Physiological Factors Influencing Bacillus thuringiensis Susceptibility in Laboratory and Field-Collected Bollworm, Helicoverpa zea (Lepidoptera: Noctuidae)

Abstract

Field-evolved resistance of Helicoverpa zea to crops expressing Cry insecticidal proteins from Bacillus thuringiensis (Bt) is widespread across the United States. To comparatively evaluate physiological factors associated with Bt susceptibility, we analyzed two laboratory strains (Benzon and SIMRU) and one field colony obtained from a commercial corn field near Pickens, Arkansas. Biochemical assays of larval midgut extracts showed that Pickens exhibited significantly altered activities of chymotrypsin-like proteases, aminopeptidase N (APN), and alkaline phosphatase (ALP) compared with the SIMRU or Benzon colonies, with differences varying by larval instar. In contrast, trypsin-like protease activities did not differ significantly among the three colonies. Gene expression analyses of ten serine protease genes and seven candidate Cry receptor genes (including cadherin, ATP-binding cassette family C2, ALP, and four APN genes) revealed significant transcriptional differences in the Pickens relative to the lab colonies. Collectively, these results suggest that chymotrypsin-like proteases may play an important role in the activation of Cry toxins in H. zea. Altered chymotrypsin and APN activities, together with differential gene expressions in the Pickens population, likely contribute to reduced Bt susceptibility. The biochemical and molecular differences provide insight into potential physiological factors underlying reduced Bt susceptibility and may inform future Bt resistance monitoring and management strategies.

1. Introduction

The corn earworm or cotton bollworm (Helicoverpa zea Boddie; Lepidoptera: Noctuidae) is a major agricultural pest in the United States, capable of causing substantial damage to economically important crops such as cotton, corn, and soybean [1,2]. Since the commercial introduction of genetically engineered crops expressing insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) Berliner in 1996, Bt crops played a critical role in managing lepidopteran pests, including H. zea [3,4]. These crops have significantly reduced reliance on conventional insecticides, enhanced biological control by natural enemies, improved farm profitability, and lowered environmental and health risks [5,6,7,8,9]. By 2019, Bt crops were planted in over 109 million hectares across 27 countries [10]. However, the evolution of resistance to Bt toxins in the target insect population reduces the advantages of transgenic crops and threatens the long-term sustainability of these benefits [11,12,13]. Documented cases of field-evolved or “practical” resistance increased from 3 in 2005 to 26 by 2020, involving 11 pest species and 9 different crystalline (Cry) toxins [13]. In the United States, the management of H. zea has proven particularly challenging due to the rapid evolution of practical resistance to 5 different Cry toxins used in Bt crops (Cry1Ab, Cry1A.105, Cry1Ac, Cry1Fa, and Cry2Ab) within just 13 years of commercialization [13]. Several factors likely contributed to this resistance, including inherent tolerance to some toxins [14] and Bt corn varieties expressing Cry toxins that fail to meet the high-dose requirement essential for effective resistance management [15].

Cry proteins are pore-forming toxins. After ingestion, Cry protoxins are solubilized and activated by proteases in the larval midgut, and after crossing the peritrophic matrix, the activated toxins bind to specific receptors on the brush border membrane of midgut epithelial cells [16,17]. Several Cry toxin receptors have been identified, including cadherins, aminopeptidases (APNs), alkaline phosphatases (ALPs), and ATP-binding cassette (ABC) transporters [17]. Effective receptor binding is conducive to pore formation, which causes ion imbalance, cell swelling, lysis, midgut leakage, and ultimately insect death [18]. In H. zea and other lepidopteran pests, resistance to Cry toxins generally arises through two primary mechanisms: reduced midgut serine protease activity hindering protoxin activation, and alterations in midgut receptors that reduce or prevent effective toxin binding [19,20]. For example, resistance to Cry1Ac in H. zea was associated with altered proteolytic processing in the midgut [21]. Practical resistance to Bt corn expressing Cry proteins has also been associated with mutations in a cadherin gene [22], and amplification of a trypsin gene cluster [23]. While not considered a major resistance pathway to Cry toxins, enzymatic detoxification has also been reported to impact susceptibility to Bt toxins [24].

The objective of this study was to comparatively evaluate physiological factors associated with Bt susceptibility in H. zea using two laboratory strains (Benzon and SIMRU) and one field-collected population obtained from a commercial corn field near Pickens, Arkansas. Specifically, we aimed to determine whether variation in midgut protease activity and putative Cry toxin binding proteins correlates with differences in Bt susceptibility among three populations. To address this objective, we examined the enzymatic activities of trypsin- and chymotrypsin-like serine proteases, as well as ALP and APN in midgut extracts from multiple larval instars across three populations. To further identify physiological factors potentially contributing to reduced Bt susceptibility in the field population, we conducted transcriptional analyses of serine proteases genes (four trypsin and six chymotrypsin), candidate Bt receptor genes (cadherin, ABCC2, ALP, and four APN genes), and four detoxification genes (esterase, Glutathione S-transferases (GSTs), CYP6B8, and CYP6B28) in 3rd instar larvae across three populations. Collectively, these biochemical and transcriptional assessments provide a comparative framework for identifying the physiological correlation of Bt susceptibility in H. zea and for informing future mechanistic and resistance monitoring studies.

2. Materials and Methods

2.1. Insect Colonies

A laboratory reference H. zea colony has been consecutively maintained for over 500 generations at the USDA-ARS Southern Insect Management Research Unit (SIMRU) in Stoneville (MS) since 1971. Another reference H. zea colony was purchased from Benzon Research Inc. (Carlisle, PA, USA). Both SIMRU and Benzon colonies are considered susceptible to Cry toxins and have been widely used in Bt susceptibility studies [2,25]. A field-derived H. zea colony was collected in June 2024 from a commercial corn field near Pickens (AR). Previous research has shown that field colonies frequently display reduced Bt susceptibility to Cry toxins [1,2]. All colonies were reared in the laboratory using an artificial diet containing soy and wheat germ [26], following the procedures described previously [27]. All colonies were reared under laboratory conditions (26 ± 1 °C, 30 ± 5% relative humidity, 14 h light:10 h dark).

2.2. Insecticides and Chemicals

All the reagents, including Triton X-100, Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA), N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAAPFpNA), leucine-p-nitroanilide (LpNA), and p-nitrophenyl phosphate disodium (pNPP), were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Bradford protein assay kit with a bovine serum albumin standard was purchased from Thermo Scientific (Waltham, MA, USA).

2.3. Midgut Extracts

Midgut tissues were dissected from 3rd, 4th, and 5th instar H. zea larvae collected from three different populations: SIMRU, Benzon, and Pickens. For each developmental stage, midgut samples were pooled as follows: 4 larvae for the 3rd instar, 3 for the 4th instar, and 2 for the 5th instar. Pooled tissue was rinsed and homogenized in 0.3 mL of ice-cold buffer (100 mM Tris-HCl, pH 8.0) using beads and a homogenizer (Fisherbrand Bead Mill 24, Waltham, MA, USA). The homogenized tissue was then centrifuged at 14,000× g for 15 min at 4 °C, and the resulting supernatants were collected for enzymic assays. Protein concentration of each enzyme extract was measured using a Bradford protein assay kit with a bovine serum albumin standard [28] (ThermoScientific, Waltham, MA, USA). Prior to enzymatic testing, samples were diluted 10-fold and assayed for protease, APN, and ALP activity. Three replicates (distinct pools) were performed per strain and instar, and all enzyme and protein assays were conducted in triplicate using a microplate reader (Agilent BioTek Synergy H1 Multimode Microplate Reader, Winooski, VT, USA).

2.4. Midgut Protease Activity

Trypsin and chymotrypsin activities were determined using BApNA and SAAPFpNA as the respective substrates, following slightly modified protocols from Zhu et al. (2007) [29]. Substrate working solutions were prepared by diluting 0.1 mL of BApNA (50 mg/mL in DMSO) or SAAPFpNA (50 mg/mL in Universal buffer) into 4.9 mL of Universal buffer (Britton–Robinson Buffer: Phosphoric acid, Acetic acid, Boric acid, and adjusted to pH 8.5 with sodium hydroxide). For each assay, 50 μL of diluted midgut extract was mixed with 50 μL of substrate solution (final concentration: 0.5 mM) in a 96-well microplate. After 30 s of incubation, the absorbance at 405 nm was recorded every 15 s for 15 min using a BioTek Synergy H1 microplate reader.

2.5. Alkaline Phosphatase (ALP) and Aminopeptidase (APN) Activity Assays

Specific activities of APN and ALP were assessed using LpNA and pNPP as substrates, respectively [29]. Working substrate solutions were prepared by diluting LpNA (80 mM in DMSO) or pNPP (51.25 mM in 100% Ethanol) with Tris-HCl (pH 8.5). For each assay, 45 μL of the appropriate working stock LpNA (final concentration: 0.8 mM in Tris-HCl) or pNPP (final concentration: 1.025 mM in Tris-HCl) and 5 μL of midgut extract were added into each well. Absorbance at 405 nm was recorded every 15 s for 15 min using a BioTek Synergy H1 microplate reader.

2.6. Quantitative Real-Time PCR (qPCR)

Midgut samples from 3rd instar larvae were dissected and preserved in RNAlater, then stored at −80 °C until RNA was extracted. Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), with a minimum of three replicates prepared per colony (each replicate consisting of three pooled midguts). First-strand of cDNA was synthesized from 1 µg of total RNA using the SuperScriptTM IV Reverse Transcriptase System (Invitrogen, Carlsbad, CA, USA). Gene-specific primers (Supplemental Table S1) were designed using Beacon Designer™ to amplify 100–400 bp fragments of 10 serine protease genes (four trypsin and six chymotrypsin genes), 7 candidate Cry receptor genes (cadherin, ABCC2, ALP, and four APN genes), and 4 detoxification genes (esterase, GST, CYP6B8, and CYP6B28). Elongation factor 1 alpha (EF-1α) and actin were used as internal reference transcript genes for normalizing, EF-1α for serine protease and Cry receptor genes, and actin for detoxification enzyme gene expression. Quantitative PCR (qPCR) reaction was performed in 10 µL volumes containing 1 µL of 1:10 diluted cDNA, 2 µL of 10 µM gene-specific primer, and 5 µL Fast SYBRTM Green Master Mix (Applied Biosystems, Foster City, CA, USA). Amplification was carried out on a Bio-Rad CFX Connect real-time system (Bio-Rad Laboratories Inc., Hercules, CA, USA) using the following protocol: 40 cycles of 95 °C for 15 s followed by 60 °C for 60 s. Gene expression of each gene was measured in at least three replicates (distinct pools) per colony, with each measured in triplicate. Gene expression was analyzed using the ΔCT method, and relative expression levels were estimated using the 2−ΔΔCT method [30].

2.7. Data Analysis

Enzyme activities (trypsin, chymotrypsin, APN, and ALP) and gene expression levels were analyzed using JMP software Version 17.0 (SAS Institute, Cary, NC, USA). Differences among treatments were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s HSD test, with significance determined at p < 0.05.

3. Results

3.1. Activity of Midgut Trypsin and Chymotrypsin

Protease activities (U per mg ± SE) of trypsin and chymotrypsin were examined in midgut extract from 3rd, 4th, and 5th instar H. zea larvae from the SIMRU, Benzon, and Pickens colonies. No significant difference in trypsin activity was detected among populations within the same developmental stage (Figure 1a). However, trypsin activity significantly decreased with larval development in both Benzon and Pickens populations (df = 8, 100, F = 12.18, p < 0.001) (Figure 1a). Chymotrypsin activity showed no significant variation among colonies in the 3rd and 4th instar. In contrast, 5th instar larvae from the Pickens colony exhibited significantly reduced chymotrypsin activity compared to the SIMRU colony (df = 8, 93, F = 5.29, p < 0.001), with no significant differences between the Benzon and Pickens populations (Figure 1b).

3.2. Activity of Midgut Alkaline Phosphatase (ALP) and Aminopeptidase N (APN)

ALP and APN activities (U per mg ± SE) were assessed in midgut extracts from 3rd, 4th, and 5th instar larvae across all three populations. APN activity did not differ significantly among populations in the 3rd and 5th instar. However, 4th instar larvae from Pickens showed significantly elevated APN activity, approximately 1.97- and 2-fold higher than SIMRU and Benzon colonies, respectively (df = 8, 99, F = 10.94, p < 0.001) (Figure 2a). In contrast, no significant differences in ALP activity were detected among SIMRU, Benzon, and Pickens in the 4th and 5th instar larvae, but significantly reduced ALP activity in 3rd instar Pickens larvae, compared to Benzon (df = 8, 104, F = 13.83, p < 0.001) (Figure 2b).

3.3. Expression Levels of Protease, Cry Toxin Receptor, and Detoxification Genes

In the Pickens population, the expression of one trypsin gene was significantly increased (Trypsin-4: df = 2, 21, F = 242.22, p < 0.001). In contrast, three of the chymotrypsin genes were significantly downregulated, compared to the SIMRU or Benzon colonies (Chymo-2: df = 2, 21, F = 7.59, p = 0.003; Chymo-4: df = 2, 15, F = 20.28, p < 0.001; Chymo-6: df = 2, 15, F = 6.97, p = 0.007) (Figure 3a). Between the two laboratory colonies, one chymotrypsin gene was significantly downregulated (Chymo-1, df = 2, 18, F = 5.81, p = 0.011), while Chymo-2 was upregulated (Chymo-2: df = 2, 21, F = 7.59, p = 0.003) (Figure 3a).

No significant differences in transcript levels were found among the three colonies for cadherin (df = 2, 11, F = 0.41, p = 0.673), ABCC2 (df = 2, 11, F = 3.53, p = 0.065), ALP (df = 2, 11, F = 1.31, p = 0.311), and one APN gene (APN1: df = 2, 11, F = 2.759, p = 0.107) (Figure 3b). Among the remaining three APN genes, one was significantly downregulated, while another two were significantly upregulated in Pickens compared to SIMRU (APN2: df = 2, 11, F = 13.18, p = 0.001; APN3: df = 2, 21, F = 81.93, p < 0.001; APN4: df = 2, 21, F = 81.93, p < 0.001). Between the SIMRU and Benzon colonies, no significant differences in expression were found between the four Cry toxin receptor genes.

In addition, no significant differences in expression were detected among the three populations for esterase (df = 2, 11, F = 1.75, p = 0.219), GST (df = 2, 11, F = 1.35, p = 0.300), CYP6B8 (df = 2, 11, F = 1.80, p = 0.210), and CYP6B28 (df = 2, 11, F = 3.95, p = 0.052) (Figure 4).

4. Discussion

Previous studies have shown that field-collected H. zea colonies frequently display significant tolerance to Cry toxins [1,2]. For example, Yang et al. (2022) reported that 93 out of 95 colonies collected from non-crop, non-Bt, and Bt crop hosts between 2016 and 2021 exhibited resistance ratios greater than 10 for at least one Cry toxin [2]. In the present study, we examined enzymatic activities and gene expression levels of midgut proteins involved in Cry toxin proteolytic processing and receptor binding in midgut extracts from two reference colonies and one field-derived population of H. zea. Enzyme activity assays revealed significant differences in activities of chymotrypsin, ALP, and APN at specific developmental stages in the Pickens field population compared with the SIMRU or Benzon laboratory colonies. In contrast, trypsin activity remained consistent among colonies within the same developmental stage. Transcriptional analyses of serine protease and Cry toxin receptor genes revealed significant alterations in the expression of several chymotrypsin and APN genes in the Pickens population. No significant differences were detected in the transcript levels of other receptors (cadherin, ABCC2, and ALP) or in the detoxification-related genes examined (esterase, GST, CYP6B8, and CYP6B28). These findings suggest that altered protease activity in the Pickens population may influence its susceptibility to Cry toxins compared to the two laboratory colonies.

Altered proteolytic activation by trypsin and chymotrypsin has been widely reported in multiple insect populations exhibiting resistance to Cry toxins. Proper enzymatic processing of Bt Cry protoxins is critical for mediating toxicity, and improper processing can reduce or eliminate toxin activity, resulting in resistance [31]. In some cases, resistant insects may express alternative proteases that degrade Cry toxins rather than activate them, thereby preventing effective binding to midgut receptors [19,20]. Alterations in midgut proteases have been documented in multiple species, including Plodia interpunctella [32,33], Helicoverpa armígera [34,35], Plutella xylostella [36,37], Ostrinia nubilalis [38,39,40], Heliothis virescens [41,42], Mythimna unipuncta [43], Spodoptera frugiperda [44], Spodoptera littoralis [45] and Leptinotarsa decemlineata [46]. In the present study, reduced chymotrypsin activity and downregulation of multiple chymotrypsin genes in the Pickens population may have impaired Cry toxin processing and activation, potentially contributing to reduced susceptibility commonly observed in field populations [1,2]. Notably, we also detected significant upregulation of one trypsin gene in the Pickens population. Similar upregulation of serine proteases (e.g., HzSP3) has been reported in Cry1Ac-resistant H. zea strains [21] and in field-resistant populations [23]. Such proteolytic alterations may confer cross-resistance to multiple Cry toxins that share similar activation pathways [20,47,48].

The Pickens population exhibited reduced ALP activity and elevated APN activity in 3rd and 4th instars, along with significantly up- or downregulation of three APN family genes in the 3rd instar relative to at least one laboratory colony. These enzymatic activities and transcriptional changes may reduce the efficacy of Cry toxins under field conditions. Consistent with these findings, Cry1Ac-selected H. zea larvae have previously shown 2- and 3-fold reduction in APN and ALP activities, respectively [49]. Similarly, decreased ALP activity has been reported in Cry-resistant strains of H. virescens [50], even when APN activity remained unchanged. Cry1Ac-resistant P. xylostella displayed coordinated downregulation of APN and ALP [51,52,53,54]. In Cry1Ac-resistant Trichoplusia ni, altered expression of TnAPN1 and TnAPN6 was reported, with only APN1 downregulation genetically linked to resistance [55]. Downregulation of APN and ALP has also been documented in other Bt-resistant insect species, including S. exigua [56], Diatrea saccharalis [57] and Ostrinia nubilalis [58]. Collectively, these studies underscore alterations in ALP and APN expression and activity in mediating Cry toxin resistance. While transcriptional variations in the expression of ABCC2 and cadherin have been reported in some Cry toxin-resistant lepidopteran species [19,20], no significant differences in transcript levels of these genes were detected between lab and field-derived H. zea in this study. This is consistent with previous evidence indicating that ABCC2 is not a major functional receptor for Cry1Ac in H. zea [59].

Transcript levels of four midgut detoxification enzyme genes (esterase, GST, CYP6B8, and CYP6B28) did not differ significantly among the three H. zea colonies examined in this study. Likewise, whole-body enzyme assays showed no significant differences in esterases, GST, and P450 activities among colonies, even though the Pickens population has also exhibited reduced susceptibility to λ-cyhalothrin [60]. Although differential expressions of CYP genes have been reported in Cry1Ac-resistant H. zea [61], the absence of variation in the selected detoxification genes in the present study suggests that these enzymes are unlikely to be a major contributor to the reduced Bt susceptibility in the Pickens population.

No significant differences in trypsin and chymotrypsin activity or APN activity were detected between the two laboratory-susceptible colonies within the same larval instar. Similarly, transcript levels of four Cry receptor genes and trypsin did not differ significantly between the Benzon and SIMRU colonies. However, expression of three chymotrypsin genes was significantly altered in Benzon compared to SIMRU, with one gene upregulated and two genes downregulated. We assessed the activities of trypsin and chymotrypsin proteases, APN, and ALP in midgut extracts from 3rd, 4th, and 5th instar larvae across all three populations. In the Pickens population, a significant reduction in chymotrypsin activity was detected in 5th instar larvae, whereas APN activity was elevated in 4th instar larvae. Despite these instar-specific differences, no consistent differences in overall APN and ALP activity were detected across larval stages. Transcript levels of seven known Cry toxin receptor genes (cadherin, ABCC2, ALP, and four APN genes) were evaluated in the Pickens field population, and future work will involve cloning these genes for sequence and protein analysis. Functional characterization using Xenopus laevis oocytes is also planned to evaluate pore formation and morphological changes in response to Cry toxin exposure.

Overall, our findings underscore the need for comprehensive biochemical and molecular investigations to better understand the reduced Bt susceptibility in the field population. Ongoing monitoring of Bt susceptibility in the field population is essential for sustaining the effectiveness of Bt crops and ensuring long-term pest management. Expanding surveillance to include multiple geographically diverse populations and integrating biochemical data with phenotypic resistance assessments will be critical for advancing and optimizing future insect resistance management (IRM) strategies.