Bioactivity of seed extracts from different genotypes of Jatropha curcas (Euphorbiaceae) against Spodoptera frugiperda (Lepidoptera: Noctuidae)

2.1 Collection of seeds

Wild plant seed samples were collected from three sites in two regions in the state of Puebla in September 2022. One site was in the municipality of Ahuehuetzingo (18.4876722 °N, 98.6374472 °W), and two sites were in the Technological University of Izucar of Matamoros: Utim 1 (18.6184222 °N, 98.4499833 °W) and Utim 2 (18.6180278 °N, 98.4501944 °W). In addition, another genotype was collected in the same month at the Center for the Development of Biological Products, Yautepec, Morelos (Ceprobi, 18.8264722 °N 99.0947222 °W). The seeds were allowed to dry under shade for a period of 24 h. Seeds were disinfected in 1 % (v/v) sodium hypochlorite, washed with distilled water, and ground in a mill (KMF 10 basic IKA WERKE; Wilmington, USA) to obtain a fine powder.

2.2 Seed extractions

Extractions were performed using reagent-grade hexane, acetone, methanol, and water representing solvents with different polarities (J.T. Bayer^® Aguascalientes, México) (Figueroa-Brito et al. 2021). One liter of solvent was placed in a beaker and 500 g of seed powder was added. The beakers were covered with aluminum foil and were allowed to stand for 72 h in a fume hood. This mixture was filtered (Whatman No. 1 filter paper) into a conical flask, and a volume of 150 mL of the mixture was poured into a 1 L flask, which was transferred to a Büchi rotary evaporator (rotavapor^® R-300 Meierseggstrasse, Germany) to remove the solvent under reduced pressure. The extracts were placed in a laminar flow fume hood for 72 h to completely remove the solvent. After this time, these extracts were redissolved in 1 mL of the extraction solvent and stored in the refrigerator for later use in bioassays.

2.3 Insect rearing

The initial origin of the S. frugiperda colony was from larvae collected in corn crops in the areas surrounding the Center for the Development of Biotic Products of the Polytechnic Institute (Yautepec, Morelos). The larvae collected from fields were analyzed for taxonomic characteristics to confirm the species was S. frugiperda (Higo et al. 2022). Diseased and parasitized larvae were eliminated. Healthy larvae were fed and developed on corn leaves in Petri dishes, where they pupated and then eclosed as adults. These adults were placed in waxy paper bags with a container with cotton moistened with a sugary substance (water + honey) for feeding. The adults copulated and laid masses of eggs that were placed in Petri dishes with moistened cotton swabs. When the larvae hatched, they were placed on an artificial diet. The diet formula was 800 mL of distilled water, 60 g diet (Product No. F0635; S.W. Corn Borer, Bio-Serv, Frenchtown, New Jersey, USA), 20 g sterile corn spike, 100 g ground corn, 40 g brewer’s yeast, 10 g vitamins (Lepidoptera fortification mix, Bio-Serv, Flemington, New Jersey), 10 g agar, 1.7 g sorbic acid (dissolved in 17 mL ethanol), 2.5 mL of formaldehyde, 1.7 g of methyl p-hydroxybenzoate, and 0.6 g of neomycin sulfate (Burton and Perkins 1972). Insects were maintained in a climatic chamber set to 25 ± 2 °C, 60 ± 5 % RH, and 12:12 h L:D.

2.4 Insect bioassays with seed extracts

The lethal concentration of each extract, causing 50 % and 90 % mortality in the population (LC₅₀ and LC₉₀, respectively) was determined through a diet ingestion bioassay. The concentrations of each treatment were 0, 100, 500, 1,000, 2,500 and 5,000 mg/kg of artificial diet, which were equivalent to 0, 100, 500, 1,000, 2,500 and 5,000 ppm. The concentrations used were determined from previous studies (Figueroa-Brio et al. 2021; Ramos-López et al. 2010). In addition, we carried out a preliminary bioassay to determine the use of these concentrations. The concentrations, extracts and genotypes were evaluated in a simple randomized experimental design.

Each extract was diluted in its extraction solvent (hexane, acetone, methanol or water) to achieve the desired concentration when it was mixed with the artificial diet and 5 ml of this mixture was added to plastic containers with adjustable lids measuring 3.0 × 3.5 cm in height and diameter and allowed to solidify for 24 h.

One larva was placed in the same plastic container and incubated at 25 ± 2 °C, 70 % relative humidity and 12:12 h L:D. A total of 30 neonate larvae were used per treatment where each larva was the experimental unit and each treatment had 30 repetitions. The dependent variables were the decrease in weight gain of larvae, pupal weight, the proportion of larvae that failed to pupate (larval mortality), the proportion of pupae that failed to eclose as moths (pupal mortality), and the duration of development of larvae and pupae (days).

2.5 Statistical analysis

A test for normality (Shapiro–Wilk W) and homoscedasticity (Bartlett test) was conducted for all the measured variables. One-way analysis of variance (ANOVA) and Tukey’s test (p < 0.05) were performed to identify potential differences among the treatments using Statistix 8.0 (Analytical Software, Florida, USA) (Analytical Software 2003). Probit analysis was used to calculate lethal concentration to 50 % larval mortality (LC₅₀) and lethal concentration to 90 % larval mortality (LC₉₀) values, using the BioStat statistical analysis program (version 5.8.1) (Ayres et al. 2007).

3.1 Biological activity of seed extracts of the Utim 1 genotype

Regarding larval weight gain, after seven days of growth, the weight gain of S. frugiperda fed on diet with 2,500 and 5,000 ppm concentrations of all the Utim1 seed extracts of J. curcas decreased. The acetonic extracts at 2,500 and 5,000 ppm decreased the larval weight gain of S. frugiperda by 57.1 % and 66.6 %, respectively compared with the control larvae weight gain (2.10 mg; F = 19.21; df = 20; p ≤ 0.0001; Table 1). In addition, the hexanic, aqueous, and methanolic extracts at 5,000 ppm also decreased larval weight gain by 57.1, 50.9, and 46.2 %, respectively (Table 1).

After 14 days, the larval weight gain of S. frugiperda was slowed down by all extracts at concentrations of 1,000, 2,500, and 5,000 ppm, except for the methanolic extract at 1,000 ppm. The acetonic, hexanic, and aqueous extracts at 5,000 ppm continued to be the most active by decreasing the larval weight gain by more than 70 % compared with larval weight gain of the control (40.81 mg; F = 125.33; df = 20; p ≤ 0.0001; Table 1).

The larval development time of S. frugiperda increased by 13 and 11 days with 5,000 and 2,500 ppm of all the extracts (except 2,500 ppm of methanolic extract), respectively, compared with the development of the control larvae (25 d; F = 2.63; df = 20; p ≤ 0.0039; Table 1).

Regarding pupal weight, there was a minor reduction in weight of 7.07 %, 5.8 %, 4.9 % and 2.6 % with 5,000 ppm of all extracts in relation to the control (158.48 mg; F = 25.74; df = 20, p < 0.0035). Also, the hexanic extract at 5,000, 2,500 and 1,000 ppm prolonged the development of the pupae by 4, 4, and 3 days in relation to the control, respectively (11 d; F = 2.05; df = 20; p ≤ 0.0042). Furthermore, the acetonic extract at 5,000 ppm prolonged the development of the S. frugiperda pupae by 3 days.

Larval mortality with all extracts (1,000 and 5,000 ppm) exceeded 56 % compared to the control (8 %; F = 113.74; df = 20; p ≤ 0.0001; Table 1). The methanolic and acetonic extracts at 5,000 ppm were the most toxic causing 72 % larval mortality, followed by the extracts: methanolic (1,000 and 2,500 ppm) and aqueous (5,000 ppm) with 68 % larval mortality.

Similarly, all the extracts from 1,000 ppm were toxic to pupae, causing ≥70 % mortality (control = 8 %; F = 125.60; df = 20; p ≤ 0.0001; Table 1). The methanolic extract at 5,000 and 2,500 ppm was the most active, causing pupal mortality of 84 and 80 %, respectively. The acetonic extract at 5,000 ppm caused 84 % mortality of S. frugiperda pupae (Table 1). The larval lethal concentration values, LC₅₀ and LC₉₀, were 1,775 and 3,740 ppm for the methanolic extract, 1,984 and 3,624 ppm for the aqueous extract, 2,191 and 4,740 ppm for the acetonic extract, and 2,398 and 5,205 ppm for the hexanic extract, respectively (Table 1).

3.2 Biological activity of seed extracts of the Utim 2 genotype

Genotype Utim 2 did not cause insectistatic effects on the insect by decreasing weight gain nor prolongation of development. On the contrary, concentrations of 100, 500 and 1,000 ppm of all the extracts increased the weight gain of the larvae. Of which the acetonic extract at 100, 500 and 1,000 ppm increased the weight gain of the larvae by more than or equal to 719 % when compared with the weight gain of the control larvae (40.81 mg; F = 353.62; df = 20; p ≤ 0.0001; Table 2).

Similarly, hexanic and aqueous extracts (100 and 500 ppm), methanolic (100, 500 and 1,000 ppm) and acetonic (100, 500, 1,000, 2,500 and 5,000 ppm) increased the weight of S. frugiperda pupae. Of which, the acetonic extract increased the pupal weight by more than 102 % in relation to the weight of the control pupae (158.48 mg; F = 296.17; df = 20; p ≤ 0.0001; Table 2).

Regarding larval mortality, concentrations between 1,000 and 5,000 ppm of all extracts (except the acetonic extract at 1,000 ppm) caused mortality ≤54 % compared with the control (8 %; F = 113.74; df = 20; p ≤ 0.0001; Table 2). The hexanic and methanolic extracts at 5,000 ppm were the most effective, causing 80 % and 72 % larval mortality, respectively (Table 2). Similarly, the hexanic extract, like the acetonic extract at 5,000 ppm caused the highest mortality of S. frugiperda pupae at 80 %, followed by the methanolic and aqueous extracts at 5,000 ppm with 78 % pupal mortality (control 8 %; F = 125.60; df = 20; p ≤ 0.0001; Table 2).

The larval lethal concentration values, LC₅₀ and LC₉₀, were 1,804 and 4,617 ppm for the methanolic extract, 2,051 and 5,647 ppm for the aqueous extract, 2,616 and 5,726 ppm for the hexanic extract, and 3,021 and 6,393 ppm for the acetonic extract, respectively (Table 2).

3.3 Biological activity of seed extracts of the Ahuehuetzingo genotype

In the 14-day-old larvae, lower weight gains (≤25.60 mg) of larvae treated with the Ahuehuetzingo hexanic and aqueous extracts at 5,000 ppm were observed compared with the control (40.81 mg; F = 1490.04; df = 20; p ≤ 0.0001; Table 3). These same hexanic and aqueous extracts at 5,000 ppm, also prolonged development of the S. frugiperda larvae by 11 days and the development of the S. frugiperda pupae by 5 days compared with the control (larvae 25 d and pupae 11 d; F = 89.05; df = 19; p ≤ 0.0001; Table 3). Also, the hexanic and acetonic extracts at 2,500 ppm, like the methanolic extract at 5,000 ppm, prolonged the development of S. frugiperda larvae by 10 days (Table 3).

In contrast, the acetonic, methanolic and aqueous extracts (100, 500 and 1,000 ppm), and hexanic extract (100, 500, 1,000 and 2,500 ppm) increased the weight gain of the 14-day-old larvae compared to the control group (40.81 mg; Table 2). The concentration of 100 ppm of all the extracts was the most active, increasing the weight gain of the larvae by more than 370 %. The same result was recorded with the pupal weight, which was increased due to the 100, 500 and 1,000 ppm of all the extracts (Table 2), with the concentration of 100 ppm being the most active, increasing pupal weight by more than 119 % (Table 2).

Regarding S. frugiperda mortality, it was observed that all the concentrations of the extracts caused larval mortality ≥50 % compared with the control (except the minimum concentration) and the acetonic extract at 5,000 ppm was the most toxic causing 100 % larval mortality (F = 113.74; df = 20; p ≤ 0.0001; Table 3). All extracts at 5,000 ppm exhibited toxicity, resulting in larval mortality ranging from 76 % to 100 %, and pupal mortality between 87 % and 100 % (Table 3).

This genotype registered larval LC₅₀ and LC₉₀ values of 858 and 1,683 ppm for the acetonic extract, 1,208 and 2,856 ppm for the hexanic extract, 1,306 and 4,321 ppm for the methanolic extract, and 1,397 and 4,180 ppm for the aqueous extract, respectively (Table 3).

3.4 Biological activity of seed extracts of the Ceprobi genotype

At 7 days, the Ceprobi extracts resulted in significant differences in larval weight gain compared with the control (2.1 mg; F = 353.04; df = 20; p ≤ 0.0001; Table 4). The hexanic extract at 5,000 and 2,500 ppm decreased the larval weight gain by 47.62 % and 28.57 %, followed by the acetonic extract at 5,000 ppm, which decreased the weight gain of the S. frugiperda larvae by 23.8 % (Table 4). After 14 days, the hexanic (5,000 and 2,500 ppm) and acetonic (5,000 ppm) extracts were the most active, decreasing the larval weight gain of S. frugiperda by 74.36 %, 53.02 % and 45.96 % compared to the weight of the control larvae (40.81 mg; F = 1728.80; df = 20; p ≤ 0.0001; Table 4).

These same extracts: hexanic at 5,000 and 2,500 ppm and acetonic at 5,000 ppm increased the larval development time of S. frugiperda by 11 days compared to the development of the control larvae (25 d; F = 85.04; df = 20; p ≤ 0.0001; Table 4). Also, the concentrations of 2,500 and 1,000 ppm of the acetonic extract and 1,000 ppm of hexanic extracts increased the development time of the larvae by 10, 9 and 8 days, respectively (Table 4).

Additionally, the hexanic extract at 5,000 ppm decreased the pupal weight of S. frugiperda by 6 % compared with the weight of the control pupae (158.49 mg; F = 24.40; df = 20; p ≤ 0.0001; Table 4). In the development of the pupae, 1,000, 2,500 and 5,000 ppm of all the extracts increased the pupal development time of S. frugiperda by 3, 4 or 5 days compared with the control (11 d; F = 15.75; df = 20; p ≤ 0.0001; Table 4).

On the other hand, the minimum concentrations (100, 500 and 1,000 ppm) of the extracts stimulated feeding, with the acetonic extract at 100 ppm increasing the weight gain of the larvae the most (357 %; Table 4). Also, this same concentration (100 ppm) of all extracts resulted in the heaviest S. frugiperda pupae (Table 4).

All higher concentration extracts of this genotype (1,000, 2,500 and 5,000 ppm) caused ≥52 % and 59 % mortality of the larvae and pupae, respectively (Table 4). The maximum concentration used (5,000 ppm) of the acetonic and aqueous extract resulted in 76 % and 80 % mortality of the larvae and the pupae, respectively (control 8 %; F = 154.26; df = 20; p ≤ 0.0001; Table 4).

The larval lethal concentrations to 50 % and 90 % mortality, LC₅₀ and LC₉₀, were 1,714 and 3,929 ppm for the aqueous extract, 1,772 and 4,049 ppm for the acetonic extract, 2,449 and 5,211 ppm for the methanolic extract, and 2,798 and 5,779 ppm for the hexanic extract, respectively (Table 4).