Efficacy of Cry1Ac toxin from Bacillus thuringiensis against the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

Transgenic Bacillus thuringiensis (Bt) cotton is engineered to express delta endotoxin (Cry toxin) proteins in lepidopteran midgut when ingested by lepidopteran larvae such as the beet armyworm, Spodoptera exigua (Hübner). The efficacy of Bt technology depends on stage, age, and susceptibility of the tested population to Cry proteins. The baseline susceptibility bioassay test for first, second, and third larval instars of S. exigua, collected from Faisalabad, Multan, and Bahawalpur, compared with susceptible laboratory population was carried out in 2015 and 2016. The LC₅₀ ranged from 0.45 to 2.52 μg ml⁻¹, 1.08 to 5.74 μg ml⁻¹, and 2.01 to 7.85 μg ml⁻¹ for first, second, and third larval instars, respectively. The Bahawalpur population was highly resistant and showed 5.63, 5.30, and 3.89 variations than the susceptible population, followed by Multan 3.01, 3.71, 3.10, and Faisalabad 1.93, 2.41, 2.31 population for first, second, and third larval instars, respectively. The molt inhibitory concentration (MIC₅₀) ranged from 0.04 to 0.56 μg ml⁻¹, 0.08 to 0.99 μg ml⁻¹, and 0.10 to 1.42 μg ml⁻¹ for the three instars, respectively. The trend in lethal concentration and its respective resistance level was higher in 2016 than in 2015.

The beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), is a polyphagous pest of various field crops including vegetables, cotton, and ornamentals. S. exigua attacks the cotton plants throughout the whole growing season causing yield losses (Osoria et al. 2008). The widespread adoption of Bt cotton has led to several non-target insect pests such as mirids (Lu et al. 2010) aphids (Zhang et al. 2012), and armyworms including the beet armyworm that becomes one of the major pests in Bt cotton fields (Arshad and Suhail, 2011). However, Bt cotton expressing only CrylAc proteins are unable to provide effective control against Spodoptera spp. (Ponsard et al. 2002; Hofs et al. 2004; Selvi et al. 2012). Farmers have to spray insecticide even in Bt crops for effective control of S. exigua (Smith, 1997). The susceptibility of tested insects to Cry toxins and changes in the baseline, due to the selection pressure of Cry1Ac crops, can be monitored through regular bioassays of the field populations (Heckel, 1993).

Insecticidal protein such as Bt protein is one of the integral parts of good management practices (GMP). It has been regarded as intra-complementary approach for natural enemies establishment when chemical control has been based on selective insecticides. Bt Cry proteins solubilize the midgut receptors and insert toxin to apical membrane and epithelium. This action causes death of treated larvae. These management tactics enable the insect to establish its colony. But the level of the toxin may vary in consistency due to adulteration and impurity. This action of breeders made the lepidopteran insects possible to infest the Bt plants and to establish their population. Under these circumstances, the beet armyworm gained substantial importance by regularly damaging the Bt plants.

A susceptibility test in view of baseline bioassay has been addressed in few parts of the world including Indian and Chinese Spodoptera population to Cry1Ac. The scientists studied monitoring of resistance in Pakistan but still Cry1Ac susceptibility on S. exigua is lacking. Therefore, the present study was carried out to evaluate the efficacy of Cry1Ac toxin from Bt against the beet armyworm, S. exigua, under laboratory conditions.

Larval collection

The larvae of S. exigua were collected from Faisalabad, Bahawalpur, and Multan to develop laboratory colonies in 2015 and 2016. The field-collected populations were assigned as Faisalabad (FSD), Bahawalpur (BWP), and Multan (MLT). The collected larvae were placed in glass vials provided with semi-synthetic diet and transported to the Insect Biodiversity and Biosystematics Laboratory, Department of Entomology, University of Agriculture Faisalabad. The susceptible laboratory population was obtained from the Nuclear Institute of Agriculture and Biotechnology (NIAB), Faisalabad.

Artificial diet

The field and susceptible populations were reared on artificial diet as reported by (Shorey and Hale, 1965; Hamed and Nadeem, 2008)

Rearing procedure

Larvae were reared at 25 ± 5 °C and 75 ± 5% RH under 16 h/ 8 h L/D cycle on the artificial diet and allowed to pupate. Pupae were collected and placed in a Petri plate. The pupae were disinfected in a 0.2% sodium hypochlorite solution. Petri plates containing pupae were placed in plastic containers until adult emergence. Adults were kept in open plastic containers covered with white netting. Adult diet (10% (v/v) honey solution) was provided by soaking the cotton wool pad in the honey solution and placed on the top of the netting cage. Every 48 h, egg-laden netting was replaced, and this process was repeated until egg production decreased or no further eggs were required. The netting was cut into small squares, and the netting containing eggs was surface sterilized in 0.05% sodium hypochlorite solution before being placed in a 250-ml round plastic cups sealed with plastic lids. The cups were placed in a controlled environment room until eggs hatching. From the first (F1) generation neonates, three larval instars (L₁), (L₂), and (L₃) were used for bioassays.

Bacillus thuringiensis toxin

Bt Cry1Ac toxin was obtained from Genralpest Biotech Research Co. Ltd, Beijing, China and was stored at − 20 °C. It had been expressed as crystalline inclusions in Escherichia coli, with the protoxins purified by sonication and successive washes with 0.5 M NaCl and water as described by Sayyed et al. (2008). The toxin was freshly prepared in distilled water for diet incorporation.

Diet incorporates bioassays

Diet incorporation method was used for conducting bioassay, which is similar to the methods established for the baseline susceptibility of Helicoverpa armigera to B. thuringiensis toxins (Wei et al. 2017). Seven concentrations (0.25, 0.50, 1, 2, 4, 8, 16 μg ml⁻¹) of Cry1Ac and a control were prepared. The artificial diet preparation was similar to that as described earlier except for the exclusion of ampicillin and a 10% reduction in a distilled water. Approximately 5 ml of the diet containing a toxin concentration was placed into a small aerated cup. Four replications were used for each bioassay. All bioassays were carried out under controlled environment in a growth chamber at 25 ± 2 °C, 65 ± 10% (RH) with a 16:8 (light/dark) cycle.

Data statistical analysis

The molt inhibitory and mortality rates were recorded after 7 days of bioassay. Molt inhibition was recorded when the larvae were unable to molt to the next larval instar. These counts were considered dead larvae. The mortality rate was recorded when larvae failed to respond with a gentle touch of a fine brush and considered as dead. The molt inhibition and mortality data were corrected from control mortality by Abbott formula (Abbott 1925), where needed. Probit analyses were done with PoloPlus (LeOra Software, 2003). The LC₅₀ and MIC₅₀ values, with their related fiducial limits at 95%, were assessed. The resistance ratios were determined by dividing LC₅₀/MIC₅₀ of field with susceptible laboratory population. LC₅₀ and MIC₅₀ values were considered significantly different when they did not overlap each other with their respective 95% fiducial limits.

Lethality of Cry1Ac toxin against S. exigua in 2015

L₁, L₂, and L₃ larvae of S. exigua collected from FSD, MLT, and BWP exhibited variable responses to different levels of Cry1Ac toxin in terms of obtained lethal concentration values (LC₅₀). The BWP population showed a maximum LC₅₀ value for all instars, followed by MLT while the lowest value was estimated for FSD population. The susceptible population showed the least LC₅₀ (Table 1). The lethal concentrations ranged from 0.45 to 2.52 μg ml⁻¹ for L₁, 1.08 to 5.74 μg ml⁻¹ for L₂ and 2.01 to 7.85 μg ml⁻¹ for L₃ (Table 1). The field populations showed a variation in susceptibility levels across all populations up to a 5.63-fold for L₁, 5.30-fold for L₂ and 3.89-fold for L₃. The molt inhibitory concentration ranged from 0.04 to 0.56 μg ml⁻¹, 0.08 to 0.99 μg ml⁻¹ and 0.10 to 1.42 μg ml⁻¹ for L₁, L₂, and L₃. The data also recorded 12.51-fold (L₁), 12.92-fold (L₂), and 13.66-fold (L₃) variations in susceptibility levels among all the tested populations (Table 2).

Lethality of Cry1Ac toxin against S. exigua in 2016

The collected S. exigua population in 2016 showed a considerable variability in LC₅₀ basis of all bio-assayed instars (Table 3). The LC₅₀ values ranged from 0.45 to 2.83 μg ml⁻¹, 1.10 to 5.91 μg ml⁻¹, and 2.01 to 7.85 μg ml⁻¹ for L₁, L₂, and L_3, respectively. L₁ showed 6.25-fold variations, followed by L₂ (5.35-fold) and L₃ (3.89-fold) in susceptibility across all populations. The molt inhibitory concentrations ranged from 0.05 to 0.63 μg ml⁻¹, 0.08 to 1.23 μg ml⁻¹, and 0.11 to 1.71 μg ml⁻¹, for L₁, L₂, and L_3, respectively. The data also identified 13.43-fold (L₁), 15.63-fold (L₂), and 15.44-fold (L₃) variations in susceptibility levels among all the tested populations (Table 4). The results also clearly indicated that BWP population was highly resistant to Cry1Ac toxin than MLT and FSD populations. The level of resistance increased with time as depicted through 2015 and 2016 analysis.

The toxicological studies (Kashyap and Amla, 2007), by applying the Bt against American and armyworms, resulted in LC₅₀ for S. litura by 0.11 μg. Bernardi et al. (2014) determined the baseline susceptibility test against S. frugiperda and Diatraea saccharalis. The LC₅₀ mortality was 61.18 and 367.86 ng Vip3Aa20 cm⁻² for the 6 populations of D. saccharalis, and between 92.38 to 611.65 ng Vip3Aa20 cm⁻² for 16 populations of S. frugiperda. Moreover, the effective concentration (EC₅₀) ranged from 48.65 to 163.60 and 21.76 to 70.09 ng Vip3Aa20 cm⁻² for D. saccharalis and S. frugiperda, respectively. Obtained results are in agreement with those of Hernandez-Martınez et al. (2008) who recorded toxic effects of different toxins from Bt on the S. exigua. Likewise, Moar et al. (1995) and Ashfaq et al. (2000) concluded that Cry1Ac was the most effective against lepidopterans and reported approximately 500-fold resistance than the susceptible strain. The variability of Bt toxin in different cotton genotypes forced the farming community to shift towards continuous spraying of chemical insecticides in southern Pakistan. The repeated sprays resulted to develop other insect pests. The present study showed that good management of S. exigua under field conditions should be used only these varieties, which have higher Bt expression or dual toxin genotypes. Resistance in S. exigua to these types of Cry toxins can be overcome by planting new and better expression genotypes. The penetration of single Cry toxin varieties has been huge that might induce more serious risk and ultimately reflected in control failure.

The present study established a benchmark for the susceptibility of S. exigua to Bt Cry1Ac toxin. The results provided a background of developing a high-level resistance of transgenic Bt cotton varieties against Spodoptera spp. The data is important to policy-makers and technology providers; those develop strategies for the exploitation of transgenic Bt-cotton varieties as a component of IPM strategy.

Data will not be shared

Bt:

Bacillus thuringiensis

BWP:

Bahawalpur

Cry:

Crystalline

FSD:

Faisalabad

GMP:

Good management practices

IPM:

Integrated pest management

L:D:

Light and dark

L₁ :

First instar larva

L₂ :

Second instar larva

L₃ :

Third instar larva

LC:

Lethal concentration

MIC:

Molt inhibitory concentration

MLT:

Multan

NaCl:

Sodium chloride

NIAB:

Nuclear Institute of Agriculture and Biotechnology

POP:

Population

RH:

Relative humidity

RR:

Resistance ratio

SS:

Susceptible

UK:

United Kingdom

The authors are thankful to the Higher Education Commission of Pakistan for providing funding facilities to conduct research study.

Funding

Higher Education Commission, Pakistan, provided funding for conducting research study.

Affiliations

1. Department of Entomology, University of Agriculture Faisalabad, Faisalabad, 38040, Pakistan

   • Muhammad Ibrahim Shahid
   • , Muhammad Arshad
   •  & Mansoor ul Hasan

2. Department of Plant Pathology, University of Agriculture Faisalabad, Faisalabad, Pakistan

   • Muhammad Aslam Khan

Contributions

All authors read and approved the final manuscript.

Corresponding author

Correspondence to Muhammad Arshad.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

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