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The rise of RNAi-based biopesticides marks a turning point in pest control, prompting reflection on the future role of Bacillus thuringiensis (Bt). Far from being outdated, Bt remains a cornerstone of sustainable agriculture, with proven efficacy, safety, and public trust. This opinion piece advocates for Bt’s continued relevance in an RNAi-driven future, urging a complementary, not competitive, view of both technologies within integrated pest management frameworks.

The fall armyworm, Spodoptera frugiperda, a lepidopteran pest from the family Noctuidae, has become a major invasive pest since 2016. Using RNAi methods to control S. frugiperda is currently under investigation. This study is the first to target the V-ATPaseD gene of S. frugiperda using RNAi. Injection of dsRNA-V-ATPaseD into the hemolymph of 4th-instar larvae significantly suppressed gene expression at 24 and 48 h post-injection. Treated larvae showed delayed development and reduced pupation after 7 days. Subsequently, V-ATPaseD silencing was achieved through topical or oral administration of chitosan/dsRNA-V-ATPaseD nanoparticles. Larvae fed these nanoparticles exhibited significant reductions in V-ATPaseD mRNA at 72 h, persisting until 96 h before normalizing. Additionally, the treated larvae displayed disrupted molting and impaired pupation. Furthermore, larvae fed chitosan/dsRNA-V-ATPaseD were more susceptible to emamectin benzoate–lufenuron at LC30 concentrations, resulting in 68% mortality—27% higher than the pesticide alone—72 h post-exposure. Combining chitosan/dsRNA-V-ATPaseD nanoparticles with emamectin benzoate–lufenuron significantly enhanced pest control efficacy, providing new insights into pesticide reduction and sustainable pest control methods for this invasive species.

Peritrophins are associated with structural and functional integrity of peritrophic membranes (PM), structures composed of chitin and proteins. PM lines the insect midgut and has roles in digestion and protection from toxins. We report the full-length cDNA cloning, molecular characterization and functional analysis of SfPER, a novel PM peritrophin A protein, in Spodoptera frugiperda . The predicted amino acid sequence indicated SfPER’s domain structure as a CMCMC-type, consisting of a signal peptide and three chitin-binding (C) domains with two intervening mucin-like (M) domains. Phylogenetic analysis determined a close relationship between SfPER and another S. frugiperda PM peritrophin partial sequence. SfPER transcripts were found in larvae and adults but were absent from eggs and pupae. Chitin affinity studies with a recombinant SfPER-C1 peritrophin A-type domain fused to SUMO/His-tag confirmed that SfPER binds to chitin. Western blots of S. frugiperda larval proteins detected different sized variants of SfPER along the PM, with larger variants found towards the posterior PM. In vivo suppression of SfPER expression did not affect susceptibility of larvae to Bacillus thuringiensis toxin, but significantly decreased pupal weight and adult emergence, possibly due to PM structural alterations impairing digestion. Our results suggest SfPER could be a novel target for insect control.

Phoretic mites attach to different body parts of the red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier, 1790), to disperse. However, the question of how the patterns of attachment sites are formed remains intriguing. Here, we examined RPW‐associated phoretic mites in four districts in Northern Portugal (macrohabitat), and investigated the patterns of mite distribution on six body parts of RPW (microhabitat). At the macrohabitat level, we detected seven phoretic mite taxa using the RPW host in each of the four studied districts, all documented for the first time in association with this invasive exotic species in Portugal. However, their relative abundance (species evenness) varied between districts, as did species diversity. All examined weevils carried mites, and the prevalence of the different taxa did not differ between districts or sex of weevils. Measured by mean abundance and degree of aggregation, Centrouropoda sp. proved to be the dominant taxon, while Acarus sp. and Curculanoetus rhynchophorus were considered common subordinate taxa and Uroovobella sp., Mesostigmata, Nenteria extremica and Dendrolaelaps sp. sparse taxa. At the microhabitat level, all taxa were present on all body parts of the RPW; the highest abundance was in a region encompassing the inner surface of the elytra and the membranous hind wings (subelytral space). Analysis of niche overlap revealed that the distribution patterns of phoretic mite taxa on the RPW were not randomly structured. In the subelytral space, interspecific coexistence of mites increased as a function of body size difference with the dominant Centrouropoda sp. We found that in the subelytral space the large dominant species Centrouropoda sp. displaced the larger species Uroobovella sp. and the similarly sized species Nenteria extremica, but coexisted with smaller taxa. We report a high species richness of up to seven phoretic mite taxa, including five unspecified taxa, actively using the red palm weevil as a host in the first study of these organisms in Portugal. We also found body size‐dependent effects on the coexistence or exclusion of phoretic mite taxa that ultimately determine their distribution patterns on the weevil body.

Background Chemical fertilisers are extensively used for crop production, which may cause soil deterioration and water pollution. Endophytic bacteria with plant-growth-promoting (PGP) activities may provide a solution to sustainably improve crop yields, including in-demand staples such as wheat. However, the diversity of the PGP endophytic bacteria in wheat across plant organs and growth stages has not been thoroughly characterised. Results Here, we report the isolation of endophytic bacteria from root, stem, leaf and seed of three winter wheat varieties at tillering, jointing, heading and seed-filling growth stages that were identified via 16S rRNA gene sequence analysis. Strains were screened for indole-3-acetic acid (IAA) production, potassium and phosphate solubilisation and the ability to grow on a nitrogen-free medium. Strain's capacity to stimulate various plant growth parameters, such as dry root weight, dry above-ground parts weight and plant height, was evaluated in pot trials. A total of 127 strains were randomly selected from 610 isolated endophytic bacterial cultures, representing ten genera and 22 taxa. Some taxa were organ-specific; others were growth-stage-specific. Bacillus aryabhattai , B. stratosphericus , Leclercia adecarboxylata and Pseudomonas oryzihabitans were detected as wheat endophytes for the first time. The IAA production, inorganic phosphorous solubilisation, organic phosphorus solubilisation, potassium solubilisation and growth on N-free medium were detected in 45%, 29%, 37%, 2.4% and 37.8% of the 127 strains, respectively. In pot trials, each strain showed variable effects on inoculated wheat plants regarding the evaluated growth parameters. Conclusions Wheat endophytic bacteria showed organ- and growth-stage diversity, which may reflect their adaptations to different plant tissues and seasonal variations, and differed in their PGP abilities. Bacillus was the most predominant bacterial taxa isolated from winter wheat plants. Our study confirmed wheat root as the best reservoir for screening endophytic bacteria with potential as biofertilisers.

Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, 'Candidatus Liberibacter asiaticus'. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.

The red palm weevil (RPW; Coleoptera: Curculionidae) is a destructive pest affecting palms worldwide, capable of causing significant economic losses and ecological damage in managed palm ecosystems. Current management heavily relies on synthetic insecticides, but their overuse fosters resistance. Bacillus thuringiensis (Bt) offers a promising alternative, producing toxins selective against various insect orders, including Coleoptera. However, no specific Bt toxin has yet been identified for RPW. This study investigates the toxicity against RPW larvae of the Bt Cry1Ia protoxin, known for its dual activity against Lepidoptera and Coleoptera. A laboratory RPW colony was reared for two generations, ensuring a reliable insect source for bioassays. Cry1Ia was expressed as a 6xHis-tagged fusion protein in Escherichia coli and purified using nickel affinity. Incubation with RPW larval gut proteases for 24 h produced a stable core of ~65 kDa. Diet-incorporation bioassays revealed high Cry1Ia toxicity in neonate larvae. In contrast, the lepidopteran-active Cry1Ac protoxin, used as a robust negative control, was completely degraded after 24 h of in vitro proteolysis and showed no toxicity in bioassays. Cry1Ia-fed larvae exhibited significant midgut cell damage, characteristic of Bt intoxication. These findings highlight Cry1Ia’s strong potential for integration into RPW management programs.

The Gram-negative rod-shaped bacterium Serratia marcescens is an opportunistic pathogen of many organisms, including insects. We report the identification and optimal in vitro chitinase production conditions of a novel chitinolytic S. marcescens strain TC-1 isolated from a naturally infected white grub (Anomala corpulenta) collected from a peanut field at Nanyang city, Henan province, China. Strain identification was conducted by morphological, physiological, biochemical and molecular analyses. The amplified 16S rRNA gene of TC-1 showed a similarity greater than 99% with multiple strains of S. marcescens. Based on Neighbor-joining phylogenetic tree analysis of bacterial 16S rRNA gene sequences, TC-1 formed a clade with S. marcescens, clearly separated from other Serratia spp. The strain TC-1 showed larvicidal activities against five insect species (A. corpulenta, Plutella xylostella, Spodoptera exigua, Helicoverpa armigera, Bombyx mori) and the nematode Caenorhabditis elegans, but not against S. litura. The operating parameters of chitinase production by TC-1 were optimized by response surface methodology using a three-factor, three-level Box-Behnken experimental design. The effects of three independent variables i.e. colloidal chitin concentration (7–13 g l−1), incubation time (24–72 h) and incubation temperature (24–32 °C) on chitinase production by TC-1 were investigated. A regression model was proposed to correlate the independent variables for an optimal chitinase activity predicted as 20.946 U ml−1, using a combination of colloidal chitin concentration, incubation time and incubation temperature of 9.06 g l−1, 63.83 h and 28.12 °C, respectively. The latter agreed well with a mean chitinase activity of 20.761 ± 0.102 U ml−1 measured in the culture supernatants of TC-1 grown under similar conditions with a colloidal chitin concentration, incubation time and incubation temperature of 9 g l−1, 64 h and 28 °C, respectively. Our study revealed the S. marcescens strain TC-1 with potential as a biocontrol agent of insect pests and nematodes and demonstrated the proposed regression model's potential to guide chitinase production by this strain.

Silkworm ovary-derived BmN4 cells rely on chromatin-induced spindle assembly to form microtubule-based square mitotic spindles that ensure accurate segregation of holocentric chromosomes during cell division. The chromosome passenger protein Aurora B regulates chromosomal condensation and segregation, spindle assembly checkpoint activation, and cytokinesis; however, its role in holocentric organisms needs further clarification. This study examined the architecture and dynamics of spindle microtubules during prophase and metaphase in BmN4 cells and those with siRNA-mediated BmAurora B knockdown using immunofluorescence labeling. Anti-α-tubulin and anti-γ-tubulin antibodies revealed faint γ-tubulin signals colocalized with α-tubulin in early prophase during nuclear membrane rupture, which intensified as prophase progressed. At this stage, bright regions of α-tubulin around and on the nuclear membrane surrounding the chromatin suggested the start of microtubules assembling in the microtubule-organizing centers (MTOCs). In metaphase, fewer but larger γ-tubulin foci were detected on both sides of the chromosomes. This resulted in a distinctive multipolar square spindle with holocentric chromosomes aligned at the metaphase plate. siRNA-mediated BmAurora B knockdown significantly reduced the γ-tubulin foci during prophase, impacting microtubule nucleation and spindle structure in metaphase. Spatiotemporal BmAurora B expression analysis provided new insights into the regulation of this mitotic kinase in silkworm larval gonads during gametogenesis. Our results suggest that BmAurora B is crucial for the formation of multipolar square spindles in holocentric insects, possibly through the activation of γ-tubulin ring complexes in multiple centrosome-like MTOCs.