The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera.

The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.

piggyBac-based transgenic Helicoverpa armigera expressing the T92C allele of the tetraspanin gene HaTSPAN1 confers dominant resistance to Bacillus thuringiensis toxin Cry1Ac.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized pest control. However, the evolution of resistance by target pests poses a significant threat to the long-term success of Bt crops. Understanding the genetics and mechanisms underlying Bt resistance is crucial for developing resistance detection methods and management tactics. The T92C mutation in a tetraspanin gene (HaTSPAN1), resulting in the L31S substitution, is associated with dominant resistance to Cry1Ac in a major pest, Helicoverpa armigera. Previous studies using CRISPR/Cas9 technique have demonstrated that knockin of the HaTSPAN1 T92C mutation confers a 125-fold resistance to Cry1Ac in the susceptible SCD strain of H. armigera. In this study, we employed the piggyBac transposon system to create two transgenic H. armigera strains based on SCD: one expressing the wild-type HaTSPAN1 gene (SCD-TSPANwt) and another expressing the T92C mutant form of HaTSPAN1 (SCD-TSPANmt). The SCD-TSPANmt strain exhibited an 82-fold resistance to Cry1Ac compared to the recipient SCD strain, while the SCD-TSPANwt strain remained susceptible. The Cry1Ac resistance followed an autosomal dominant inheritance mode and was genetically linked with the transgene locus in the SCD-TSPANmt strain of H. armigera. Our results further confirm the causal association between the T92C mutation of HaTSPAN1 and dominant resistance to Cry1Ac in H. armigera. Additionally, they suggest that the piggyBac-mediated transformation system we used in H. armigera is promising for functional investigations of candidate Bt resistance genes from other lepidopteran pests.

Synergism of Cry1 Toxins by a Fusion Protein Derived from a Cadherin Fragment and an Antibody Peptide.

Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 VL) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli. The HaCad-TBR-2D3 VL enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 VL over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.

Exploratory comparative transcriptomic analysis reveals potential gene targets associated with Cry1A.105 and Cry2Ab2 resistance in fall armyworm (Spodoptera frugiperda).

Genetically modified (GM) crops, expressing Bacillus thuringiensis (Bt) insecticidal toxins, have substantially transformed agriculture. Despite rapid adoption, their environmental and economic benefits face scrutiny due to unsustainable agricultural practices and the emergence of resistant pests like Spodoptera frugiperda, known as the fall armyworm (FAW). FAW's adaptation to Bt technology in corn and cotton compromises the long-term efficacy of Bt crops. To advance the understanding of the genetic foundations of resistance mechanisms, we conducted an exploratory comparative transcriptomic analysis of two divergent FAW populations. One population exhibited practical resistance to the Bt insecticidal proteins Cry1A.105 and Cry2Ab2, expressed in the genetically engineered MON-89Ø34 - 3 maize, while the other population remained susceptible to these proteins. Differential expression analysis supported that Cry1A.105 and Cry2Ab2 significantly affect the FAW physiology. A total of 247 and 254 differentially expressed genes were identified in the Cry-resistant and susceptible populations, respectively. By integrating our findings with established literature and databases, we underscored 53 gene targets potentially involved in FAW's resistance to Cry1A.105 and Cry2Ab2. In particular, we considered and discussed the potential roles of the differentially expressed genes encoding ABC transporters, G protein-coupled receptors, the P450 enzymatic system, and other Bt-related detoxification genes. Based on these findings, we emphasize the importance of exploratory transcriptomic analyses to uncover potential gene targets involved with Bt insecticidal proteins resistance, and to support the advantages of GM crops in the face of emerging challenges.

A transient brain endothelial translatome response to endotoxin is associated with mild cognitive changes post-shock in young mice.

Sepsis-associated encephalopathy (SAE) is associated with increased risk of long-term cognitive impairment. SAE is driven, at least in part, by brain endothelial dysfunction in response to systemic cytokine signaling. However, the mechanisms driving SAE and its consequences remain largely unknown. Here, we performed translating ribosome affinity purification and RNA-sequencing (TRAP-seq) from the brain endothelium to determine the transcriptional changes after an acute endotoxemic (LPS) challenge. LPS induced a strong acute transcriptional response in the brain endothelium that partially correlates with the whole brain transcriptional response and suggested an endothelial-specific hypoxia response. Consistent with a crucial role for IL-6, loss of the main regulator of this pathway, SOCS3, leads to a broadening of the population of genes responsive to LPS, suggesting that an overactivation of the IL-6/JAK/STAT3 pathway leads to an increased transcriptional response that could explain our prior findings of severe brain injury in these mice. To identify any potential sequelae of this acute response, we performed brain TRAP-seq following a battery of behavioral tests in mice after apparent recovery. We found that the transcriptional response returns to baseline within days post-challenge, but reductions in gene expression regulating protein translation and respiratory electron transport remained. We observed that mice that recovered from the endotoxemic shock showed mild, sex-dependent cognitive impairment, suggesting that the acute brain injury led to sustained effects. A better understanding of the transcriptional and non-transcriptional changes in response to shock is needed in order to prevent and/or revert the devastating consequences of septic shock.

Silence of Aminopeptidase N 2 gene reveals the trade-offs for acquiring Cry1Ac resistance in Plutella xylostella.

Bacillus thuringiensis (Bt) is widely used as a biopesticide worldwide. To date, at least eight pest species have been found to be resistant to Bt in the field. As the first pest that was reported having resistance to Bt in the field, considerable research has been done on the mechanisms of Bt resistance in Plutella xylostella. However, whether the acquisition of Bt resistance by P. xylostella comes at a fitness cost is also a valuable question. In this study, Aminopeptidase-N 2 (APN2), a Cry toxin receptor gene of P. xylostella, was knocked down by RNA interference, resulting in improved resistance to Cry1Ac. It was also found that larval mortality of APN2 knockdown P. xylostella was significantly higher than that of the control, while the pupation rate, pupal weight, eclosion rate, fecundity (egg/female), hatchability, and female adult longevity were significantly lower in APN2 knockdown P. xylostella than in the control. These results illustrate that if Cry1Ac resistance was obtained only through the reduction of APN2 expression, P. xylostella would need to incur some fitness costs for it.

The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.

Fall armyworm (Lepidoptera: Noctuidae): practical resistance of 2 Brazilian populations to Cry1A.105†+†Cry2Ab and Cry1F Bt maize.

In the Americas, transgenic crops producing insecticidal proteins from Bacillus thuringiensis Berliner (Bt, Bacillales: Bacillaceae) have been used widely to manage fall armyworm (FAW, Spodoptera frugiperda [J.E. Smith]). As resistance to Cry1 single-gene Bt maize (Zea mays L.) rapidly evolved in some FAW populations, pyramided Bt maize hybrids producing Cry1, Cry2, or Vip3Aa proteins were introduced in the 2010s. We examined field-evolved resistance to single- and dual-protein Bt maize hybrids in 2 locations in southeastern Brazil, where plant damage by FAW larvae far exceeded the economic threshold in 2017. We collected late-instar larvae in Cry1A.105 + Cry2Ab and Cry1F maize fields and established 2 FAW populations in the laboratory. The F1 offspring reared on the foliage of Bt and non-Bt maize plants (Cry1A.105 + Cry2Ab and Cry1F) showed neonate-to-adult survival rates as high as 70% for both populations. There was no significant difference in the life-table parameters of armyworms reared on non-Bt and Bt maize foliage, indicating complete resistance to Cry1A.105 + Cry2Ab maize. Larval survival rates of reciprocal crosses of a susceptible laboratory strain and the field-collected populations indicated nonrecessive resistance to Cry1F and a recessive resistance to Cry1A.105 + Cry2Ab maize. When relaxing the selection pressure, the armyworm fitness varied on Cry1A.105 + Cry2Ab and non-Bt maize; the resistance was somewhat stable across 12 generations, without strong fitness costs, although one of the lines died confounded by a depleted-quality, artificial rearing diet. To our knowledge, this is the first report documenting the practical resistance of FAW to a pyramided Bt crop. We discuss the implications for resistance management.

The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response.

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation.

Characterization of the mode of action of eCry1Gb.1Ig, a fall armyworm (Spodoptera frugiperda) active protein, with a novel site of action.

Insect pests cause immense agronomic losses worldwide. One of the most destructive of major crops is the Fall Armyworm (Spodoptera frugiperda, FAW). The ability to migrate long distances, a prodigious appetite, and a demonstrated ability to develop resistance to insecticides, make it a difficult target to control. Insecticidal proteins, for example those produced by the bacterium Bacillus thuringiensis, are among the safest and most effective insect control agents. Genetically modified (GM) crops expressing such proteins are a key part of a successful integrated pest management (IPM) program for FAW. However, due to the development of populations resistant to commercialized GM products, new GM traits are desperately needed. Herein, we describe a further characterization of the newly engineered trait protein eCry1Gb.1Ig. Similar to other well characterized Cry proteins, eCry1Gb.1Ig is shown to bind FAW midgut cells and induce cell-death. Binding competition assays using trait proteins from other FAW-active events show a lack of competition when binding FAW brush border membrane vesicles (BBMVs) and when utilizing non-pore-forming versions as competitors in in vivo bioassays. Similarly, insect cell lines expressing SfABCC2 and SfABCC3 (well characterized receptors of existing commercial Cry proteins) are insensitive to eCry1Gb.1Ig. These findings are consistent with results from our previous work showing that eCry1Gb.1Ig is effective in controlling insects with resistance to existing traits. This underscores the value of eCry1Gb.1Ig as a new GM trait protein with a unique site-of-action and its potential positive impact to global food production.

Larval density-dependent mortality of western corn rootworm (Coleoptera: Chrysomelidae) in Bt and non-Bt maize and implications on dose calculations†.

BACKGROUND: Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) have been used to manage insect pests for nearly 30 years. Dose of a Bt crop is key to assessing the risk of resistance evolution because it affects the heritability of resistance traits. Western corn rootworm (Diabrotica virgifera virgifera, LeConte), a major pest of maize, has evolved resistance to all commercially available Bt traits targeting it, and threatens resistance to future transgenic traits. Past research shows the dose of Bt maize targeting western corn rootworm can be confounded by larval density-dependent mortality. We conducted a 2-year field study at two locations to quantify larval density-dependent mortality in Bt and non-Bt maize. We used these results to calculate dose for our method and compared it to three previously published methods. Additionally, adult emergence and root injury were analyzed for predicting initial egg density. RESULTS: Increased pest density caused greater proportions of larvae to die in Bt maize than in non-Bt maize. All methods for calculating dose produced values less than high-dose, and stochastic variation had the greatest impact on dose at high and low pest densities. Our method for calculating dose did not produce values positively correlated with pest density while the three other methods did. CONCLUSION: To achieve the most accurate calculation of dose for transgenic maize targeting western corn rootworm, density-dependent mortality should be taken into account for both transgenic and non-transgenic maize and assessed at moderate pest densities. © 2024 Society of Chemical Industry.

Urea Cycle of Bacillus thuringiensis Affects Its Survival under UV Stress.

Bacillus thuringiensis (Bt) is widely used to produce biological pesticides. However, its persistence is limited because of ultraviolet (UV) rays. In our previous study, we found that exogenous intermediates of the urea cycle were beneficial to Bt for survival under UV stress. To further explore the effect of the urea cycle on the resistance mechanism of Bt, the rocF/argG gene, encoding arginase and argininosuccinate synthase, respectively, were knocked out and recovered in this study. After the target genes were removed, respectively, the urea cycle in the tested Bt was inhibited to varying degrees. The UV stress test showed that the urea cycle disorder could reduce the resistance of Bt under UV stress. Meanwhile, the antioxidant enzyme activities of Bt were also decreased to varying degrees due to the knockout of the target genes. All of these results revealed that the urea cycle can metabolically regulate the stress resistance of Bt.

Nitric oxide and tumor necrosis factor-⍺ levels are negatively correlated in endotoxin tolerance recovery in vitro.

Endotoxin tolerance (ET) is the hyporesponsiveness to lipopolysaccharide (LPS) after prior exposure. It is characterized by the downregulation of pro-inflammatory cytokine levels. Although ET protects against inflammation, its abolishment or recovery is critical for immunity. Nitric oxide (NO) plays various roles in the development of ET; however, its specific role in ET recovery remains unknown. To induce ET, RAW264.7 cells (a murine macrophage cell line) were pre-exposed to LPS (LPS1, 100 ng/mL for 24 h) and subsequently re-stimulated with LPS (LPS2, 100 ng/mL for 24 h). Expression of cytokines, NO, nitrite and inducible NO synthase (iNOS) were measured after 0, 12, 24, and 36 h of resting after LPS1 treatment with or without the iNOS-specific inhibitor, 1400W. LPS2-induced tumor necrosis factor-⍺ (TNF-⍺) and interleukin-6 (IL-6) were downregulated after LPS1 treatment, confirming the development of ET. Notably, TNF-⍺ and IL-6 levels spontaneously rebounded after 12-24 h of resting following LPS1 treatment. In contrast, levles of NO, nitrite and iNOS increased during ET development and decreased during ET recovery. Moreover, 1400W inhibited ET development and blocked the early production of NO (<12 h) during ET recovery. Our findings suggest a negative correlation between iNOS-induced NO and cytokine levels in the abolishment of ET.

Clinical use of progesterone in human sperm preparation media for increasing IVF success.

RESEARCH QUESTION: Can the addition of progesterone and neurotensin, molecular agents found in the female reproductive tract, after sperm washing increase the fertilization potential of human spermatozoa? DESIGN: (i) Cohort study of 24 men. Spermatozoa selected by swim-up were incubated in either progesterone or neurotensin (0.1-100 µM) for 1-4 h, and hyperactive motility and binding to hyaluronan (0.1-100 µM) were assessed. The effect of progesterone 10 µM on sperm function was assessed in a blinded manner, including: hyperactive motility, binding to hyaluronan, tyrosine phosphorylation, acrosome reaction and oxidative DNA damage. (i) Embryo safety testing [one-cell mouse embryo assay (MEA), endotoxin and sterility counts (n = 3)] in preclinical embryo models of IVF (murine and porcine, n = 7 each model) and a small preliminary human study (n = 4) of couples undergoing standard IVF with oocytes inseminated with spermatozoa ± 10 µM progesterone. RESULTS: Progesterone 10 µM increased sperm binding to hyaluronan, hyperactive motility and tyrosine phosphorylation (all P < 0.05). Neurotensin had no effect (P > 0.05). Progesterone 10 µM in human embryo culture media passed embryo safety testing (MEA, endotoxin concentration and sterility plate count). In preclinical models of IVF, the exposure of spermatozoa to progesterone 10 µM and oocytes to progesterone 1 µM was not detrimental, and increased the fertilization rate in mice and the blastocyst cell number in mice and pigs (all P ≤ 0.03). In humans, every transferred blastocyst that had been produced from spermatozoa exposed to progesterone resulted in a live birth. CONCLUSION: The addition of progesterone to sperm preparation media shows promise as an adjunct to current methods for increasing fertilization potential. Randomized controlled trials are required to determine the clinical utility of progesterone for improving IVF outcomes.

Radiation-Detoxified Form of Endotoxin Effectively Activates Th1 Responses and Attenuates Ragweed-Induced Th2-Type Airway Inflammation in Mice.

Urbanization with reduced microbial exposure is associated with an increased burden of asthma and atopic symptoms. Conversely, environmental exposure to endotoxins in childhood can protect against the development of allergies. Our study aimed to investigate whether the renaturation of the indoor environment with aerosolized radiation-detoxified lipopolysaccharide (RD-LPS) has a preventative effect against the development of ragweed-induced Th2-type airway inflammation. To explore this, cages of six-week-old BALB/c mice were treated daily with aerosolized native LPS (N-LPS) or RD-LPS. After a 10-week treatment period, mice were sensitized and challenged with ragweed pollen extract, and inflammatory cell infiltration into the airways was observed. As dendritic cells (DCs) play a crucial role in the polarization of T-cell responses, in our in vitro experiments, the effects of N-LPS and RD-LPS were compared on human monocyte-derived DCs (moDCs). Mice in RD-LPS-rich milieu developed significantly less allergic airway inflammation than mice in N-LPS-rich or common environments. The results of our in vitro experiments demonstrate that RD-LPS-exposed moDCs have a higher Th1-polarizing capacity than moDCs exposed to N-LPS. Consequently, we suppose that the aerosolized, non-toxic RD-LPS applied in early life for the renaturation of urban indoors may be suitable for the prevention of Th2-mediated allergies in childhood.

A novel insecticide, isocycloseram, shows promise as an alternative to chlorpyrifos against a direct pest of peanut, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae).

Larvae of the southern corn rootworm (SCR) Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae) are primary pests of peanut in the Virginia-Carolina region of the United States, and are relatively sporadic pests in southern states such as Georgia, Alabama, and Florida. Peanuts have strict quality standards which, when they are not met, can diminish crop value by more than 65%. Management of direct pests like SCR is therefore crucial to maintaining the economic viability of the crop. The soil-dwelling nature of SCR larvae complicates management due to difficulties associated with monitoring and predicting infestations. Nonchemical management options are limited in this system; preventative insecticide applications are the most reliable management strategy for at-risk fields. Chlorpyrifos was the standard product for larval SCR management in peanut until its registration was revoked in 2022, leaving no effective chemical management option for larvae. We tested a novel insecticide, isocycloseram, for its ability to reduce pod scarring, pod penetration, and non-SCR pod damage in field studies conducted in Suffolk, Virginia in 2020-2022. Overall injury was low in 2020 and 2022, and in 2022 there was not a significant effect of treatment. In 2021, 2 simulated chemigation applications of isocycloseram in July significantly reduced pod scarring and overall pod injury relative to chlorpyrifos and the untreated control. Our results suggest that isocycloseram may become an effective option for managing SCR in peanut, although more work is needed to understand the mechanisms by which it is effective as a soil-applied insecticide.

Mutation in PgABCC2 confers low-level resistance to Cry1Ac in pink bollworm.

BACKGROUND: With the increasing incidence of pest resistance to transgenic crops producing Bacillus thuringiensis (Bt) proteins in the field, elucidating the molecular basis of resistance is important for monitoring, delaying and countering pest resistance. Previous work revealed that mutation or down-regulated expression of the cadherin gene (PgCad1) is associated with pink bollworm (Pectinophora gossypiella) resistance to Cry1Ac, and 20 mutant PgCad1 alleles (r1-r20) were characterized. Here, we tested the hypothesis that the ABC transporter PgABCC2 is a functional receptor for the Bt toxin Cry1Ac and that a mutation is associated with resistance. RESULTS: We identified and characterized the first resistance allele (rC2) of PgABCC2 in the laboratory-selected Cry1Ac-resistant strain AQ-C2 of pink bollworm. The rC2 allele had a one-base deletion in exon20, resulting in a frameshift and the introduction of a premature stop codon. This resulting PgABCC2 protein had a truncated C-terminus, including the loss of the NBD2 domain. AQ-C2 exhibited 20.2-fold greater resistance to Cry1Ac than the susceptible strain, and its inheritance of Cry1Ac resistance was recessive and genetically linked to PgABCC2. When produced in cultured insect cells, recombinant wild-type and rC2 mutant PgABCC2 proteins localized within the cell plasma membrane, although substantial cytoplasmic retention was also observed for the mutant protein, while the mutant PgABCC2 caused a 13.9-fold decrease in Cry1Ac toxicity versus the wild-type PgABCC2. CONCLUSIONS: PgABCC2 is a functional receptor of Cry1Ac and the loss of its carboxyl terminus (including its NBD2 domain) confers low-level resistance to Cry1Ac in both larvae and in cultured cells. © 2024 Society of Chemical Industry.

MiR-2b-3p Downregulated PxTrypsin-9 Expression in the Larval Midgut to Decrease Cry1Ac Susceptibility of the Diamondback Moth, Plutella xylostella (L.).

Crystal (Cry) toxins, produced by Bacillus thuringiensis, are widely used as effective biological pesticides in agricultural production. However, insects always quickly evolve adaptations against Cry toxins within a few generations. In this study, we focused on the Cry1Ac protoxin activated by protease. Our results identified PxTrypsin-9 as a trypsin gene that plays a key role in Cry1Ac virulence in Plutella xylostella larvae. In addition, P. xylostella miR-2b-3p, a member of the micoRNA-2 (miR-2) family, was significantly upregulated by Cry1Ac protoxin and targeted to PxTrypsin-9 downregulated its expression. The mRNA level of PxTrypsin-9, regulated by miR-2b-3p, revealed an increased tolerance of P. xylostella larvae to Cry1Ac at the post-transcriptional level. Considering that miR-2b and trypsin genes are widely distributed in various pest species, our study provides the basis for further investigation of the roles of miRNAs in the regulation of the resistance to Cry1Ac and other insecticides.

Peritrophins are involved in the defense against Bacillus thuringiensis and nucleopolyhedrovirus formulations in Spodoptera littoralis (Lepidoptera: Noctuidae).

The peritrophic matrix (or peritrophic membrane, PM) is present in most insects where it acts as a barrier to mechanical insults and pathogens, as well as a facilitator of digestive processes. The PM is formed by the binding of structural PM proteins, referred to as peritrophins, to chitin fibrils and spans the entire midgut in lepidopterans. To investigate the role of peritrophins in a highly polyphagous lepidopteran pest, namely the cotton leafworm (Spodoptera littoralis), we generated Insect Intestinal Mucin (IIM-) and non-mucin Peritrophin (PER-) mutant strains via CRISPR/Cas9 mutagenesis. Both strains exhibited deformed PMs and retarded developmental rates. Bioassays conducted with Bacillus thuringiensis (Bt) and nucleopolyhedrovirus (SpliNPV) formulations showed that both the IIM- and PER- mutant larvae were more susceptible to these bioinsecticides compared to the wild-type (WT) larvae with intact PM. Interestingly, the provision of chitin-binding agent Calcofluor (CF) in the diet lowered the toxicity of Bt formulations in both WT and IIM- larvae and the protective effect of CF was significantly lower in PER- larvae. This suggested that the interaction of CF with PER is responsible for Bt resistance mediated by CF. In contrast, the provision of CF caused increased susceptibility to SpliNPV in both mutants and WT larvae. The study showed the importance of peritrophins in the defense against pathogens in S. littoralis and revealed novel insights into CF-mediated resistance to Cry toxin.

RNA m6 A Methylation Suppresses Insect Juvenile Hormone Degradation to Minimize Fitness Costs in Response to A Pathogenic Attack.

Bioinsecticides and transgenic crops based on the bacterial pathogen Bacillus thuringiensis (Bt) can effectively control diverse agricultural insect pests, nevertheless, the evolution of resistance without obvious fitness costs has seriously eroded the sustainable use of these Bt products. Recently, it has been discovered that an increased titer of juvenile hormone (JH) favors an insect host (Plutella xylostella) to enhance fitness whilst resisting the Bt pathogen, however, the underlying regulatory mechanisms of the increased JH titer are obscure. Here, the involvement of N6 -methyladenosine (m6 A) RNA modification in modulating the availability of JH in this process is defined. Specifically, it is found that two m6 A methyltransferase subunit genes, PxMettl3 and PxMettl14, repress the expression of a key JH-degrading enzyme JH esterase (JHE) to induce an increased JH titer, mitigating the fitness costs associated with a robust defense against the Bt pathogen. This study identifies an as-yet uncharacterized m6 A-mediated epigenetic regulator of insect hormones for maintaining fitness during pathogen defense and unveils an emerging Bt resistance-related m6 A methylation atlas in insects, which further expands the functional landscape of m6 A modification and showcases the pivotal role of epigenetic regulation in host-pathogen interactions.