Metabolic Adaptations in Phosphine-Resistant Tribolium castaneum Driven by Mitochondrial Enzyme Variability and Gene Expression.

Phosphine fumigation is essential for controlling storage pests like Tribolium castaneum, but its frequent application has resulted in resistance, primarily due to mutations in the Dihydrolipoamide dehydrogenase (DLD) gene associated with the rph2 allele. This study demonstrates that the Patiala population exhibits homozygous resistance variations across populations, contrasting with the susceptibility observed in laboratory cultures. Our assessment of mitochondrial DLD and Cytochrome c oxidase (COX) activities showed significantly elevated levels in the Patiala population, with increases of approximately sevenfold for DLD and 6.92-fold for COX, indicating mitochondrial adaptations for enhanced energy production. Kinetic analyses of DLD in the resistant Patiala population showed a higher Vmax (0.005 mmol/min) and a significantly increased Km (16.66 mM), indicating variations in maximal enzyme activity and substrate affinity. Furthermore, resistant T. castaneum populations displayed substantial upregulation of DLD and COX gene expression, with DLD expression increasing by 10.53-fold and COX expression peaking at 102.57-fold in Patiala. Pearson correlation analysis indicated strong positive correlations (r > 0.8) between enzymatic activity and gene expression for both DLD and COX, suggesting a coordinated role in resistance mechanisms. The PCA biplot illustrated distribution patterns of enzymatic activity and gene expression among field-resistant populations, highlighting the association between increased resistance and elevated enzymatic activity and gene expression levels. Therefore, the upregulation of DLD and COX activities in resistant populations underscores their critical roles in counteracting phosphine, reflecting metabolic reprogramming for improved energy production under stress.

Comparison of phenotypic and genotypic frequency of phosphine resistance in select field populations of Tribolium castaneum from India.

BACKGROUND: Tribolium castaneum causes substantial damage to stored grains, leading to economic losses. The present study evaluates phosphine resistance in adult and larval stages of T. castaneum from north and northeast India, where continuous and long-term phosphine use in large-scale storage conditions intensifies resistance, posing risks to grain quality, safety, and industry profitability. METHODS AND RESULTS: This study utilized T. castaneum bioassays and CAPS markers restriction digestion methodology to assess resistance. The phenotypic results indicated a lower LC50 value in larvae compared to adults, while the resistance ratio remained consistent across both stages. Similarly, the genotypic analysis revealed comparable resistance levels regardless of the developmental stage. We categorized the freshly collected populations based on resistance ratios, with Shillong showing weak resistance, Delhi and Sonipat displaying moderate resistance, and Karnal, Hapur, Moga, and Patiala exhibiting strong resistance to phosphine. Further validation by accessing findings and exploring the relationship between phenotypic and genotypic variations using Principal Component Analysis (PCA). This comprehensive study enhances our understanding of T. castaneum resistance levels, providing valuable insights for the development of targeted pest management strategies. CONCLUSION: This study provides insights into the current phenotypic and genotypic resistance levels of T. castaneum in North and North East India. Understanding this is crucial for developing effective pest management strategies and future research on biological and physiological aspects of phosphine resistance in insects, enabling the formulation of effective management practices. Addressing phosphine resistance is vital for sustainable pest management and the long-term viability of the agricultural and food industries.

Electrophysiological and Behavioral Responses of Spotted Stem Borer, Chilo partellus, to Sex Pheromone Components and Their Blends.

The spotted stem borer, Chilo partellus (Lepidoptera: Crambidae) is the major insect pest of maize and sorghum crops during the rainy season. The pheromone released by adult virgin females has been reported to consist of (Z)-11-hexadecenal (Z11-16:Ald) as major component and (Z)-11-hexadecenol (Z11-16OH) as minor component. The latter has been reported to reduce the efficiency of major component to trap the male moths. We studied the electrophysiological and behavioral response of male C. partellus moths to Z11-16:Ald, Z11-16OH, their E-isomers (E)-11-hexadecenal and (E)-11-hexadecen-1-ol, and blends. Electroantennogram (EAG) studies revealed that male C. partellus antennae elicited significantly greater responses to both (Z)-11-hexadecenal and (Z)-11-hexadecenol compared to their respective E-isomers. Behavioral response studies through wind tunnel and cage bioassay showed that blends of Z11-16:Ald and Z11-16OH in the proportion of 100:100 and 100:95 elicited significantly higher responses from male moths. The attractiveness of these pheromone components and blends in field also revealed that traps baited with 100:100 proportion was most effective. Our studies clearly showed that the minor alcohol component Z11-16OH is important for enhancing attractiveness of the pheromone and provides a more effective blend for monitoring of this pest.

Novel moisture retaining dustable powder containing Steinernema abbasi effectively controls damage of subterranean termite in wheat and chickpea.

The application of biocontrol agents in farm operations for pest control programs is gaining priority and preference globally. Effective delivery, infectivity of the biocontrol agents, and quality shelf-life products containing these bioagents are vital parameters responsible for the success of biopesticides under field conditions. In the present study, moisture-retaining bio-insecticidal dustable powder formulation (SaP) of Steinernema abbasi (Sa) infective juveniles (IJs) was developed and assessed for its shelf life, physicochemical profile, and bio-efficacy against subterranean termite under field conditions. Formulation exhibited free-flowing character, with pH of 6.50-7.50, and apparent density in the range 0.50-0.70 g cm-3. The bioefficacy study for two rabi seasons (2020-2021, and 2021-2022) in wheat and chickpea grown in an experimental farm heavily infested with subterranean termites (Odontotermes obesus) revealed a significant reduction in plant damage due to pest attack in formulation-treated plots, monitored in terms of relative number of infested tillers in wheat and infested plants in chickpea fields. The reduced damage to the crop caused by termite was reflected in the relative differences in the growth and yield attributes as well. The study establishes the potential of the developed product as a biopesticide suitable for organic farming and integrated pest management operations.

Revealing the Role of CYP346 Family Genes on Phosphine Resistance in Indian Populations of Tribolium castaneum.

BACKGROUND: Phosphine resistance in Tribolium castaneum challenges grain storage. This study investigates the impact of cytochrome P450 (CYP) enzymes and CYP346 family genes on phosphine resistance in Indian Tribolium castaneum populations. METHODS: Seven field populations of T. castaneum were compared with Lab- susceptible population for their resistance to phosphine. The levels of cytochrome P450 enzyme and expression of certain CYP346 family genes were tracked in these populations. RESULTS: The highly resistant Patiala population showed significantly increased CYP450 activity (11.26 ± 0.14 nmol/min/mg protein, 7.41-fold higher) compared to the lab-susceptible population (1.52 ± 0.09 nmol/min/mg protein) when assayed using 8 mM p-nitroanisole as the substrate. The mRNA expression was measured relative to the standard gene RPS18 and revealed significant upregulation of CYP346B1 and CYP346B3 in highly resistant populations Moga and Patiala (CYP346B1: 12.09 ± 2.19 to 21.74 ± 3.82; CYP346B3: 59.097 ± 10.265 to 50.148 ± 8.272). Patiala's CYP346B1 exhibited an impressive 685.76-fold change, and Moga's CYP346B3 showed a 361.893-fold change compared to lab-susceptible. Linear regression confirmed robust fits for each gene (R2: 0.693 to 0.756). Principal component analysis (PCA) demonstrated a strong positive correlation between CYP346 genes expression; and cytochrome P450 activity. Patiala, Moga, and Hapur populations showed conformity, associating higher resistance with increased P450 activity and CYP346 gene expression. Cluster analysis highlighted a potential correlation between CYP346B1, CYP346B2, and CYP346B3 and P450 activity, with Patiala and Moga clustering together. CONCLUSIONS: Variability in CYP346B1 and CYP346B3 in strong resistance populations may contribute to adaptation and resistance mechanisms. The study provides insights into specific CYP346 family genes associated with phosphine resistance, emphasizing the intricate interaction between CYP450 detoxifying enzymes, CYP346 family genes, and resistance mechanisms. The upregulation of CYP346 genes suggests a survival advantage for T. castaneum against phosphine, diminishing phosphine's efficacy as a pest control measure.

Current status of phosphine resistance in Indian field populations of Tribolium castaneum and its influence on antioxidant enzyme activities.

Resistance to phosphine is widely reported in several stored product insect pests globally. However, knowledge of its prevalence and the association of antioxidant enzymes with phosphine resistance is limited. Herein, we assessed the levels of phosphine susceptibility and estimated the antioxidant enzyme activities viz., superoxide dismutase (SOD), peroxidase (POX), and catalase (CAT) in selected Indian populations of red flour beetle Tribolium castaneum (Herbst). Dose-response probit assays revealed that the LC50 values ranged from 0.038 to 1.277 mg L-1 showing 2.11 to 70.94-fold resistance to phosphine compared to susceptible check. Activities of antioxidant enzymes varied significantly between the T. castaneum populations following phosphine exposure. The magnitude of SOD activity ranged from 8.77 to18.82 U mg-1 protein, while, the activities of POX and CAT varied between 52.42 and 408.32 and 61.11 to 247.49 µM H2O2 reduced min-1 mg-1 of protein, respectively. The correlation analysis revealed a significant positive association of SOD (r = 0.89) and POX (r = 0.98) with increased resistance ratio, while the CAT (r = - 0.98) is negatively linked with resistance to phosphine. A principal component analysis identified phosphine resistance was closely associated with POX and SOD activities but was unrelated to the CAT activity. Our results throw light on the varied association of antioxidant enzyme activities in response to phosphine fumigation in field populations of T. castaneum. Further studies on the biochemical and molecular basis of phosphine stress in insects may help to devise suitable strategies to safeguard storage commodities and ensure a sustainable environment.

Dynamic changes in virus-induced volatiles in cotton modulate the orientation and oviposition behavior of the whitefly Bemisia tabaci.

Manipulation of insect vector behavior by virus-induced plant volatiles is well known. But how the viral disease progression alters the plant volatiles and its effect on vector behavior remains less explored. Our studies tracked changes in volatile profile in progressive infection stages of cotton leaf curl virus (CLCuV) infected plants and their effect on B. tabaci behavior. Significant differences in virus titers were noticed between progressive infection stages showing distinct symptoms. Whiteflies initially settled on CLCuV infected plants, but their preference was shifted to healthy plants over time. GC-MS analysis revealed subtle quantitative/qualitative changes in volatile organic compounds (VOCs) between the healthy and selected CLCuV infection stages. VOCs such as hexanal, (E)-2-hexen-1-ol, (+)-α-pinene, (-)-ß-pinene, (Z)-3-hexen-1-ol, (+)-sylvestrene, and (1S,2E,6E, 10R)-3,7,11,11-tetramethylbicycloundeca-2,6-diene (Bicyclogermacrene) were associated with the infection stage showing upward curling of leaves; (E)-2-hexen-1-ol, ß-myrcene, ß-ocimene, and copaene were associated with the infection stage showing downward curling. Validation studies with eight synthetic VOCs indicated that γ-terpinene elicited attraction to B. tabaci (Olfactometric Preference Index (OPI) = 1.65), while ß-ocimene exhibited strong repellence (OPI = 0.64) and oviposition reduction (66.01%-92.55%). Our studies have demonstrated that progression of CLCuV disease in cotton was associated with dynamic changes in volatile profile which influences the behavioural responses of whitefly, B.tabaci. Results have shown that VOCs such as (+)-α-pinene, (-)-ß-pinene γ-Terpinene, α-guaiene; 4- hydroxy- 4 methyl-2- pentanone and ß-ocimene emitted from Begomovirus infected plants could be the driving force for early attraction and later repellence/oviposition deterrence of B. tabaci on virus-infected plants. The findings of this study offer scope for the management of whitefly, B. tabaci through semiochemicals.

A CRISPR way for accelerating cereal crop improvement: Progress and challenges.

Humans rely heavily on cereal grains as a key source of nutrients, hence regular improvement of cereal crops is essential for ensuring food security. The current food crisis at the global level is due to the rising population and harsh climatic conditions which prompts scientists to develop smart resilient cereal crops to attain food security. Cereal crop improvement in the past generally depended on imprecise methods like random mutagenesis and conventional genetic recombination which results in high off targeting risks. In this context, we have witnessed the application of targeted mutagenesis using versatile CRISPR-Cas systems for cereal crop improvement in sustainable agriculture. Accelerated crop improvement using molecular breeding methods based on CRISPR-Cas genome editing (GE) is an unprecedented tool for plant biotechnology and agriculture. The last decade has shown the fidelity, accuracy, low levels of off-target effects, and the high efficacy of CRISPR technology to induce targeted mutagenesis for the improvement of cereal crops such as wheat, rice, maize, barley, and millets. Since the genomic databases of these cereal crops are available, several modifications using GE technologies have been performed to attain desirable results. This review provides a brief overview of GE technologies and includes an elaborate account of the mechanisms and applications of CRISPR-Cas editing systems to induce targeted mutagenesis in cereal crops for improving the desired traits. Further, we describe recent developments in CRISPR-Cas-based targeted mutagenesis through base editing and prime editing to develop resilient cereal crop plants, possibly providing new dimensions in the field of cereal crop genome editing.

Unraveling Microbial Volatile Elicitors Using a Transparent Methodology for Induction of Systemic Resistance and Regulation of Antioxidant Genes at Expression Levels in Chili against Bacterial Wilt Disease.

Microbial volatiles benefit the agricultural ecological system by promoting plant growth and systemic resistance against diseases without harming the environment. To explore the plant growth-promoting efficiency of VOCs produced by Pseudomonas fluorescens PDS1 and Bacillus subtilis KA9 in terms of chili plant growth and its biocontrol efficiency against Ralstonia solanacearum, experiments were conducted both in vitro and in vivo. A closure assembly was designed using a half-inverted plastic bottle to demonstrate plant-microbial interactions via volatile compounds. The most common volatile organic compounds were identified and reported; they promoted plant development and induced systemic resistance (ISR) against wilt pathogen R. solanacearum. The PDS1 and KA9 VOCs significantly increased defensive enzyme activity and overexpressed the antioxidant genes PAL, POD, SOD, WRKYa, PAL1, DEF-1, CAT-2, WRKY40, HSFC1, LOX2, and NPR1 related to plant defense. The overall gene expression was greater in root tissue as compared to leaf tissue in chili plant. Our findings shed light on the relationship among rhizobacteria, pathogen, and host plants, resulting in plant growth promotion, disease suppression, systemic resistance-inducing potential, and antioxidant response with related gene expression in the leaf and root tissue of chili.

The induced knockdown of GmCAD receptor protein encoding gene in Galleria mellonella decreased the insect susceptibility to a Photorhabdus akhurstii oral toxin.

Photorhabdus bacteria secrete a repertoire of protein toxins that can kill the host insect. Among them, toxin complex (Tc) proteins have gained significant attention due to their wider conservation across the different bacterial genera. In our laboratory, a C-terminal domain of TcaB protein was characterized from P. akhurstii bacterium that conferred the potent oral insecticidal effect on Galleria mellonella. However, the role of insect gut receptors in the TcaB intoxication process was yet to be investigated. In the current study, we examined the transcription of candidate midgut receptors in TcaB-infected larvae and subsequently cloned a cadherin-like gene, GmCAD, from G. mellonella. GmCAD was highly transcribed in the fourth-instar larval stage and specifically in the midgut tissues. Our ligand blot and binding ELISA assays indicated that TcaB binds to the truncated peptides from the GmCAD transmembrane-proximal region with greater affinity than that from the transmembrane-distal region. Oral administration of bacterially expressed GmCAD dsRNA in G. mellonella severely attenuated the expression of target mRNA, which in turn alleviated the negative effect of TcaB on insect survival (TcaB-induced mortality in CAD dsRNA pretreated larvae reduced by 72-83% compared to control), implying the association of GmCAD in the TcaB intoxication process. Present findings form a basis of future research related to the insect gut receptor interactions with Photorhabdus toxins.