Molecular profiling of resistance alleles in Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) collected from different locations

Abstract

Background

After the commercialization of insect-resistant transgenic Bt cotton Bollgard I & II, India ranks first in the world in cotton production. Cotton insecticide consumption was drastically reduced as nearly 95% of the cotton area was replaced with Bollgard II. However, the benefits of transgenic cotton appear to have been diminished as the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) developed field resistance to Cry1Ac expressing Bt cotton in India in 2008. In 2015, there was an unusual survival of P. gossypiella on Bollgard II cotton in some parts of Gujarat and Maharashtra, which is a cause for concern.

Results

In the present study, PCR analysis and gel visualization of BGII resistant field population of P. gossypiella showed that the presence of r1, r2, r3, r1s, r2s, r3s, and ss mutated cadherin alleles, which produced amplicon sizes of 750 bp, 700 bp, 730 bp, 12,700 bp, 750 bp, 980 bp, 600 bp and 1600 bp, respectively, when seven different types of cadherin allele(s) specific primers were used. The r1 mutant allele was detected in Amaravati, Wardha, Yavatmal, and Nagpur, but not in Akola and Buldhana, using primers r1disfor and Int-540. The r2 mutant allele was detected in Akola, Wardha, and Nagpur, but not in Buldhana, Amaravati, and Yavatmal, using primers r2disback and Cad2366. The r3 mutant allele was detected in all locations using primers r3disback and Cad3221. Amplicons of sizes 750 bp, 700 bp and 730 bp were obtained for r1, r2, and r3 mutant alleles, respectively. The sizes of the amplicons were 1270 bp for r1s, 750 bp for r2s, and 980 bp for r3s.The absence of bands for r1, r2, and r3 cadherin alleles in individuals indicated the possibility of having the ss allele, which was confirmed using primers Cad3324 and Int-651. The presence of ss mutant allele was observed in field-collected P. gossypiella populations from BG II cotton in various locations, with a 600 bp and 1600 bp size amplicons produced using the same primers.

Conclusions

PCR analysis revealed the presence of r1, r2, r3, r1s, r2s, r3s, and ss mutated cadherin alleles in field-collected populations of Vidarbha which provide evidence to field-evolved resistance of P. gossypiella to BGII cotton.

Background

India is the largest cotton cultivator globally, and prior to the use of genetically modified Bt cotton, cotton yields were impacted by insect pests such as the bollworm complex. The adoption of Bt cotton hybrids, which produce Cry1Ac (BG I), has significantly impacted the management of bollworms in India, making it the world's largest producer of cotton. Bacillus thuringiensis(Bt) is a bacterium that produces Cry proteins or endotoxins, and scientists have introduced genes from Bt into cotton plants to create transgenic Bt-cotton. The Cry genes from Bt var. kurstaki were transferred to cotton using Agrobacterium and CaMV 35S promoter. These genes produce crystalline endotoxins that disrupt the osmotic balance of susceptible insects' midgut, causing epithelial cell lysis, starvation, and ultimately death. Currently, there are over 700 Cry gene sequences that have been identified, coding for 78 different Cry proteins and 323 Cry protein holotypes (Mathew et al. 2018). Pectinophora gossypiella (Saunders) is a common pest of Bt cotton in India and worldwide, causing damage ranging from 10 to 50%. Cry1Ac and Cry2Ab are the Bt toxins widely used in insect-resistant transgenic crops. Bt cotton expressing the Cry1Ac gene (BG I) has controlled all bollworms, including P. gossypiella, since 2002. To delay bollworm resistance to BG I, a second-generation Bt cotton (BGII) expressing Cry1Ac + Cry2Ab was introduced, replacing over 95% of traditional cotton cultivation in India and reducing pesticide use. P. gossypiella has become more aggressive due to the development of insecticide and Cry toxin resistance. Survivability of P. gossypiella on Bollgard cotton was observed in Gujarat, (Monsanto Report, 2010) and larval survival on Bollgard II was significantly higher in some districts of Saurashtra. An outbreak of P. gossypiella in Maharashtra in 2016 (Naik et al. 2017) caused significant damage and yield losses in Bt cotton, as well as increased management costs in several states. Cry1Ac-resistant P. gossypiella has a higher survivability rate on BG I compared to dual-stacked cotton (Kranthi 2016). P. gossypiella populations in India have developed resistance to Cry1Ac, with an LC₅₀ of 0.04 g (Muralimohan et al. 2009). Cry2Ab-resistant P. gossypiella also showed cross-resistance to Cry1Ac, but Cry1Ac-resistant strains had low cross-resistance to Cry2Ab. After 5 years of exposure to Cry1Ac, the frequency of resistant individuals increased to 56% in Gujarat state. Cry toxins bind to specific receptors in the midgut of lepidopteran insects, causing cell lysis and death. Cadherin, aminopeptidase N, and alkaline phosphatase are crucial receptors for Cry toxin toxicity (Zhao et al. 2017). Insects can develop resistance to Cry toxins by altering their binding sites (Ferre and van Rie 2002). Despite a decade of exposure, P. gossypiella populations in the United States did not develop resistance to Bt cotton (Tabashink et al. 2006). Mutations in the cadherin gene confer Cry1Ac resistance in P. gossypiella, and 13 cadherin-resistant alleles have been identified in resistant P. gossypiella populations from the United States, India, and China (Morin et al. 2003). Alternative splicing of mRNA in India also results in cadherin mutations that confer Cry1Ac resistance in P. gossypiella (Fabrick et al. 2014). A mutation in the trans-membrane region of cadherin was found to induce Cry1Ac resistance in P. gossypiella (Wang et al. 2018). Current study aims to profile molecular resistance alleles in P. gossypiella populations from different locations.

Methods

Sample collection

Pink bollworm-infested mature green cotton bolls were collected at random from commercial/farmer cotton fields of BG-II in the Vidarbha districts of Akola, Buldhana, Amaravati, Wardha, Yavatmal, and Nagpur during October–November 2020, representing all cotton growing zones of Vidharba.

Genomic DNA extraction protocol

To extract DNA from the P. gossypiella larvae, 200 µl of ice-cold homogenization buffer was added to crush insects. Twenty-five µl of lysis buffer was then added and the mixture was incubated at room temperature for 20 min. Following this, 25 µl of 20% Sodium dodecyl sulfate (SDS) was added and the mixture was incubated at 65º C for 50 min. RNAase was then added at a concentration of 5 µl per tube and the mixture was incubated at 37º C for 15 min. Subsequently, 30 µl of potassium acetate buffer was added and the mixture was incubated at 4ºC for 30 min (3 M for Potassium and 5 M for acetate). An equal volume of Phenol: Chloroform 1:1 [Phenol (5 ml); Chloroform Isoamylalcohol (5 ml)] and 500 µl Phenol: Chloroform Isoamylalcohol was added, and the mixture was centrifuged at 12,000 rpm for about 1 min. The aqueous layer was then collected and mixed with ice-cold 600 µl Isoproponal and incubated at -20º C overnight. The mixture was centrifuged at 12,000 rpm for 10 min and the supernatant was removed. The resulting pellet was washed with 500 µl of 70% ethanol, air-dried, and dissolved in 50 µl of TE buffer. Before being utilized in a PCR reaction, the DNA was diluted to a concentration of 100 ng/ml. The DNA concentration was measured using a NanoDrop® ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE) and the quality was verified through 0.8% agarose gel electrophoresis. This ensured that the DNA was suitable for use as a template in the PCR reaction.

Quantification of genomic DNA

The isolated genomic DNA was quantified using the ethidium bromide fluorescent quantification method, as described by Maniatis et al. (1982). The band intensity of the isolated DNA in agarose, dissolved in 40 ml of 1XTAE buffer containing ethidium bromide at a concentration of 0.5 μg per ml, was compared to the band intensity of a reference λ DNA Hind III digest. In addition to this method, the quality and concentration of the DNA were also determined, using a spectrophotometer and by gel electrophoresis with 1.2% agarose and known concentrations of uncut λ DNA.

$${\text{DNA}}\;\left( {{\mu g}/{\mu l}} \right) = \frac{{{\text{OD}}260 \times 50\left( {{\text{Dilution}}\;{\text{factor}}} \right) \times 50\;{\mu l}/{\text{ml}}}}{1000}$$

The purity of the DNA was determined by measuring the ratio of OD260 to OD280. A ratio of 1.6 or less indicates that the sample may contain proteins and/or other UV absorbers, while a ratio greater than 2.0 suggests the presence of contamination from chloroform or phenol.

PCR amplification

Mutations in the cadherin gene were amplified from 6 populations of P. gossypiella, using the cadherin gene-specific primers (Table 1) provided below as described by Morin et al. (2003).

Table 1 PCR Primers used for determining cadherin genotypes

PCR reactions were performed with a total volume of 20 μl, comprising 2.5 μl of 10 × PCR buffer, 0.4 μl of dNTPs, 0.5 μl of each primer, 0.25 μl of Taq DNA polymerase (M/S Bangalore Genei Pvt. Ltd., Bangalore), 14.85 μl of water, and 1 μl of genomic DNA. A touchdown thermal cycling protocol, covering a range of annealing temperatures from 55 to 50°C, was also used for amplification in a Thermal Cycler (Bio-Rad, Hercules, CA). The cycling began with an initial denaturation at 94°C for 5 min. The cycling parameters were then set at 39 cycles of 94°C for 20 s, 55°C for 1 min, and 72°C for 1 min (Table 2), followed by a final cycle of 1 min at 55°C and a 7-min extension step at 72°C.

Table 2 Different cycles of DNA amplification of pink bollworm population through PCR Primers

Results

Detection of r alleles using allele-specific cadherin gene primers

Three allele-specific PCR reactions were performed, each with an allele-specific primer, to selectively amplify the respective r allele. Different allele-specific primers namely; r1disfor and Int-540 for r1, r2disback, Cad2366 for r2 and r3disback, and Cad3221 for r3 were used during the present investigation. After the PCR reaction, electrophoresis was carried out to see the presence of r1, r2, and r3 mutant allele(s) in field-collected P. gossypiella population collected from BG II cotton. Visualization of gel images states the presence of r1, r2, and r3 mutant alleles in BG II resistant field populations of collected P. gossypiella. A 750 bp size amplicons were obtained when primers r1disfor and Int-540 were used to detect the presence of the r1 mutant allele. The r1 mutant allele is detected in locations of Amaravati, Wardha, Yavatmal, and Nagpur except for Akola and Buldhana (Fig. 1). A 700 bp size amplicons were produced when primers r2disback and Cad2366 were used to detect the presence of r2 mutant allele. r2 mutant allele is detected in locations of Akola, Wardha, and Nagpur, except for Buldhana, Amaravati, and Yavatmal (Fig. 2). A 730 bp size amplicons were produced when primers r3disback and Cad3221 were used to detect the presence of r3 mutant allele. r3 mutant allele was detected in locations of Akola, Buldhana, Amaravati, Wardha, Yavatmal, and Nagpur (Fig. 3).

Fig. 1

PCR amplified r1 cadherin gene in BG II resistant populations of Pectinophora gossypiella using r1disfor &Int-540. M = 1 Kb plus DNA ladder, Lane 1 = Akola, Lane 2 = Buldana, Lane 3 = Amravati, Lane 4 = Warda, Lane 5 = Yavatmal,Lane 6 = Nagpur

Fig. 2

PCR amplified r2 cadherin gene in BG II resistant populations of Pectinophora gossypiella using Cad2366 & r2disback. M = 1 Kb plus DNA ladder, Lane 1 = Akola, Lane 2 = Buldana, Lane 3 = Amravati, Lane 4 = Warda, Lane 5 = Yavatmal, Lane 6 = Nagpur

Fig. 3

PCR amplified r3 cadherin gene in BG II resistant populations of Pectinophora gossypiella using Cad3221 &r3disback. M = 1 Kb plus DNA ladder, Lane 1 = Akola, Lane 2 = Buldana, Lane 3 = Amravati, Lane 4 = Warda, Lane 5 = Yavatmal, Lane 6 = Nagpur

Discrimination between resistant homozygotes and heterozygotes using allele-specific cadherin gene primers

PCR reactions were done to discriminate between homozygous resistant (r1r1, r2r2, or r3r3) alleles and heterozygous resistant (r1s, r2s, or r3s) alleles. Primers used were Cad3386 and Int-651, Cad2366 and r2allback and Int-651and Cad3221 and Int-651. After the PCR reaction, electrophoresis was carried out to see the presence of r1s, r2s and r3s mutant allele(s) in the field-collected P. gossypiella population collected from BG II cotton. Visualization of gel images states the presence of r1s, r2s, and r3s mutant allele in BG II resistant field populations of P. gossypiella. A 1270 bp size amplicon was produced when primers Cad3324 and Int-651 were used to detect the presence of the r1s mutant allele. r1s mutant allele was detected in locations of Akola, Amaravati, Yavatmal, and Nagpur, except for Buldhana and Wardha (Fig. 4). A 750 bp size amplicons were produced when primers Cad2366 and r2allback were used to detect the presence of r2s mutant allele. r2s mutant allele was detected in locations of Akola, Amaravati, Wardha, Yavatmal, and Nagpur except for Buldhana (Fig. 5). A 980 bp size amplicons were produced when primers Cad3221 and Int-651 were used to detect the presence of r3s mutant allele. r3s mutant allele was detected in locations of Akola, Amaravati, Wardha, Yavatmal, and Nagpur except for Buldhana (Fig. 6).

Fig. 4

PCR amplified r1s cadherin gene in BG II resistant populations of Pectinophora gossypiella using Cad3386 &Int-651. M = 1 Kb plus DNA ladder, Lane 1 = Akola, Lane 2 = Buldana, Lane 3 = Amravati, Lane 4 = Warda, Lane 5 = Yavatmal, Lane 6 = Nagpur

Fig. 5

PCR amplified r2s cadherin gene in BG II resistant populations of Pectinophora gossypiella using Cad2366 & r2allback. M = 1 Kb plus DNA ladder, Lane 1 = Akola, Lane 2 = Buldana, Lane 3 = Amravati, Lane 4 = Warda, Lane 5 = Yavatmal, Lane 6 = Nagpur

Fig. 6

PCR amplified r3s cadherin gene in BG II resistant populations of Pectinophora gossypiella using Cad3221 &Int-651. M = 1 Kb plus DNA ladder, Lane 1 = Akola, Lane 2 = Buldana, Lane 3 = Amravati,Lane 4 = Warda, Lane 5 = Yavatmal, Lane 6 = Nagpur

Individuals that produce no bands when tested for their presence of r1, r2, and r3 cadherin alleles could be ss. However, it was again reconfirmed using primers Cad3324 and Int-651. After the PCR reaction, electrophoresis was carried out to see the presence of ss mutant allele(s) in the field-collected P. gossypiella population from BG II cotton. Visualization of gel images states the presence of ss mutant allele in BG II resistant field-collected populations of P. gossypiella in Akola, Buldhana, Amaravati, Wardha, Yavatmal, and Nagpur locations. A 600 bp and 1600 bp size amplicons were produced when primers Cad3324 and Int-651 were used to detect the presence of the ss mutant allele (Fig. 7).

Fig. 7

PCR amplified ss cadherin gene in BG II resistant populations of Pectinophora gossypiella using Cad3324 & Int-651. M = 1 Kb plus DNA ladder, Lane 1 = Akola, Lane 2 = Buldana, Lane 3 = Amravati, Lane 4 = Warda, Lane 5 = Yavatmal, Lane 6 = Nagpur

Discussion

Findings of the present study suggested that mutations in the cadherin receptor had a major role in BG II resistance in populations of P. gossypiella. The findings are consistent with the presence of the same resistant alleles in Cry1Ac-resistant Amreli populations of P. gossypiella in India reported by Dhurua and Gujar (2011). Similarly, Morin et al. (2004) found that in many insects, reduced binding of Cry toxins to different receptors such as cadherin, alkaline phosphatase, and aminopeptidase N is the most accepted Mode 1 type of resistance to Cry1Ac toxin. P. gossypiella resistance to Cry1Ac toxin in the United States has been linked to mutations in cadherin receptors r1, r2, and r3. Fabrick et al. (2014) discovered eight different mutated cadherin alleles in Cry1Ac resistance, namely r5 to r12, they further found that these alleles are associated with mutations disrupting the extracellular domain of cadherin receptors that bind to Cry1Ac toxin. Mechanisms underlying P. gossypiella resistance against Cry2Ab and thereby to BG II were unknown. Similarly, Wang et al. (2018) found P. gossypiella to be 220 times more resistant to Cry1Ac and 2.1 times more resistant to Cry2Ab in China. In Cry1Ac-resistant P. gossypiella, the new mutant allele r13 disrupts cadherin cellular trafficking in the trans-membrane region. As a result, single-point mutations in H. armigera mislocalized cadherin receptors in Cry1Ac-resistant individuals, affecting membrane receptor trafficking. Although different alleles are responsible for Cry1Ac resistance in P. gossypiella studies on trans-membrane mutations in cadherin receptors need to be prioritized to elucidate the mechanism of Cry2Ab and BG II resistance in P. gossypiella in India. Similarly, Wang et al. (2019) discovered the most common resistance allele (r13) in the United States, accounting for more than 71% of all resistance alleles detected. Correspondingly Wang et al. (2019) discovered that the r16 allele, which disrupts cadherin localization mediates Cry1Ac resistance in P. gossypiella. Likewise, Wang et al. (2020) discovered that the r14 allele of the P. gossypiella cadherin gene (PgCad1) was recessive and was tightly linked to r14, which reduced translation, increased degradation and/or mislocalized cadherin. Host specificity and toxicity of different groups of Bt Cry toxin vary. Although the molecular interactions of Cry toxins are not fully understood, Cry toxins' binding to specific midgut proteins varies depending on the toxins and hosts. To be effective against insects, Bt Cry toxins must bind to GPI-anchored receptors found in brush border membrane vesicles in the insect's midgut. The most widely accepted theory in the laboratory and field-selected Cry1Ac-resistant strains of many insects were that toxin binding to the midgut epithelium was reduced (Wu 2014). Caccia et al. (2010) discovered that binding site alteration is a mechanism of resistance in both Cry1A and Cry2A class proteins in many insects. Previous researchers reported mutations in cadherin receptors for Cry1Ac resistance, but little work has been reported on enzymatic activity in various insects. Earlier reduced membrane-bound alkaline phosphate levels in Cry1Ac-resistant Helicoverpa virescens were linked to resistance development, according to Jurat-Fuents and Adang (2004). Cry1Ac resistance was conferred in Indian populations of P. gossypiella by reduced binding of Cry1Ac to larval brush border membrane vesicles (BBMV) (Ojha et al. 2014). Previous and present data on the mechanism of resistance in P. gossypiella to Cry2Ab and thereby to BG II suggest that more research is needed to identify the new sights and more emphasis may be placed on the role of different resistant alleles, alkaline phosphatases, ABC transporters and gut inhabiting symbionts to accelerate monitoring and management of field-evolved resistance to Cry2Ab.

Conclusion

PCR analysis of the field population of P. gossypiella revealed the presence of various mutated cadherin alleles (r1, r2, r3, r1s, r2s, r3s, and ss) that produced different amplicon sizes when seven different types of cadherin allele-specific primers were used. The gel visualization of the results suggested that the field population has evolved resistance to BGII cotton.