samples was injected twice. The spectra were collected at 71 eV ionization voltages, and the analyzed mass range was 15 to 600 m per z. The identification of the components was confirmed by comparison of the retention indices with those of authentic compounds using the Kovats index (Kovats 1958), based on n-alkanes C6 to C26, and by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 11 mass spectral database. The relative percentage of the individual components in the crude extract from leaves of S. crotalarioides was expressed as percentage based on the peak areas obtained.
Data are expressed as the means ± standard errors of the mean for 4 replicates. Results were excluded from analysis if the mortality rate in the control samples was above 20%. In addition, if the percentage of larvae killed during each time interval in the control samples ranged between 5 and 20%, the mortality of treated samples was corrected using Abbott's formula (Abbott 1925).
Insecticidal activities of the chloroform extract of Senna crotalarioides leaves to control Spodoptera frugiperda.
where x = percentage mortality in the treated sample, and y = percentage mortality in the control.
The SYSTAT (vers. 9) analysis program (SYSTAT Software Inc., San Jose, California, USA) (Stein et al. 1997) was used to fit treatment concentration–response, and for calculating the LC50, 95%, lower and upper fiducial limits, and chi-square values by Probit analysis. Accumulated mortality at each concentration was expressed as the sum of the percentage of larval mortality plus the percentage of pupal mortality. The differences in the mean values were evaluated by analysis of variance (ANOVA). The Tukey test was used for all pair-wise multiple comparisons of groups.
ChemBioDraw Ultra 13 molecule editor (PerkinElmer, Waltham, Massachusetts, USA) was used for drawing chemical structures.
INSECTICIDAL AND JUVENOMIMETIC ACTIVITIES OF SENNA CROTALARIOIDES
The chloroform extract of S. crotalarioides caused an increase in S. frugiperda mortality (P < 0.001) during the development of the insects (Table 1). Comparison of the mean mortality at each concentration with the 0 ppm concentration (control) showed a concentration-dependent effect (Fig. 1).
Higher rates of mortality were obtained when higher concentrations of the chloroform extract were used. From 1,000 ppm and above, the chloroform extract of S. crotalarioides significantly affected S. frugiperda larvae, hindering their pupation. At a concentration of 2,000 ppm of chloroform extract, 45% of the larvae completed the larval stage, but only 30% were able to pupate and develop into adults. The chloroform extract of S. crotalarioides at 4,000 ppm caused 100% accumulated mortality. At the pupal stage, a concentration-dependent effect could not be observed.
The exposure to the chloroform extract of S. crotalarioides extends the duration of the larval stage of S. frugiperda, including the prepupal period. In particular, larvae exposed to 2,000, 4,000, and 5,000 ppm of chloroform extracts of S. crotalarioides took longer to reach the pupal stage compared to the control insects (Table 2). In the pupal stage, the most marked effects were observed when the larvae were exposed to 4,000 and 5,000 ppm of the extract, observing statistically significant differences when comparing the duration of the stages in relation to the control. On the other hand, exposure of larvae to the chloroform extract at concentrations higher than 1,000 ppm reduced the body weight of the pupae.
GAS CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRY ANALYSIS OF THE CHLOROFORM EXTRACT OF SENNA CROTALARIOIDES
A total of 22 compounds were identified in the chloroform extract of S. crotalarioides leaves using gas chromatography-mass spectrometry, by comparing their retention indices and mass spectra fragmentation patterns with the reference spectra of the National Institute of Standards and Technology library. Among these compounds were fatty acids, terpenes, aldehydes, esters, and primary aliphatic alcohols, which constitute 99.2% of the chemical components present in the extract. The retention times, peak areas (%), and retention indexes of these compounds are presented in Table 3.
Insectistatic activities of the chloroform extract of Senna crotalarioides leaves to control Spodoptera frugiperda.
INSECTICIDAL AND JUVENOMIMETIC ACTIVITIES OF 1-OCTACO-SANOL
The 1-octacosanol caused an increase in S. frugiperda mortality (P < 0.001) during the development of the insects (Table 4). Higher rates of mortality were obtained when higher 1-octacosanol concentrations were used. From 1,000 ppm and above, 1-octacosanol significantly affected S. frugiperda larvae, hindering their pupation. At a concentration of 1,000 ppm of 1-octacosanol, 35% of the larvae completed the larval stage, but only 15% were able to pupate and develop into adults. At the pupal stage, a dose-dependent effect could not be observed, starting with the lowest concentration tested.
The exposure to 1-octacosanol extends the duration of the larval stage of S. frugiperda. In particular, larvae exposed to 400, 600, and 1,000 ppm of 1-octacosanol took longer to reach the pupal stage compared to the control insects of the corresponding treatment (Table 5). In the pupal stage, the most marked effects were observed when the larvae were exposed to 1,000 ppm 1-octacosanol, observing statistically significant differences when comparing the duration of the stage in relation to the control (P < 0.001). On the other hand, the exposure of the larvae to 1-octacosanol at concentrations higher than 600 ppm reduced the body weight of the pupae.
The insecticidal effect of various Senna species has been evaluated to contol coleopterans that infest stored grains, and that produce important economic losses and affect their quality and safety. The n-hexane extract of the pods of Senna italica Mill. (Fabaceae) caused 100% mortality in adults of Callosbruchus analis F. (Coleoptera: Chrysomelidae) (Yagi et al. 2013). The leaf extract from Senna obtusifolia (L.) H.S. Irwin & Barneby (Fabaceae) showed repellency activity of class II (between 20.1–40%) to control adults of Sitophilus zeamais (Motschulsky) (Coleoptera: Dryophthoridae) (de Souza Tavares et al. 2014). For its part, the ethyl acetate extract of the seeds (EtOAc) and secondary metabolites of Senna tora (L.) Roxb. (Fabaceae) (= Cassia tora) showed an LT50 (h) of 1.080 for EtOAc, 1.743 for compound 1, and 1.687 for compound 2, as well as 20% and 35% oviposition deterrence activity at 200 and 300 µg mL-1 with EtOAc, 60%, 75%, and 75% with compound 1, and 60%, 70%, and 70% with compound 2 at 100, 200, and 300 µg mL-1, respectively. The antifeedant effects were determined for EtOAc showing less than 5% at 100, 200, and 300 µg mL-1, but with compound 1, this activity was 40%, 65%, and 80%, and 55%, 70%, and 75% with compound 2 at the same concentrations to control Callosobruchus maculatus (Fabricius) (Coleoptera: Chrysomelidae) (Mbatchou et al. 2018). On the other hand, certain Senna spp. contain proteinaceous inhibitors that deregulate proteolytic processes in insects (Vasudev et al. 2015), which induces a larval food stress that can affect the growth rate, development time, body weight, survival (Fürstenberg-Hägg et al. 2013), fecundity, and percentage of larvae hatching (Vasudev et al. 2015). The results of these studies have demonstrated that this genus synthetizes metabolites with potential insecticidal activity. In this work, the insecticidal activity of S. crotalarioides was evaluated to control the fall armyworm, considering that Mexico is the fifth largest maize producer, and S. frugiperda is the main pest that attacks this crop (Blanco et al. 2014), limiting the yield and productivity (Martínez-Jaime et al. 2018). A preliminary study indicated that S. crotalarioides displayed larvicidal activity to S. frugiperda (Quintana-López et al. 2016). The chloroform extract of the leaves of S. crotalarioides exerts its toxicity at the early instar stage of S. frugiperda. The most important toxic action was observed on larvae. The early instar affected is important because Spodoptera larvae died in the larval and prepupal period, particularly between the first and second instars (Montezano et al. 2014). This is important considering that the larval stage of S. frugiperda is the stage that causes the greatest damage to crops (Tavares et al. 2013). On the other hand, the exposure to the chloroform extract of the leaves of S. crotalarioides extends the duration of the larval stage. This response has been described as a compensatory action for the larvae to recover when feeding on a low-quality host and still be able to pupate and achieve a greater weight (Silva et al. 2017).
Chemical composition of the chloroform extract of Senna crotalarioides leaves.
Insecticidal activities of 1-octacosanol to control Spodoptera frugiperda.
Insectistatic activities of 1-octacosanol against Spodoptera frugiperda.
Analysis by gas chromatography-mass spectrometry allowed the identification of various chemical compounds within the chloroform extract of the leaves of S. crotalarioides. Major chemical constituents were 1-octacosanol (C28H58O) (63.245%, Rt 23.296 min), a primary 28 carbon atom saturated alcohol (Fig. 2), followed by 1-triacontanol (C30H62O) (9.472%, Rt 25.706 min), palmitic acid (C16H32O2) (5.281%, Rt 15.587 min), and octacosanal (5.209%, Rt 23.296 min). Other identified components appeared at a proportion of less than 5%. The lowest percentage content of peak area (0.075%) was for (9E)-octadecenoic acid (Rt 17.125 min). Some components identified by gas chromatography-mass spectrometry in S. crotalarioides chloroform extract, such as phytol, tetracontane, squalene, α-tocopherol, triacontanol, octadecadienoic acid, and stigmasterol, also have been identified in the species S. italica and Senna spp. (Gololo et al. 2016; Silva et al. 2016; Madkour et al. 2017).
Exposure to 1-octacosanol, the major component of the chloroform extract of S. crotalarioides, increased mortality of the S. frugiperda in the larval stage, including the pupal stage. Also, this C28-chain alcohol caused a decrease in the body weight of S. frugiperda pupae. Although there are no data on the insecticidal activity of 1-octacosanol, previous studies indicate that some long-chain alcohols deploy antifeedant activities (Ganassi et al. 2016; Aznar-Fernández et al. 2018), besides ovicide and larvicide activities (Sinniah 1983). The second most abundant compound in the extract (triacontanol) is a plant growth regulator that partially reverses the jasmonic acid-induced proteinase inhibition (Ramanarayan & Swamy 2004). On the other hand, it has been suggested that fatty acids, such as palmitic acid, possess insecticidal activity and inhibit the growth of the related species Spodoptera littoralis (Boisdu-val) (Lepidoptera: Noctuidae) (Farag et al. 2011). Finally, the C28-aldehyde, octacosanal, suppresses aggressiveness in some insects (Mizuno et al. 2018). The obtained results suggest that the major compounds identified in the chloroform extract of S. crotalarioides contribute significantly to the larvicidal and pupicidal activities.
The chloroform extract of S. crotalarioides caused significant larval mortality and reduction of the pupal weight and adult emergence in S. frugiperda. Chromatographic analysis using gas chromatography-mass spectrometry revealed that the leaves of S. crotalarioides synthesize long chain alkanes that increase the mortality of the S. frugiperda larval stage, including the pupal stage. The insecticidal and insectistatic evaluation of 1-octa-cosanol, as the major component of S. crotalarioides chloroform extract, is presented for the first time. These results can serve as a starting point for the development of botanical insecticides based on S. crotalarioides leaf extracts to be used in integrated pest management, and to reduce the use of synthetic pesticides and their negative effects on the environment.
Part of this work was supported by the Secretaría de Educación Pública (PRODEP, Grant number: UAQ-PTC-324), and Consejo Nacional de Ciencia y Tecnología (CONACYT).
The authors declare no potential conflicts of interest with respect to the research, authorship, and publication of this article.
All mandatory laboratory health and safety procedures were followed at all times during the experiment. The handling of the insects was conducted following the “Recommendations concerning insect handling and insect allergies,” https://www.biology.lu.se/internal/employment/work-environment/insect-handling.