Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo. Tomo 57(1). ISSN (en línea) 1853-8665. Año 2025.

Original article

 

Impact of Cry1Ac soybean (Glicine max) on biological and reproductive cycles and herbivory capacity of Spodoptera cosmioides and Spodoptera eridania (Lepidoptera: Noctuidae)

Impacto de la soja (Glicine max) Cry1Ac sobre el ciclo biológico, reproductivo y la capacidad herbívora de Spodoptera cosmioides y Spodoptera eridania (Lepidoptera: Noctuidae)

 

Alejandra Liliana Lutz1*,

Laura Noemí Fernandez1, 2,

Verónica Eugenia Ruiz2,

María Cecilia Curis1,

Melina Soledad Buttarelli3,

Pablo Daniel Sánchez1,

María Alejandra Favaro1, 2,

Roberto Ricardo Scotta1

 

1 Universidad Nacional del Litoral. Facultad de Ciencias Agrarias. Departamento de Producción Vegetal. Kreder 2805 (3080). Esperanza. Santa Fe. Argentina.

2 Universidad Nacional del Litoral. Facultad de Ciencias Agrarias. ICiAgro Litoral. CONICET, Esperanza. Argentina.

3 Estación Experimental Agropecuaria Rafaela (INTA E.E.A. Rafaela). Área de producción Vegetal. Ruta 34 km 227 (2300). Rafaela. Santa Fe. Argentina.

 

* alutz@fca.unl.edu.ar

 

Abstract

Increasing populations of Spodoptera cosmioides (Walker) and Spodoptera eridania (Stoll) have recently been detected in soybean crops in central Argentina. Besides being polyphagous, these species tolerate the Cry1Ac insecticidal toxin, expressed by genetically modified Bt soybean (MON89788 x MON87701). Consequently, when facing big populations, farmers often apply insecticides. This study aimed to determine the effects of Bt soybean on the consumption, biological cycle, and reproduction of both Spodoptera species. Larval feeding on Bt soybean led to a shorter pupal period (23% less than control) and a decreased leaf-area consumption for S. cosmioides (14% less than the non-Bt soybean). In S. eridania, the larval stage, adult longevity, larva-to-adult, and oviposition periods were reduced (11, 23, 13, and 30% shorter than control, respectively). Despite these reductions, both Lepidoptera species completed their reproductive cycles. These valuable findings help us understand the biology of these potential pests in Bt soybean crops in Argentina.

Keywords: Glicine max (L.), plant resistance, non-target pests, black armyworm, southern armyworm

 

Resumen

En los últimos años, las poblaciones de Spodoptera cosmioides (Walker) y Spodoptera eridania (Stoll) se han incrementado en los cultivos de soja de la zona central de Argentina. Además de ser polífagas, estas especies son tolerantes a la toxina insecticida Cry1Ac expresada por la soja Bt genéticamente modificada (MON89788 x MON87701), por lo que los agricultores deben recurrir al control químico con insecticidas cuando se presentan altas densidades poblacionales. Este estudio tuvo como objetivo determinar el efecto de la soja Bt sobre el consumo, ciclo biológico y reproducción de ambas especies de Spodoptera. La alimentación larval con soja Bt determinó una menor duración del período pupal (23% menos que el tratamiento control) y una disminución en el consumo de área foliar en S. cosmioides (14% menos que la soja no Bt). Spodoptera eridania registró una menor duración del estado larval, longevidad de adultos, período larva-adulto y del período de oviposición (11, 23, 13 y 30% menos que el tratamiento control, respectivamente). Sin embargo, ambas especies de Lepidoptera completaron su ciclo reproductivo con éxito. Los resultados obtenidos en este trabajo son de gran utilidad para comprender la biología de estas especies, que tienen el potencial de convertirse en plagas importantes en los cultivos de soja Bt en Argentina.

Palabras clave: Glicine max (L.), plantas resistentes, plagas no blanco, oruga cogollera negra, oruga cogollera del sur

 

Originales: Recepción: 15/02/2024- Aceptación: 18/10/2024

 

 

Introduction

 

 

Genetically modified (GM) crops exhibiting insect-resistance are valuable tools in integrated pest management (IPM) systems (25). These crops express genes derived from the entomopathogenic bacterium Bacillus thuringiensis Berliner (Bt), producing (Cry) proteins with highly selective insecticidal activity. Bt soybean expressing these insecticidal toxins is effective in controlling several major lepidopteran pests in agricultural environments, including Anticarsia gemmatalis (Hübner), Chrysodeixis includens (Walker), Helicoverpa gelotopoeon (Dyar) and Rachiplusia nu (Guenée) (44).

High efficacy of Bt soybean crops against pest populations and the consequent reduced insecticide use has significantly altered the agroecosystem. Consequently, the reduced interspecific competition after controlling Bt target species has facilitated the emergence of new phytophagous pest species; many of which could become economically significant (19, 30, 47). Recent reports mention increasing populations of Spodoptera cosmioides Walker and Spodoptera eridania Stoll (Lepidoptera: Noctuidae) in Argentinean soybean crops, including Bt cultivars (23, 29, 30, 32). Factors contributing to these phenomena include tolerance to the Cry1Ac protein, insecticide resistance and the ability to complete life cycles on the weed Amaranthus sp. (2, 5, 9, 24, 26, 30). S. eridania thrives in temperate regions like the Argentinean Pampas, with a developmental threshold of 11.9°C and an inability to complete its life cycle above 34°C. In contrast, S. cosmioides is adapted to warmer temperatures (from 13.2), prevailing in soybean and cotton crops in northern Argentina (31). Although soybean and cotton are the preferred hosts (11), caterpillars of both species are polyphagous, and develop on weeds and grain, fruit, and ornamental crops (14). These species also have greater herbivorous potential than other soybean defoliators, consuming vegetative structures, flowers, and pods (6, 21, 27, 38).

Understanding biological and reproductive pest cycles is essential for elucidating population dynamics and predicting potential crop populations. Most studies assessing the effects of the Cry1Ac protein on the development and foliar consumption of lepidopteran pests (4, 5, 8, 12) have been conducted in Brazil, with almost no equivalent in Argentina. This study hypothesized that the Cry1Ac protein affects biological performance, reproduction, and feeding behavior of both Spodoptera species. We investigated the impact of Bt soybean on foliar consumption and life cycle to assess pest potential in the central soybean region of Argentina.

 

 

Materials and methods

 

 

This study was conducted in the breeding chamber of the Plant Production Department at the Facultad de Ciencias Agrarias (Universidad Nacional del Litoral), in Esperanza City, Santa Fe province, Argentina.

 

 

Insect rearing

 

 

Spodoptera cosmioides and S. eridania larvae were collected in February 2019 from commercial soybean fields in Santa Fe province, near Franck (31°35’00” S 60°56’00” W, 31 m a. s. l.) and Santa María Norte (31°31’00” S 61°08’00” W, 44 m a. s. l.). The caterpillars were transported to the breeding chamber in containers with soybean leaves and identified using taxonomic keys (43, 46). They were reared under controlled temperature (24 ± 2°C), relative humidity (60%), and photoperiod (14:10 h, light: dark) in transparent PVC boxes (26 cm long, 17 cm wide, and 7 cm high), covered with muslin caps for air circulation. An artificial diet consisting of corn flour, wheatgerm, yeast, water, agar, nipagin, benzoic acid, and ascorbic acid was provided until pupation (33). The emerged adults were placed in oviposition cages (50 cm length, 40 cm width, and 40 cm height), with paper sheets for oviposition. They were fed daily with an artificial adult diet (10) provided through soaked cotton. Eggs were collected daily and placed in 9 cm diameter Petri dishes with artificial feed for neonate larvae. Three days after hatching, larvae were transferred to PVC boxes for large-scale rearing with an artificial diet. This process continued until the F2 generation, ensuring enough population for the study.

 

 

Plant material

 

 

Leaves for larvae feed were obtained from soybean cultivars RA 5715 IPRO (Bt) and RA 549 (non-Bt). Both cultivars are glyphosate-tolerant, but only the former expresses the Cry1Ac toxin. To ensure a continuous supply of leaves, both cultivars were periodically planted in 3x2 m plots under field conditions. Weeds were manually removed and soybean plants were kept disease-free by the eventual application of fungicides.

 

 

Effect of Bt soybean on the biological and reproductive cycle of S. cosmioides and S. eridania

 

 

A second instar (L2) larva of either S. cosmioides or S. eridania was placed on two soybean leaflets (from Bt or non-Bt soybean plants, depending on the treatment) inside 9 cm diameter Petri dishes lined with absorbent paper. Petioles were wrapped in cotton saturated with distilled water, maintaining humidity. Bt and non-Bt soybean leaflets were harvested at V6-V8 vegetative stage before anthesis, according to phenology by Fehr et al. (1977). The V6-V8 vegetative stage corresponds to maximum Cry1Ac expression in the Bt cultivar (45). Food and absorbent papers were renewed daily until pupation. We defined sex by observing the terminal portion of pupae (7) using a stereomicroscope set (Lancet Instruments, China) at 30× magnification. Thirty replicates were performed for each treatment (Bt and non-Bt soybean) and species (S. cosmioides and S. eridania). Once adults emerged, one couple was placed per oviposition container (17 cm height, 11 cm upper diameter, and 7 cm lower diameter), covered with a muslin cap facilitating air circulation and preventing adult escape. The same diet used for rearing was supplied to adults with soaked cotton (10). Fecundity was determined by daily collecting egg masses laid by females after mating. Egg masses were photographed using an Olympus SZ40 stereomicroscope (Olympus Corporation, Tokyo, Japan) at 40X for egg counting, considering any overlapping or superimposed eggs. Each egg mass was placed in a separate Petri dish (9 cm diameter) lined with absorbent paper and food for emerging neonates. Fertility (viable eggs) was estimated by the number of viable larvae hatched from each egg mass. The assays were conducted with 11 and 10 couples of S. cosmioides and 14 and 10 couples of S. eridania for the Bt and non-Bt soybean treatments, respectively.

The following variables were recorded: duration (in days) of larval, pupal and adult stages, the larva-to-adult period, pupal weight (g) using an OHAUS-PIONNER precision scale (± 0.0001 g), fecundity (number of eggs/female), fertility (number of hatched eggs), pre-oviposition (days from adult emergence to first egg laying), oviposition (days from the first to the last egg laying), and post-oviposition (days from last egg laying to death).

 

 

Effect of Bt soybean on leaf consumption by larvae of S. cosmioides and S. eridania

 

 

Leaf area consumption (cm2) was determined using the same larvae and soybean cultivars (species and treatments) as when assessing the impact of Bt soybean on the biological and reproductive cycle. Fresh leaflets were provided daily as food, and the remaining unconsumed portions were scanned using an HP Deskjet F4280 multifunction printer. The consumed leaf area was quantified by image analysis with ImageJ® software (1). Adjusted leaf area loss due to dehydration was based on data from soybean leaflets not exposed to larvae.

 

 

Statistical analysis

 

 

Bioassays for each lepidopteran species were conducted independently under a completely randomized experimental design. Since the duration of larva, pupal, and adult stages, as well as the larva-to-adult period did not meet normality, non-parametric Kruskal-Wallis test (α ≤ 0.05) was performed. Pupal weight was analyzed by ANOVA and Tukey test (α ≤ 0.05). Foliar consumption

means were compared using an independent samples T-test (α ≤ 0.05). All statistical analyses were conducted using InfoStat software (13).

 

 

Results

 

 

Effect of Bt soybean on the biological and reproductive cycle of S. cosmioides and S. eridania

 

 

Feeding S. cosmioides larvae with Bt soybean leaves did not significantly affect larval duration compared to control, with 20.46 and 19.48 days, respectively (H= 0.64; p= 0.4160) (table 1). However, in S. eridania, significant differences were evidenced for larval length of 28.13 days when fed with Bt soybean leaves and 31.69 days when supplied with non-Bt soybean leaves (H= 7.43; p= 0.0062) (table 1).

Significant differences in the S. cosmioides pupal stage duration showed 10.65 and 13.96 days (H= 14.72; p= 0.0001), with Bt and non-Bt soybean, respectively. In S. eridania, differences were not significant (H= 3.77; p= 0.0434) (table 1). Regarding the adult stage, no significant differences were found in S. cosmioides (H= 0.04; p= 0.8485). However, S. eridania adults lived significantly longer on non-Bt soybean leaves (12.87 days), compared to Bt soybean (9.90 days) (H= 11.70; p= 0.0005) (table 1).

 

Table 1. Days of larval, pupal, and adult stages, larva-to-adult period, and pupae weight (g) (Mean ± SD) of Spodoptera cosmioides and S. eridania, fed Bt and non-Bt soybean leaves under controlled conditions.

Tabla 1. Duración (días) de los estadios larval, pupal y adulto, el período de larva a adulto y el peso de las pupas (g) (Media ± DE) de Spodoptera cosmioides y S. eridania, alimentadas con hojas de soja Bt y no-Bt en condiciones controladas.

* Different letters indicate significant differences among treatments. (Test: Kruskal Wallis, α ≤ 0.05). Pupal weight (Test: Tukey, α ≤ 0.05).

*Diferentes letras en las columnas indican diferencias significativas entre tratamientos (Prueba: Kruskal Wallis, α ≤ 0,05). Peso de pupa (Prueba: Tukey, α ≤ 0,05).

 

The larva-to-adult period for S. cosmioides was 44.31 days on Bt soybean and 46.70 days on non-Bt soybean, with no significant differences (H= 0.48; p= 0.4881). In contrast, S. eridania had a larva-to-adult period significantly longer on non-Bt soybean (57.45 days) compared to Bt soybean (49.97 days) (H= 13.89; p= 0.0002) (table 1).

Considering pupal weight, no significant differences were found for either species: S. cosmioides (F= 0.95; p= 0.3359) and S. eridania (F= 3.77; p= 0.0577) (table 1).

Larval feeding did not affect average number of eggs per female in either species. S. cosmioides had 3291.82 eggs on Bt soybean leaves and 3049.0 on non-Bt soybean leaves (H= 0.08; p= 0.7782). S. eridania had lower fecundity than S. cosmioides, with 841.79 eggs on Bt soybean and 830.70 eggs on non-Bt soybean (H= 0.01; p= 0.9068) (table 2). The Cry1Ac protein ingested by larvae did not affect fecundity in the studied species (table 2). Similarly, no significant differences were observed in fertility (percentage of hatched eggs) between treatment species (H= 0.04; p= 0.8327 for S. cosmioides and H= 2.14; p= 0.1432 for S. eridania) (table 2).

 

Table 2. Fecundity (number of eggs) and Fertility (% of hatched eggs) (Mean ± SD) of Spodoptera cosmioides and S. eridania fed Bt and non-Bt soybean leaves under controlled conditions.

Tabla 2. Fecundidad (número de huevos) y fertilidad (% de huevos eclosionados) (Media ± DE) de Spodoptera cosmioides y S. eridania alimentadas con hojas de soja Bt y no-Bt en condiciones controladas.

*Different letters in columns indicate significant differences between treatments. (Test: Kruskal Wallis α ≤ 0.05).

* Diferentes letras en las columnas indican diferencias significativas entre tratamientos (Prueba: Kruskal Wallis α ≤ 0,05).

 

Pre and post-oviposition periods were similar for both species under both larval feeding treatments (table 3), (H= 1.32; p= 0.2395 and H= 0.85; p= 0.34 for S. cosmioides, respectively; H= 0.51; p= 0.4460 and H= 2.25; p= 0.1238 for S. eridania, respectively). However, significant differences were found in the oviposition period for S. eridania, with 4.29 and 6.13 days in adults emerging from larvae fed Bt and non-Bt soybean leaves, respectively (H= 4.62; p= 0.0293).

 

Table 3. Pre-oviposition, oviposition and post-oviposition periods of Spodoptera cosmioides and S. eridania (Mean ± SD) fed Bt and non-Bt soybean leaves under controlled conditions.

Tabla 3. Períodos de preoviposición, oviposición y postoviposición de Spodoptera cosmioides y S. eridania (Media ± DE) alimentados con hojas de soja Bt y no Bt en condiciones controladas.

* Different letters in columns indicate significant differences between treatments. (Test: Kruskal Wallis α ≤ 0.05).

* Diferentes letras en las columnas indican diferencias significativas entre tratamientos (Prueba: Kruskal Wallis α ≤ 0,05).

 

 

Effect of Bt soybean on leaf consumption by S. cosmioides and S. eridania

 

 

Total leaf area consumption by S. cosmioides was lower when larvae were fed with Bt soybean (T= -2.77; p= 0.0081). In contrast, S. eridania showed no significant differences in leaf area consumption between Bt and non-Bt soybean leaves (T= 0.05; p= 0.9585) (table 4).

 

Table 4. Leaf area consumption of Spodoptera cosmioides and S. eridania (Mean ± SD) fed Bt and non-Bt soybean leaves under controlled conditions.

Tabla 4. Consumo de área foliar de Spodoptera cosmioides y S. eridania (Media ± DE) alimentados con hojas de soja Bt y no Bt en condiciones controladas.

*Different letters in columns indicate significant differences between treatments. (Test: T, α ≤ 0.05).

* Diferentes letras en las columnas indican diferencias significativas entre tratamientos. (Test: T, α ≤ 0,05).

 

 

Discussion

 

 

gM crops expressing Cry proteins are crucial for pest control. Besides killing susceptible species, these crops can have sublethal effects on tolerant species, through direct or indirect exposure, leading to broader ecological changes (40). Spodoptera cosmioides and S. eridania exhibit tolerance against the Cry1Ac protein (2) due to the type and quantity of receptor proteins in larval midgut membranes, low receptor affinity, or rapid protein degradation (35). Thus, insect exposure to stress factors like Cry1Ac protein expressed by Bt soybean may enhance fitness of the exposed population (17, 18). This explains why S. eridania individuals exhibited shorter durations in both larval and adult stages, and a reduced larva-to-adult period when fed soybean leaves expressing the Cry1Ac protein. In contrast, S. cosmioides only experienced a decrease in the pupal period when fed on insect-resistant GM soybeans (Bt).

Regarding larval cycle, our results for S. cosmioides agree with Bernardi et al. (2014) and Silva et al. (2019), who observed a similar duration for the last larval stage under the same treatments. In contrast, S. eridania showed significant differences between treatments with an average duration of 28.13 days on Bt soybeans and 31.69 days on non-Bt soybeans. These results are consistent with those reported by Bortolotto et al. (2014) and Rabelo et al. (2020), who observed a significant reduction of 2 days in the larval stage of S. eridania when fed GM soybeans expressing Cry1Ac.

Our results showed that S. eridania adults from Bt soybeans live 3 days less than those from the control group. In contrast, Silva (2013) and Bortolotto et al. (2014) reported a significant 3 days-increase in longevity of S. eridania males when reared on Bt soybean leaves. This discrepancy suggests that Cry1Ac might induce asynchronous adults’ emergence between the two cultivars, potentially reducing mating chances in natural conditions. According to Jakka et al. (2014) and Murúa et al. (2019), the non-simultaneous emergence of adults in both cultivars could compromise the refuge strategy to avoid or delay resistance emergence. On the other hand, we found a shortened life cycle of S. eridania (7.48 days) when larvae were fed soybeans expressing Cry1Ac, as seen by Ramírez & Gómez (2010), who reported an average life cycle of 51.72 days for S. eridania with artificial diet.

Several studies have demonstrated that noctuid pupae weight can vary with temperature, host plants, and exposure to sublethal insecticide concentrations or Bt crop toxins (22). However, our results indicate that Bt protein did not affect pupal weight of either species. Additionally, feeding larvae with Bt soybean leaves did not affect the reproductive capacity of either Spodoptera species, as observed by Silva et al. (2016) and Sosa et al. (2020), in S. cosmioides for larvae fed with Bt soybean leaves. However, Páez Jerez et al. (2022) reported more eggs per female in S. cosmioides individuals fed Bt soybean. According to Specht & Roque-Specht (2019), fecundity in S. cosmioides is highly variable, with females capable of producing up to 5000 eggs/female, higher than for S. eridania, S. albula, S. frugiperda and S. littoralis (26, 27). In our study, average egg number per S. eridania female is consistent with Silva (2013), who reported similar fecundity in females reared on both Bt and non-Bt soybeans during larval stage, with averages of 881.35 and 911.85 eggs per female, respectively.

We found that exposure to the insecticidal protein Cry1Ac during larval stage shortened the oviposition period in S. eridania. Although the literature lacks specific data on the oviposition period of S. eridania fed on Bt cultivars, previous studies have reported variable oviposition periods ranging from 4.2 days to 6. 75 days when larvae were reared on non-Bt soybean leaves (14).

Biological fitness is the ability of an organism to compete successfully, pass on its genes to subsequent generations and influence population density and the potential to become a pest. However, insecticide exposure can have variable effects, enhancing or reducing performance, potentially leading to adverse impacts on survival, developmental rate, reproduction, and adult longevity (3). This phenomenon has been documented in several Lepidoptera species exposed to Bt protein (16). In our study, we observed a reduced pupal period in S. cosmioides and shortened larval, adult, and larval-to-adult cycles and oviposition periods in S. eridania when fed Bt soybean leaves.

Food quantity and quality directly influence host plant preference affecting biological, physiological, and behavioral features (11). While some studies have found no effects of Cry toxins on foliar consumption in lepidopterans (11), other research reports less leaf consumption due to Cry proteins in corn (5), as we found for S. cosmioides. According to Zurbrügg et al. (2010), glyphosate-resistant soybeans expressing the Cry1Ac toxin have more carbohydrates and lower protein content than non-transgenic cultivars. This variation in nutritional composition may influence insect food preference as seen in S. cosmioides when fed on Cry1Ac-expressing soybean.

 

 

Conclusion

 

 

Transgenic crops expressing Cry insecticidal proteins are valuable tools for controlling susceptible pests. However, they may also induce changes in life cycles, population dynamics, reproductive stages, feeding behavior, or longevity of non-target species. Understanding developmental and reproductive parameters of these non-target pests is essential for predicting population growth and species dynamics within agricultural systems. Our findings shed light on the biology of S. cosmioides and S. eridania in Bt soybean crops in Argentina, considering foliar consumption and herbivorous capacity. Since our experiments were conducted under controlled conditions, these investigations should further assess actual field damage caused by Spodoptera species in soybean crops.

 

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Funding

Financial support for this study was provided by the Universidad Nacional del Litoral (Argentina), through the Curso de Acción para la Investigación y Desarrollo (CAI + D) Program (50120150100131LI).