Insecticidal Activity of Bacillus thuringiensis Strains on the Nettle Caterpillar, Euprosterna elaeasa (Lepidoptera: Limacodidae)

In the present work, we evaluated the insecticidal activity of Bacillus thuringiensis (Bt) strains on Euprosterna elaeasa as an alternative for the organophosphate insecticide use in oil palm plantations in the Americas. The toxic effects of four Bt-strains (HD-1 var. kurstaki, SA-12 var. kurstaki, ABTS-1857 var. aizawai, and GC-91 var. aizawai) were evaluated against E. elaeasa caterpillars for toxicity, survival, anti-feeding, and mortality in field-controlled conditions. The Bt-strains, ABTS-1857 var. aizawai (LC50 = 0.84 mg mL−1), GC-91 var. aizawai (LC50 = 1.13 mg mL−1), and HD-1 var. kurstaki (LC50 = 1.25 mg mL−1), were the most toxic to E. elaeasa. The caterpillar survival was 99% without exposure to Bt-strains, and decreased to 52–23% in insects treated with the LC50 and 10–1% in insects exposed to LC90 after 48 h. Furthermore, Bt-strains decreased significantly the consumption of oil palm leaves of E. elaeasa 3 h after exposure. Mortality of E. elaeasa caterpillars caused by Bt-strains had similar lethal effects in the laboratory and in field conditions. Our data suggest that Bt-strains have insecticidal activity against E. elaeasa and, therefore, have potential applications in oil palm pest management schemes.

In Colombia, chemical insecticides such as acephate, methamidophos, and monocrotophos are used on oil palm crops to control E. elaeasa [1,5]. Due to the high level of infestation and the rapid spread of E. elaeasa in oil palm trees, the use of insecticides is common practice [6,7]. However, recent studies have shown the presence of these insecticides in minimal quantities in palm oil [8,9]. Conventional insecticides are expensive and cause environmental pollution [10], atmosphere ozone-depletion [11], Insects 2020, 11, 310 2 of 10 residual long [12], and insecticide resistance [13]. New alternatives that are more sustainable, different from organophosphates, are needed to replace the main insecticides historically used against E. elaeasa for the past 50 years [1,6]. The search for alternatives for E. elaeasa control is important, considering the impact generated by the use of insecticides in this agroecosystem [6]. Thus, the use of natural enemies, such as viruses, bacteria, and fungi, can be an alternative for oil palm pest control [14][15][16].
Bacillus thuringiensis (Bt) is a biocontrol agent for defoliating pests worldwide, and individual strains are specific to a small group of insect targets without effects on animals and environment [17]. Bt is a gram-positive spore-forming bacterium with entomopathogenic properties. In the sporulation, Bt produces crystalline or "Cry" inclusions, called δ-endotoxins, biosynthesized during the second phase of the growth cycle [18]. In this cycle, the Cry proteins are converted in active toxins upon insect ingestion [19]. Several Cry proteins displaying activity on insects have been identified: the Cry1 proteins are toxic to Lepidoptera [20], while the Cry3 proteins are toxic to Coleoptera [21,22]; also, a high number of different subgroups (Cry1Ac, Cry1Ba, Cry8Ca, Cry1Eb, Cry1J, etc.) are active against mosquitoes, Coleoptera, Diptera, Hemiptera, and Hymenoptera [21,23,24].
Bt was reported as a biological control agent for oil palm pests [6,25]. Different oil palm lepidopteran species may have different levels of susceptibility to a specific Cry protein that occurs in Metisa plana Walker (Psychidae) [16], Opsiphanes cassina Felder (Nymphalidae) [26], and Tirathaba rufivena Walker (Pyralidae) [27]. This microbiological agent provides biodiversity in agroecosystems and the delivery of ecosystem services to agricultural production, especially in pest population regulation [28]. Since the entomopathogenic bacterium infects their host through the midgut, they hold greater potential as biocontrol agents for E. elaeasa; however, the use of Bt on this insect has not been carried out.
This study evaluated the insecticidal activity of Bt strains as potential agents to control E. elaeasa, explained in different experiments: (i) toxicity test, (ii) survivorship, (iii) anti-feeding effect, and (iv) mortality in field conditions. The objective was to contribute to the development of strategies for controlling E. elaeasa, as a replacement for organophosphate insecticides.

Insects
In the field, 2527 adults of E. elaeasa (males = 1284, females = 1243) were captured manually during the day, in 5-yr-old commercial plantations of oil palm in the county of Puerto Wilches, Santander, Colombia (N 07 • 20', W 73 • 54'). The insects were transferred in plastic trays (30 × 50 × 50 cm) with perforated lids for ventilation to the Entomology Laboratory of the Oil Palm Monterrey Plantation (Puerto Wilches, Santander, Colombia) to establish a breeding colony in laboratory conditions. Adults were fed a honey solution daily (15 mL of honey and distilled water, in a 2:1 ratio) applied with a sponge. Males and females of E. elaeasa were isolated in glass containers (30 × 30 × 30 cm) covered with a nylon mesh and containing E. guineensis leaves. For egg development, 9800 eggs oviposited on the surface of the leaves were collected every 24 h and placed in Petri dishes (90 × 15 mm high) containing a paper towel saturated with water. After hatching, first-instar caterpillars (n = 7550) were placed individually in glass vials (5 × 25 cm) covered with cotton and fed every 24 h with E. guineensis leaves. Eggs and caterpillars were maintained in incubators at 27 ± 1 • C, with 75 ± 5% RH and 12:12 (L:D) photoperiod. Newly third instar E. elaeasa caterpillars were used in the laboratory and field condition bioassays.

Concentration-Mortality Bioassay
Commercial Bt formulations commonly used to control Lepidoptera were used in all bioassays and selected for quality, high-efficiency, and non-toxicity (toxicity Class IV) [18]. The following Bt strains, HD-1 var. kurstaki (Dipel ® , Abbott Laboratories, North Chicago, IL, USA), SA-12 var. kurstaki (Thuricide ® , Certis USA LLC, Columbia, MD, USA), ABTS-1857 var. aizawai (XenTari ® , Insects 2020, 11, 310 3 of 10 Valent Bioscience Corporation, Osage, Iowa, USA), and GC-91 var. aizawai (Agree ® , Certis USA LLC, Columbia, MD, USA), were prepared in an aqueous solution with 0.1% Triton X-100 (strains and distilled water) to obtain a stock suspension (100 g L −1 ), from which dilutions were prepared as needed. Six concentrations (0.156, 0312, 0.625, 1.25, 2.5, and 5 mg mL −1 ) were used to evaluate the toxicity of each Bt-strain to E. elaeasa caterpillars, construct concentration-mortality curves, and estimate the lethal concentrations (LC 50 and LC 90 ). Distilled water with 0.1% Triton X-100 was used as control. The application of the concentrations was carried out by the feeding method using oil palm leaves. Pieces (10 × 10 mm) of oil palm leaves were cut, sterilized with 5% sodium hypochlorite with three successive series of distilled water, and dried at room temperature. Then, pieces of oil palm leaf were dipped in solutions of different concentrations of each Bt-strain for 10 s and allowed to air dry for a period of 1 h. Caterpillars were placed individually in Petri dishes, and a piece of oil palm leaf treated with Bt-strain was provided. Three replicates with 50 insects of each were used in concentration testing, and the experimental design was completely randomized. The dead insects were counted after 48 h Bt-strain exposure.

Time-Mortality Bioassay
Caterpillars of E. elaeasa were placed individually in Petri dishes and exposed to the lethal concentrations (LC 50 and LC 90 ) of each of the Bt-strains determined in the dose-response relationship. A control was performed using distilled water with 0.1% Triton X-100. Exposure procedures and conditions were the same as described above for the concentration-mortality bioassay Section 2.2. The number of live insects was recorded every 6 h for 2 d. Three replicates of 50 insects were used by each Bt-strain and the experimental design was completely randomized.

Anti-Feeding Effect
Caterpillars of E. elaeasa were placed individually in Petri dishes with a piece of oil palm leaf (10 × 10 mm) treated with LC 50 or LC 90 of each Bt-strains and distilled water as control. Caterpillars were in contact with E. guineensis leaves for 3 h and, after this, the pieces were photographed with a digital photographic camera (D40, 18D55 mm, Nikon Corporation, Tokyo, Kantō, Japan) with a 15 cm macro focus in natural and flourishing light (SB-700 Nikon Corporation). The images were analyzed using the digital analysis software, UTHSCSA Image Tool v. 2.0 (University of Texas, Austin, TX, USA). The leaf area consumed by the caterpillar was measured in mm 2 , with pixels based on the RGB (red, 213 nm; green, 111 nm; blue, 56 bits) histogram. Twenty repetitions for each of the Bt-strain concentrations (LC 50 and LC 90 ) and control were carried out in a completely randomized design.

Mortality in Semi-Controlled Test
The bioassay was conducted in 5-yr-old commercial oil palm plantations (cv 'Tenera' × 'Deli Ghana') in the county of Puerto Wilches (Santander, Colombia), with an average temperature of 27.98 • C, 81-93% relative humidity, 1455 to 2258 h of sunshine per year, and 2189 mm of annual rainfall. In these conditions, fifty palm trees were selected and E. elaeasa caterpillars were used for each Bt-strain in the controlled field test. For each palm tree, 50 caterpillars were placed on the leaf No. 17, according to the rules of phyllotaxy [29] and isolated with a nylon trap (0.5 × 0.5 × 1.20 m). Treatments consisted of adding each Bt-strain at the calculated LC 90 concentration, and distilled water with 0.1% Triton X-100 as the control, with ten replications per treatment. Applications of 100 mL of Bt-strain per leaf were made by a manual pump (Royal Condor ® , 5 L capacity, Soacha, Cundinamarca, Colombia), and the number of dead caterpillars was counted after 15 d Bt-strain exposure.

Statistical Analysis
The concentration-mortality data were submitted to Probit analysis to obtain a dose-response curve [30]. The time-mortality data were analyzed for survival analysis (Kaplan-Meier estimators, log-rank test) with the Origin Pro 9.1 software (OriginLab Corporation, Northampton, MA, USA).
Insects 2020, 11, 310 4 of 10 Anti-feeding effect data were arcsine-transformed and submitted to one-way ANOVA, and a Tukey's honestly significance difference (HSD) (p < 0.05) test was also used for comparison of means. Mortality data in semi-controlled conditions were summarized in percentages and submitted to one-way ANOVA and a Tukey's HSD (p < 0.05); also, all values presented as mean ± SEM. Statistical procedures were analyzed by SAS 9.0 software (SAS Institute, Campus Drive Cary, NC, USA).

Concentration-Mortality Bioassay
The dose-response model provided a good fit to the data (p > 0.05), allowing the determination of toxicological endpoints, and confirms the toxicity of each Bt-strain to E. elaeasa Table

Discussion
The use of various Bt-strains was effective in causing mortality, compromising survivorship, and reducing the consumption rate of the nettle caterpillar, E. elaeasa. The Bt-strains HD-1 var. kurstaki, SA-12 var. kurstaki, ABTS-1857 var. aizawai, and GC-91 var. aizawai were toxic to E. elaeasa caterpillars and have a strong effect through oral exposure. Bt-strains caused mortality in E. elaeasa in a concentration-dependent manner, as demonstrated in other defoliating pests [19,31,32]. Euprosterna elaeasa caterpillars exposed to high concentrations of Bt-strains displayed muscle contractions, oral or anal secretions, and consequently, septicemia. In this context, symptoms in E. elaeasa caterpillars were consistent with the known effects of microbial disruption of insect midgut membranes. A set of results point to the effects on the digestive system of lepidopterous pests, such as Diatraea saccharalis Fabricius (Crambidae) [33], Plutella xylostella Linnaeus (Plutellidae) [34], and Spodoptera frugiperda JE Smith (Noctuidae) [35], after Bt-strains oral exposure. In general, few Btstrains are effective against E. elaeasa at different concentrations and reinforce their use as an alternative to organophosphate insecticides on this species.
In this study, the survival time of E. elaeasa decreases mainly with HD-1 var. kurstaki and ABTS-1857 var. aizawai. However, periods of Bt-strains, from 24 to 48 h, were necessary to induce mortality in E. elaeasa. Survivorship of this insect is associated with the quick action in the midgut of several Cry proteins produced by Bt-strains and observed in other lepidopteran pests [36,37]. In this study, the compared effects of the Bt-strains on E. elaeasa occur at various periods. These time differences

Discussion
The use of various Bt-strains was effective in causing mortality, compromising survivorship, and reducing the consumption rate of the nettle caterpillar, E. elaeasa. The Bt-strains HD-1 var. kurstaki, SA-12 var. kurstaki, ABTS-1857 var. aizawai, and GC-91 var. aizawai were toxic to E. elaeasa caterpillars and have a strong effect through oral exposure. Bt-strains caused mortality in E. elaeasa in a concentration-dependent manner, as demonstrated in other defoliating pests [19,31,32]. Euprosterna elaeasa caterpillars exposed to high concentrations of Bt-strains displayed muscle contractions, oral or anal secretions, and consequently, septicemia. In this context, symptoms in E. elaeasa caterpillars were consistent with the known effects of microbial disruption of insect midgut membranes. A set of results point to the effects on the digestive system of lepidopterous pests, such as Diatraea saccharalis Fabricius (Crambidae) [33], Plutella xylostella Linnaeus (Plutellidae) [34], and Spodoptera frugiperda JE Smith (Noctuidae) [35], after Bt-strains oral exposure. In general, few Bt-strains are effective against E. elaeasa at different concentrations and reinforce their use as an alternative to organophosphate insecticides on this species.
In this study, the survival time of E. elaeasa decreases mainly with HD-1 var. kurstaki and ABTS-1857 var. aizawai. However, periods of Bt-strains, from 24 to 48 h, were necessary to induce mortality in E. elaeasa. Survivorship of this insect is associated with the quick action in the midgut of several Cry proteins produced by Bt-strains and observed in other lepidopteran pests [36,37]. In this study, the compared effects of the Bt-strains on E. elaeasa occur at various periods. These time differences occur commonly between strains of the same subspecies (e.g., Bt var. kurstaki and Bt var. aizawai) [38], by specific variation of the δ-endotoxins originating from different Bt-strains [39], host immune responses [40], and virulent factors of strain types [41]. In this sense, Bt toxins have been reported to reduce or inhibit larval growth, development, or weight, interrupting the insect's lifecycle [42]. The rapid effect against E. elaeasa suggests that the insecticidal activity of Bt-strains causes detrimental effects on neonates, with appreciable population reduction during the first days of infestation and can be essential for protecting oil palm trees.
The decrease in the consumption of oil palm leaves treated with LC 50 and LC 90 of Bt-strains suggests an anti-feeding effect on E. elaeasa, represented by different rates of intoxication and, consequently, cessation of feeding. On the other hand, the concentration-dependent effect on total food consumption by both lethal concentrations indicates that intoxication of Bt-strains is cumulative. Effects of Bt-strains after 24 h exposure on Helicoverpa armigera Hübner (Noctuidae), Phyllocnistis citrella Stainton (Gracillariidae), and Tuta absoluta Meyrick (Gelechiidae) were observed [43][44][45], causing a dramatic reduction in initial leaf consumption. In E. elaeasa, the reduced consumption (in both lethal concentrations) exposure regimes demonstrated that intoxication has serious deleterious effects that may translate into metabolic costs associated with the repair of midgut epithelium damage in caterpillar survivors. For instance, altered permeability and damage of the midgut interfere with food uptake, affect activity enzymes associated with digestion, and influence energy metabolism. Additionally, intoxication alters hemolymph pH and suppresses the immune response [19,39,40]. Our results demonstrate that the interplay of concentration and exposure regimen can produce anti-feeding effects, indicating that Bt-strains intoxication of E. elaeasa caterpillars is cumulative.
The Bt-strains, HD-1 var. kurstaki, SA-12 var. kurstaki, ABTS-1857 var. aizawai, and GC-91 var. aizawai, showed lethal effects against E. elaeasa in palm trees in the field, and results were consistent with those observed in the laboratory. However, mortality level at the larval stage was lower than those obtained under laboratory conditions. Efficacy of Bt-strains in field conditions may be due to environmental factors [46], toxins degradation [47], gut microbiota competition [48], and inactivation by the target organism [49]. The lethal effect of Bt and its effectiveness was also studied in other Limacodidae pests under field conditions as a potential biocontrol agent for Acharia apicalis Dyar [50], Acharia fusca Stoll [51], and Parasa lepida Cramer [52]. The results show that Bt-strains have a specific mode of action that affects a high number of E. elaeasa caterpillars. In particular, HD-1 var. kurstaki and ABTS-1857 var. aizawai are the most effective in the field, and that maximum efficiency from strains should be used during this life stage.

Conclusions
The insecticidal effect of four Bt-strains for controlling E. elaeasa were studied. The Bt-strains HD-1 var. kurstaki, SA-12 var. kurstaki, ABTS-1857 var. aizawai, and GC-91 var. aizawai cause mortality, reduces survivorship, and an anti-feeding effect on this insect, with the potential to control its field populations. The toxicity of the bacterium may efficiently manage E. elaeasa caterpillars and reduce the insect's damage to oil palm trees and transmission of the Pestalotiopsis fungal complex. Bt-strains have lethal and sublethal effects on E. elaeasa, and are an alternative to organophosphate insecticides in oil palm plantations, aiding efforts to manage insecticide resistance.