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Multifunctional lysozymes from the assassin bug Sycanus bifidus: Insecticidal proteins with anticoagulant and melanization inhibition properties.

BACKGROUND: Predatory hemipterans deploy complex venom cocktails to immobilize preys, yet the specific roles of many individual venom components remain poorly understood. RESULTS: Four lysozyme genes were identified from the genome of the predatory assassin bug Sycanus bifidus (Hemiptera: Reduviidae), comprising one i-type (SbLyzi) and three c-type lysozyme genes (SbLyzc1-3). Transcriptomic and quantitative (q)PCR analyses revealed that these lysozymes were expressed at different levels in various venom glands. Of them, SbLyzc1-3 with signal peptides displayed significant transcriptions in the venom glands, implicating these lysozymes as venom constituents. Functional assays found that SbLyzc1 and SbLyzc2 showed antibacterial activity against Pseudomonas aeruginosa and Enterococcus faecalis. Three lysozymes (SbLyzi, SbLyzc1 and SbLyzc3) suppressed thrombin-induced fibrin clot formation, indicating anticoagulant activity, with SbLyzc1 exhibiting the greatest potency [half-maximal inhibitory concentration (IC50) = 0.036 ± 0.003 μg μL-1]. SbLyzi and SbLyzc1 inhibited phenoloxidase activity in the hemolymph of the yellow mealworm Tenebrio molitor pupae, thereby suppressing its hemolymph melanization, with maximal inhibition rates of 78.4% and 74.3%, respectively. All four lysozymes exhibited insecticidal effects, causing >80% mortality in yellow mealworm pupae following injection of 6 μg per individual, with SbLyzc1 showing the highest insecticidal potency [half-maximal lethal dose (LD50) = 4.25 ± 0.51 μg g-1]. CONCLUSION: These findings demonstrate that lysozymes from S. bifidus possess multifunctional biological activities and SbLyzc1-3 act as significant venom components involved in capturing prey, providing new insights into the functional diversity of lysozymes in predatory bugs and their potential application in biological control strategies. © 2026 Society of Chemical Industry.

Animals

Paradigm shift for cry gene expression in Bacillus thuringiensis.

In most Bacillus thuringiensis strains, the cry genes are transcribed by RNA polymerases containing sporulation-sigma factors E or K, leading to the formation of an insecticidal crystal within the mother cell along spore development. The kurstaki HD1 strain, a parent of commercial strains, also releases the insecticidal proteins Cry1I and Vip3A in the extracellular medium. vip3A expression is activated by the transcriptional regulator VipR at the onset of the stationary phase. Here, we expanded the VipR regulon in strain HD1 by identifying the VipR-binding box upstream from the cry2Aa, cry2Ab, and cry1Ia genes, and conducting transcription assays. Unexpectedly, a VipR box was located in the promoter of a putative N-acetylmuramoyl-l-alanine amidase (ami) gene upstream from cry1Ac in strain kurstaki HD73, closely related to the HD1 but devoid of vipR. Introduction of vipR in this strain led to the expression of the ami-cry1Ac operon, resulting in an early and increased production of Cry1Ac. We demonstrated that Cry1Ac was also produced in a VipR-dependent manner in an HD73 ∆spo0A mutant. Similarly, an HD1 ∆spo0A strain produces all the insecticidal proteins encoded in its genome, including cry2Ab, previously considered unexpressed. A genomic analysis also revealed the presence of putative VipR-binding sequences in lepidopteran-active strains, upstream from cry genes such as cry1E, cry1F, cry9D, and cry9E. Overall, our results break the dogma on the regulation of cry1A and cry2A genes and provide evidence of sporulation-independent Cry toxin production in biopesticidal Bt strains.IMPORTANCEBacillus thuringiensis is a remarkably efficient entomopathogen due to its ability to produce various insecticidal proteins, such as Cry or Vip. This property has made it a highly effective biopesticide used worldwide. Our work modifies the paradigm of cry1 and cry2 genes being regulated solely by sporulation-specific sigma factors and thus exclusively expressed during this process. Indeed, we demonstrated that the VipR regulator controls the transcription of vip3Aa, cry2Aa, cry2Ab, cry1Ia, and the ami-cry1A operons encoded by a strain closely related to that of commercial biopesticides and specifically turns on their expression from the onset of the stationary phase, leading to the production of insecticidal crystals independently of sporulation. By providing new knowledge on the regulation of insecticidal protein genes, these findings bring new insight for the genetic improvement of Bt strains used as commercial biopesticides.

Bacillus thuringiensis

[Metabolism and in vitro binding of several organochlorine and organophosphate pesticides to calf thymus DNA and rat liver microsomal proteins].

The organophosphorus insecticide ethyl-parathion was transformed by rat liver microsomes into metabolites which were bound to calf thymus DNA, in large amount. When the rats were treated by phenobarbital or 3-methylcholanthrene, the metabolite binding to DNA was increased two-fold. By contrast, in the same conditions, the organochlorine insecticides, aldrin, dieldrin and gamma hexachlorocyclohexane (lindane), did not yield metabolites able to bind to DNA and to proteins.

Aldrin

[Effects of the organophosphorus insecticide Ofunack on the metabolism of cells cultured in vitro].

The effects of "ofunack (0,0-diethyl-0-(3-oxo-2-phenyl-2H-pyridazine-6-y1) phosphorothionate on the metabolism of IB-RS-2 cell line were studied. "Ofunack" stimulates cell growth and protein synthesis till 24 hours after its addition to the maintenance medium. Otherwise after this time "ofunack" induces an inhibition both on cell growth and on protein synthesis. This insecticide also inhibits the synthesis of RNA and DNA.

Animals

The transgenic Vip3A poplar plant confers high resistance against Hyphantria cunea Drury.

Poplar is severely damaged by Hyphantria cunea (fall webworm), which significantly reduces tree productivity. However, conventional pest management methods are largely ineffective against fall webworm infestation. In this study, we demonstrated that the Vip3A protein possesses high insecticidal activity against H. cunea by overexpressing a synthetic THI1-Vip3A gene in poplar plants. A dicot codon-optimized Vip3A gene, fused with the THI1 chloroplast signal peptide sequence, was chemically synthesized and introduced into the poplar cv. '741' genome via Agrobacterium-mediated transformation. PCR, RT-PCR, and ELISA analyses confirmed the integration and successful expression of the transgene at both the mRNA and protein levels. The Vip3A protein concentration in chloroplasts was approximately 4.8-fold higher than in the whole leaf extract, indicating that the Vip3A protein was successfully targeted to and accumulated within the chloroplasts by the THI1 signal peptide. Subsequently, four transgenic lines with high Vip3A expression were subjected to H. cunea infestation. Compared to wild-type plants, these four transgenic lines exhibited significantly higher resistance, resulting in pest mortality rates exceeding 95% and significantly reduced leaf damage. Together, these results indicate that Vip3A possesses high insecticidal activity against H. cunea. Therefore, transgenic THI1-Vip3A poplar plants can serve as valuable germplasm for breeding poplar cultivars with high resistance to H. cunea infestation.

Plants, Genetically Modified

Bacillus thuringiensis pathogenicity islands encode regulatory circuits controlling insecticidal Cry toxin expression during vegetative growth.

Bacillus thuringiensis (Bt) produces insecticidal toxins, including Cry and Vip3 proteins, that are widely used for biological pest control. Cry proteins are classically expressed during sporulation under the control of sporulation-specific σ factors, whereas Vip3 is produced during vegetative growth, suggesting distinct regulatory pathways. Notably, many cry and vip3A genes are clustered within pathogenicity islands (PAIs), such as BtPAI-1. However, whether these PAIs also encode regulatory mechanisms coordinating toxin expression remains unclear. Here, we identify VipR, a BtPAI-1-encoded transcriptional regulator, as an activator of insecticidal gene expression during the vegetative phase in Bt strains HD-1 and CT-43. In these strains, VipR promotes the transcription of BtPAI-1 associated insecticidal genes, including vip3A and selected cry genes, resulting in premature Cry protein accumulation and increased insecticidal activity. In addition, VipR contributes to the vegetative-phase expression of the non-BtPAI-1 cry9Aa genes in strain BGSC 4AE1. Phylogenetic analysis revealed that vipR is widely distributed in one-third of Bt strains, and is strongly associated with PAIs. Futhermore, heterologous expression of vipR in BGSC 4J5 and HD-73 was sufficient to activate vegetative-phase transcription of some cry independently of sporulation-specific σ factor cascade. These results support a role for VipR in coordinating vegetative-phase expression of insecticidal genes in the Bt strains examined and suggest that BtPAI-1 can encode both insecticidal determinants and regulatory functions that influence their expression. These findings provide new insights into the regulatory architecture of Bt pathogenicity islands and may facilitate the engineering of strains with enhanced insecticidal activity.

Bacillus thuringiensis

Degradation by Eco R1 endonuclease of DNA isolated from phages infecting Bacillus licheniformis and Bacillus thuringiensis.

DNA was isolated from lytic phages of two strains, Bacillus licheniformis, a producer of bacitracin, and Bacillus thuringiensis forming protein paracrystals with pronounced insecticidal effects. Its sensitivity to Eco R1 restriction endonuclease was determined. It was the aim of the work to find out whether these phages could serve as vectors in the transfer and possible amplification of genes of the two important industrial species of bacilli. Approximate values of the molecular weight of DNA of the two phages were determined after degradation of the phage DNA by Eco R1, followed by comparison of electrophoretic mobility of individual fragments with that of the Eco R1-degraded DNA of phage lambdab2.

Bacillus

Radiobiochemistry of phytodrugs: I. role of juvenile hormones and analogs in the biosynthesis of proteins and RNA in drosophila larvae.

The present communication concerns the investigations of the effect of Juvenile Hormone (J.H.) and its analogs (J.H.A.), such as Altosid (R) and Altozar(R), used as insecticides, on the biosynthesis of proteins and RNA in subcellular preparations of drosophila larvae with Leucine-U-14C and Uridine-2-14C respectively. The incorporation of leucine into proteins decreases progressively from crude homogenate to the microsome-free fraction in the same way as the degradation of the compounds. During RNA biosynthesis the incorporation of uridine increases from the crude homogenate up to mithocondria-free fraction and with Altosid is always below the test controls. The reduced incorporation of uridine into RNA and leucine into proteins in the presence of Altosid and Altozar indicates a genetic action of this compounds comparable to that of natural J.H. but slightly more efficient.

Animals

Genome-wide identification and expression profiling of CSP and OBP genes in Stictocephala bisonia reveals candidate genes potentially associated with insecticide response.

Stictocephala bisonia is an important invasive agricultural pest. Due to the frequent application of insecticides in its habitat, this species is under intense selection pressure. Chemosensory proteins (CSPs) and odorant-binding proteins (OBPs) are known to play key roles in insecticide resistance, but their specific functions in S. bisonia remain unclear. In this study, we identified a total of 22 SbisCSPs and 16 SbisOBPs based on the S. bisonia genome. To screen for candidate genes potentially linked to insecticide resistance, we adopted a multi-criteria screening strategy that integrated phylogenetic analysis, molecular docking with three insecticides, and tissue-specific expression profiling. Phylogenetic analysis identified several SbisCSPs and SbisOBPs clustering with genes known to be involved in insecticide resistance, serving as an initial evolutionary filter. Molecular docking results indicated that λ-Cyhalothrin exhibited the strong predicted binding affinity with most of SbisCSPs and SbisOBPs. Subsequent qPCR validation of seven prioritized candidates revealed distinct expression patterns: SbisCSP22 was highly expressed in adults and demonstrated strong binding affinity to all three insecticides tested, suggesting a potential role in mediating multi-insecticide response. Conversely, SbisCSP17 was significantly upregulated in larvae, clustered with genes known to mediate imidacloprid resistance, and exhibited strong binding affinity to imidacloprid. Given its larval-specific expression and the soil-dwelling behavior of larvae, we hypothesize that SbisCSP17 is a key candidate gene for larvae coping with soil-treated insecticides.

Animals

Absence of beta-exotoxin in Thuricide preparations.

The biological insecticide Thuricide is produced from B. thuringiensis, Berliner, var. kurstaki (serotype 3a, 3b), a bacterial strain which does not synthesize exotoxin. Thus, our product is devoid of any C-mitotic or mutagenic potentiality such as is to be found in exotoxin.

Bacillus thuringiensis

Proteomic responses of the oil palm pest Metisa plana (Psychidae) to farnesyl acetate exposure.

Metisa plana Walker (Lepidoptera: Psychidae) is a major defoliator of oil palm in Malaysia, causing substantial economic losses. Farnesyl acetate (FA), a sesquiterpenoid compound, has been proposed as a potential insecticidal agent against M. plana, yet its molecular impact on larval physiology remains poorly understood. Here, we employed label-free quantitative proteomics, functional enrichment analysis, and targeted transcript assessment to characterize the temporal proteomic response of M. plana larvae at 7 and 14 days after treatment (DAT) with FA. Principal component analysis revealed robust separation between treated and control samples at both time points, indicating sustained treatment-driven proteomic restructuring. Early exposure (7 DAT) elicited a heterogeneous response involving stress-associated proteins, redox enzymes, and cytoskeletal regulators, whereas later exposure (14 DAT) produced a consolidated profile characterized by metabolic reprogramming, downregulation of ribosomal proteins, induction of heat shock proteins, and enrichment of RNA surveillance and mitochondrial pathways. Targeted transcript analysis qualitatively supported proteomic trends for HSP83 and aldehyde dehydrogenase X, although limited amplification precluded quantitative inference. Collectively, these findings demonstrate that FA exposure drives a shift from acute proteomic perturbation toward a maintenance-oriented physiological state, prioritizing proteostasis, energy management, and stress adaptation over growth and development. This integrated molecular perspective provides mechanistic insight into the chronic effects of FA, highlighting its potential to suppress larval performance and informing the development of biorational, physiology-based pest management strategies in non-model insects.

Animals

Disruption of HaVipR1 confers Vip3Aa resistance in the moth crop pest Helicoverpa armigera.

The global reliance on Bacillus thuringiensis (Bt) proteins for controlling lepidopteran pests in cotton, corn, and soybean crops underscores the critical need to understand resistance mechanisms. Vip3Aa, one of the most widely deployed and currently effective Bt proteins in genetically modified crops, plays a pivotal role in pest management. This study investigates the molecular basis of Vip3Aa resistance in Australian Helicoverpa armigera through genetic crosses, and integrated genomic and transcriptomic analyses. We identified a previously uncharacterized gene, LOC110373801 (designated HaVipR1), as potentially important in Vip3Aa resistance in two field-derived resistant lines. Functional validation using CRISPR/Cas9 knockout in susceptible lines confirmed the gene's role in conferring high-level resistance to Vip3Aa. Despite extensive laboratory selection of Vip3Aa-resistant colonies in Lepidoptera, the biochemical mechanisms underlying resistance have remained elusive. Our research identifies HaVipR1 as a potential contributor to resistance, adding to our understanding of how insects may develop resistance to this important Bt protein. The identification of HaVipR1 contributes to our understanding of potential resistance mechanisms and may inform future resistance management strategies. Future work should explore the biochemical pathways influenced by HaVipR1 and assess its interactions with other resistance mechanisms. The approach utilized here underscores the value of field-derived resistant lines for understanding resistance in agricultural pests and highlights the need for targeted approaches to manage resistance sustainably.

Animals