Corrigendum: Dichotomous sperm in Lepidopteran insects: a biorational target for pest management.

[This corrects the article DOI: 10.3389/finsc.2023.1198252.].

Genetic Enhancement for Biotic Stress Resistance in Basmati Rice through Marker-Assisted Backcross Breeding.

Pusa Basmati 1509 (PB1509) is one of the major foreign-exchange-earning varieties of Basmati rice; it is semi-dwarf and early maturing with exceptional cooking quality and strong aroma. However, it is highly susceptible to various biotic stresses including bacterial blight and blast. Therefore, bacterial blight resistance genes, namely, xa13 + Xa21 and Xa38, and fungal blast resistance genes Pi9 + Pib and Pita were incorporated into the genetic background of recurrent parent (RP) PB1509 using donor parents, namely, Pusa Basmati 1718 (PB1718), Pusa 1927 (P1927), Pusa 1929 (P1929) and Tetep, respectively. Foreground selection was carried out with respective gene-linked markers, stringent phenotypic selection for recurrent parent phenotype, early generation background selection with Simple sequence repeat (SSR) markers, and background analysis at advanced generations with Rice Pan Genome Array comprising 80K SNPs. This has led to the development of Near isogenic lines (NILs), namely, Pusa 3037, Pusa 3054, Pusa 3060 and Pusa 3066 carrying genes xa13 + Xa21, Xa38, Pi9 + Pib and Pita with genomic similarity of 98.25%, 98.92%, 97.38% and 97.69%, respectively, as compared to the RP. Based on GGE-biplot analysis, Pusa 3037-1-44-3-164-20-249-2 carrying xa13 + Xa21, Pusa 3054-2-47-7-166-24-261-3 carrying Xa38, Pusa 3060-3-55-17-157-4-124-1 carrying Pi9 + Pib, and Pusa 3066-4-56-20-159-8-174-1 carrying Pita were identified to be relatively stable and better-performing individuals in the tested environments. Intercrossing between the best BC3F1s has led to the generation of Pusa 3122 (xa13 + Xa21 + Xa38), Pusa 3124 (Xa38 + Pi9 + Pib) and Pusa 3123 (Pi9 + Pib + Pita) with agronomy, grain and cooking quality parameters at par with PB1509. Cultivation of such improved varieties will help farmers reduce the cost of cultivation with decreased pesticide use and improve productivity with ensured safety to consumers.

Receptivity and Remating Propensity in Female Spodoptera litura (Fabricius) after Mating with an Irradiated Male or Its F1 Male Progeny.

The 'Inherited or F1 sterility technique' (IS), using sub-sterilized male moths, is a widely proposed pest management tool for Lepidoptera pests in general, and the tobacco cutworm Spodoptera litura (Fabr.) in particular. However, the multiple mating tendency of female moths and the ejaculate quality of male moths might influence the efficiency of this technique. Reduced ejaculate quality was observed in irradiated males, as evidenced by radiation's impact on certain bio-parameters, such as the weight of the spermatophores and their protein content, sperm count, the molecular expression of the sex peptide receptor (SPR) and egg fertility, with a greater impact in F1 male progeny. During the remating of females with untreated males, irrespective of the irradiation status of the first male, there was an increase in calling behavior, remating propensity and fertility in females, with a larger time gap between consecutive matings. The ability of F1 male progeny to check remating propensity in females 24 h after the initial mating was lower than that of unirradiated males. Partially sterile (130 Gy) males were as successful as unirradiated males in inducing the level of mating refractoriness in females. Decreased ejaculate quality in F1 male progeny could be associated with increased female receptivity during remating. Understanding the influence of male moth irradiation, insemination quality and post (initial)-mating intervals on the remating behavior of normal female moths and induced sterility might help in simulation modeling and optimizing IS insect programs.

Phylogenetic analysis and interactomics study unveil gene co-optive evolution of LysR-type transcription regulators across non-pathogenic, opportunistic, and pathogenic mycobacteria.

Mycobacterial species is known for inhabiting various niches ranging from soil to harsh intracellular environment of animal hosts and their survival through constant changes. For survival and persistence, these organisms must quickly adapt by bringing shift in their metabolism. Metabolic shifts are brought by sensing the environmental cues usually by membrane localized sensor molecules. These signals are transmitted to regulators of various metabolic pathways leading to post-translational modifications of regulators ultimately resulting in altered metabolic state of the cell. Multiple regulatory mechanisms have been unearthed so far that play crucial role in adapting to these situations, and among them, the signal-dependent transcriptional regulators mediated responses are integral for the microbes to perceive environmental signals and generate appropriate adaptive responses. LysR-type transcriptional regulators (LTTRs) form the largest family of transcriptional regulators, which are present in all kingdoms of life. Their numbers vary among bacterial genera and even in different mycobacterial species. To understand the evolutionary aspect of pathogenicity based on LTTRs, we performed phylogenetic analysis of LTTRs encoded by several mycobacterial species representing non-pathogenic (NP), opportunistic (OP), and totally pathogenic (TP) mycobacteria. Our results showed that LTTRs of TP clustered separately from LTTRs of NP and OP mycobacteria. In addition, LTTRs frequency per Mb of genome was reduced in TP when compared with NP and OP. Further, the protein-protein interactions and degree-based network analysis showed concomitant increased interactions per LTTRs with increase in pathogenicity. These results suggested the increase in regulon of LTTRs during evolution of TP mycobacteria.

Dichotomous sperm in Lepidopteran insects: a biorational target for pest management.

Lepidoptera are unusual in possessing two distinct kinds of sperm, regular nucleated (eupyrene) sperm and anucleate (apyrene) sperm ('parasperm'). Sperm of both types are transferred to the female and are required for male fertility. Apyrene sperm play 'helper' roles, assisting eupyrene sperm to gain access to unfertilized eggs and influencing the reproductive behavior of mated female moths. Sperm development and behavior are promising targets for environmentally safer, target-specific biorational control strategies in lepidopteran pest insects. Sperm dimorphism provides a wide window in which to manipulate sperm functionality and dynamics, thereby impairing the reproductive fitness of pest species. Opportunities to interfere with spermatozoa are available not only while sperm are still in the male (before copulation), but also in the female (after copulation, when sperm are still in the male-provided spermatophore, or during storage in the female's spermatheca). Biomolecular technologies like RNAi, miRNAs and CRISPR-Cas9 are promising strategies to achieve lepidopteran pest control by targeting genes directly or indirectly involved in dichotomous sperm production, function, or persistence.

Genome-wide association study reveals novel genomic regions governing agronomic and grain quality traits and superior allelic combinations for Basmati rice improvement.

Background: Basmati is a speciality segment in the rice genepool characterised by explicit grain quality. For the want of suitable populations, genome-wide association study (GWAS) in Basmati rice has not been attempted. Materials: To address this gap, we have performed a GWAS on a panel of 172 elite Basmati multiparent population comprising of potential restorers and maintainers. Phenotypic data was generated for various agronomic and grain quality traits across seven different environments during two consecutive crop seasons. Based on the observed phenotypic variation, three agronomic traits namely, days to fifty per cent flowering, plant height and panicle length, and three grain quality traits namely, kernel length before cooking, length breadth ratio and kernel length after cooking were subjected to GWAS. Genotyped with 80K SNP array, the population was subjected to principal component analysis to stratify the underlying substructure and subjected to the association analysis using Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) model. Results: We identified 32 unique MTAs including 11 robust MTAs for the agronomic traits and 25 unique MTAs including two robust MTAs for the grain quality traits. Six out of 13 robust MTAs were novel. By genome annotation, six candidate genes associated with the robust MTAs were identified. Further analysis of the allelic combinations of the robust MTAs enabled the identification of superior allelic combinations in the population. This information was utilized in selecting 77 elite Basmati rice genotypes from the panel. Conclusion: This is the first ever GWAS study in Basmati rice which could generate valuable information usable for further breeding through marker assisted selection, including enhancing of heterosis.

Effect of Irradiation on Reproduction of Female Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae) in Relation to the Inherited Sterility Technique.

Radiobiological investigations on the reproductive behavior of female Spodoptera litura (Fabr.) were conducted with the aim of determining the suitable radio-sterilizing dose for females in order to release them along with sub-sterile males for effective implementation of the Inherited Sterility technique against this pest. Calling and copulation duration significantly increased, while mating success, oviposition, fertility and longevity significantly decreased with increasing radiation dose (100-200 Gy) compared to control. In view of the effect of irradiation on mating behavior and reproductive viability of female S. litura, 130 Gy was identified as a suitable radio-sterilization dose. Further molecular studies were conducted to corroborate this dose for female sterilization, along with a higher dose of 200 Gy in order to validate the gradational response of ionizing radiation. GC-MS analysis indicated decreased sex pheromone titer at 130 Gy, which was more pronounced at 200 Gy. Pheromone-associated genes, PBAN and PBAN-R showed decreased expression at 130 Gy, and were drastically reduced at 200 Gy. The fertility-related Vg gene also showed a negative correlation with radiation exposure. Based on these radiation responses of female S. litura, 130 Gy might be considered a suitable dose for complete female sterility and its inclusion in sterile insect programs against S. litura.

Radiation Hormesis to Improve the Quality of Adult Spodoptera litura (Fabr.).

Mass rearing of insects of high biological quality is a crucial attribute for the successful implementation of sterile insect release programs. Various ontogenetic stages of Spodoptera litura (Fabr.) were treated with a range of low doses of ionizing radiation (0.25-1.25 Gy) to assess whether these gamma doses could elicit a stimulating effect on the growth and viability of developing moths. Doses in the range of 0.75 Gy to 1.0 Gy administered to eggs positively influenced pupal weight, adult emergence, and growth index, with a faster developmental period. The enhanced longevity of adults derived from eggs treated with 0.75 Gy and 1.0 Gy, and for larvae and pupae treated with 1.0 Gy, indicated a hormetic effect on these life stages. Furthermore, the use of these hormetic doses upregulated the relative mRNA expression of genes associated with longevity (foxo, sirtuin 2 like/sirt1, atg8) and viability/antioxidative function (cat and sod), suggesting a positive hormetic effect at the transcriptional level. These results indicated the potential use of low dose irradiation (0.75-1 Gy) on preimaginal stages as hormetic doses to improve the quality of the reared moths. This might increase the efficiency of the inherited sterility technique for the management of these lepidopteran pests.

Population Dynamics of Wide Compatibility System and Evaluation of Intersubspecific Hybrids by indica-japonica Hybridization in Rice.

The exploitation of heterosis through intersubspecific hybridisation between indica and japonica has been a major breeding target in rice, but is marred by the cross incompatibility between the genomes. Wide compatibility (WC) is a triallelic system at the S5 locus on chromosome 6 that ensures the specificity of hybridisation within and between indica and japonica. The S5n allele that favours intercrossing is sparsely distributed in the rice gene pool and therefore warrants identification of diverse WC sources to develop superior intersubspecific hybrids. In this study, we have identified several novel WC sources through the marker-assisted screening of a large set of 950 rice genotypes. Seventeen percent of the genotypes carried S5n, which fell into two subpopulations. The WC genotypes showed wide phenotypic and genotypic variability, including both indica and japonica lines. Based on phenotypic performance, the WC varieties were grouped into three clusters. A subset of 41 WC varieties was used to develop 164 hybrids, of which WC/japonica hybrids showed relative superiority over WC/indica hybrids. The multilocation evaluation of hybrids indicated that hybrids derived from WC varieties, such as IRG137, IRG143, OYR128, and IRGC10658, were higher yielding across all the three different locations. Most of the hybrids showed the stability of performance across locations. The identified diverse set of wide compatible varieties (WCVs) can be used in the development of intersubspecific hybrids and also for parental line development in hybrid rice breeding.

Radiation biology of a serious tropical pigeon pea pest, Maruca vitrata (Fabricius) (Lepidoptera: Crambidae) and potential of radiation mediated 'inherited (F1) sterility technique' for the pest suppression.

Purpose: The impact of gamma radiation on the reproductive biology of the spotted pod borer, Maruca vitrata (Fabr.) (Lepidoptera: Crambidae) was ascertained to explore the potential of the radio-genetic 'Inherited (F1) Sterility' technique (modified Sterile Insect technique) to control this serious pigeonpea pest in India.Materials and methods: Radio-biological investigations involved dose-response studies, at a range of 100-250 Gy, with respect to radiation induced adverse effects on reproductive behavior in substerilized parent (P1) moths, inheritance of sterility and associated metamorphic disruption in F1 progeny. The reproductive competence of P1 male moths and their F1 progeny was evaluated by ascertaining the survival, mating success, sperm transfer and amphimixis of gametes from irradiated male parents and their F1 progeny. Daily ovipositional and egg fertility profile were evaluated as crucial parameters for simulation modeling for this radio-genetic technique.Results: Radiation-induced check in insemination rate and sterility in P1 and F1 generation had a positive correlation with gamma dose. The metamorphic adverse effects in F1 progeny of substerilized P1 males were influenced by P1 irradiation. A higher dose of 200 Gy that resulted in 21.1% fertility in parent cross (P1♂x N♀) could form only 14.4% F1 adults that were all malformed. A gamma dose of 150 Gy (administered to P1 males) could be considered as a suitable dose for F1 sterility, that induced 57.4% control of reproduction in parent generation, followed by 72-85% control of reproduction in F1 progeny involved in three crosses -F1♂xN♀, N♂xF1♀, F1♂xF1♀. Daily fertility profile of the eggs laid indicated a marked reduction on 4th day onwards during the ovipositional span in P1 and F1 generation. High degree of mating competitiveness value (CV) was noticed for 150 Gy irradiated male P1 moths (0.78-0.93), and their F1 male progeny (0.70-0.89), released in different sex ratios in the field cages having untreated moths. Reduced relative damage to the pigeon pea plants by the released F1 progeny in the field cages was observed in comparison to control.Conclusion: The present study indicated the potential use of a dose of 150 Gy to apply the 'F1 sterility' technique for parabiological suppression of the pigeon pea pod borer. The present findings and reproductive performance of the irradiated moths in F2 generation along with other compatible biorational pest control tactics (that are in progress), might help in formulating an effective integrated pest management module, including the F1 sterility technique as an integral component.

Genomic adaptation to polyphagy and insecticides in a major East Asian noctuid pest.

The tobacco cutworm, Spodoptera litura, is among the most widespread and destructive agricultural pests, feeding on over 100 crops throughout tropical and subtropical Asia. By genome sequencing, physical mapping and transcriptome analysis, we found that the gene families encoding receptors for bitter or toxic substances and detoxification enzymes, such as cytochrome P450, carboxylesterase and glutathione-S-transferase, were massively expanded in this polyphagous species, enabling its extraordinary ability to detect and detoxify many plant secondary compounds. Larval exposure to insecticidal toxins induced expression of detoxification genes, and knockdown of representative genes using short interfering RNA (siRNA) reduced larval survival, consistent with their contribution to the insect's natural pesticide tolerance. A population genetics study indicated that this species expanded throughout southeast Asia by migrating along a South India-South China-Japan axis, adapting to wide-ranging ecological conditions with diverse host plants and insecticides, surviving and adapting with the aid of its expanded detoxification systems. The findings of this study will enable the development of new pest management strategies for the control of major agricultural pests such as S. litura.

Expression map of a complete set of gustatory receptor genes in chemosensory organs of Bombyx mori.

Most lepidopteran species are herbivores, and interaction with host plants affects their gene expression and behavior as well as their genome evolution. Gustatory receptors (Grs) are expected to mediate host plant selection, feeding, oviposition and courtship behavior. However, due to their high diversity, sequence divergence and extremely low level of expression it has been difficult to identify precisely a complete set of Grs in Lepidoptera. By manual annotation and BAC sequencing, we improved annotation of 43 gene sequences compared with previously reported Grs in the most studied lepidopteran model, the silkworm, Bombyx mori, and identified 7 new tandem copies of BmGr30 on chromosome 7, bringing the total number of BmGrs to 76. Among these, we mapped 68 genes to chromosomes in a newly constructed chromosome distribution map and 8 genes to scaffolds; we also found new evidence for large clusters of BmGrs, especially from the bitter receptor family. RNA-seq analysis of diverse BmGr expression patterns in chemosensory organs of larvae and adults enabled us to draw a precise organ specific map of BmGr expression. Interestingly, most of the clustered genes were expressed in the same tissues and more than half of the genes were expressed in larval maxillae, larval thoracic legs and adult legs. For example, BmGr63 showed high expression levels in all organs in both larval and adult stages. By contrast, some genes showed expression limited to specific developmental stages or organs and tissues. BmGr19 was highly expressed in larval chemosensory organs (especially antennae and thoracic legs), the single exon genes BmGr53 and BmGr67 were expressed exclusively in larval tissues, the BmGr27-BmGr31 gene cluster on chr7 displayed a high expression level limited to adult legs and the candidate CO2 receptor BmGr2 was highly expressed in adult antennae, where few other Grs were expressed. Transcriptional analysis of the Grs in B. mori provides a valuable new reference for finding genes involved in plant-insect interactions in Lepidoptera and establishing correlations between these genes and vital insect behaviors like host plant selection and courtship for mating.

Oral immunoglobulin for preventing necrotizing enterocolitis in preterm and low birth weight neonates.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most common emergency involving the gastrointestinal tract occurring in the neonatal period. There have been published reports that suggest that oral immunoglobulins (Ig)A and IgG produce an immunoprotective effect in the gastrointestinal mucosa. OBJECTIVES: To determine the effect of oral immunoglobulin on the incidence of necrotizing enterocolitis and other complications in preterm or low birth weight (or both) neonates. SEARCH METHODS: We used the standard search strategy of the Cochrane Neonatal Group. We searched the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library 2016, Issue 1), PubMed (1966 to January 2016), CINAHL (1982 to January 2016) and EMBASE (1980 to January 2016) and conference proceedings. SELECTION CRITERIA: All randomized or quasi-randomised controlled trials where oral immunoglobulins were used as prophylaxis against NEC in preterm (less than 37 weeks' gestation) or low birth weight (less than 2500 gram), or both, neonates. DATA COLLECTION AND ANALYSIS: We performed data collection and analysis in accordance with the standard methods of the Cochrane Neonatal Review Group. MAIN RESULTS: The search identified five studies on oral immunoglobulin for the prevention of NEC of which three met the inclusion criteria. In this review of the three eligible trials (including 2095 neonates), the oral administration of IgG or an IgG/IgA combination did not result in a significant reduction in the incidence of definite NEC (typical risk ratio (RR) 0.84, 95% confidence interval (CI) 0.57 to 1.25; typical risk difference (RD) -0.01, 95% CI -0.03 to 0.01; 3 studies, 1840 infants), suspected NEC (RR 0.84, 95% CI 0.49 to 1.46; RD -0.01, 95% CI -0.02 to 0.01; 1 study, 1529 infants), need for surgery (typical RR 0.21, 95% CI 0.02 to 1.75; typical RD -0.03, 95% CI -0.06 to 0.00; 2 studies, 311 infants) or death from NEC (typical RR 1.10, 95% CI 0.47 to 2.59; typical RD 0.00, 95% CI -0.01 to 0.01; 3 studies, 1840 infants). AUTHORS' CONCLUSIONS: Based on the available trials, the evidence does not support the administration of oral immunoglobulin for the prevention of NEC. There are no randomized controlled trials of oral IgA alone for the prevention of NEC.

Radioresistant Sf9 insect cells display efficient antioxidant defence against high dose γ-radiation.

PURPOSE: To investigate the effect of gamma radiation-induced alterations in antioxidant defence of radioresistant Sf9 insect cells. MATERIALS AND METHODS: Sf9 cells were irradiated at doses ranging from 0.5-200 Gy. Lipid peroxidation and protein carbonylation levels were observed at 4 h post-exposure along with reduced glutathione/oxidized glutathione (GSH/GSSG) profile as well as specific activities of redox active enzymes superoxide dismutase (SOD), catalase, ascorbate peroxidase (APOx), and glutathione reductase (GR). Human brain malignant glioma (BMG-1) cells were used for comparing radiation response of mammalian cells. RESULTS: Sf9 cells displayed significantly less radiation-induced reactive oxygen/nitrogen species (ROS/RNS) generation, protein carbonylation and growth inhibition as compared to mammalian cells. Sf9 cells have higher basal APOx (∼4-fold), catalase (∼1.7-fold), SOD (∼1.3-fold) activity and GSH level (∼2.2-fold) compared to mammalian cells. A radiation dose-dependent increase in SOD, Catalase and APOx activity was found in Sf9 cells at least up to 100 Gy dose, while maximum activity in mammalian cells was achieved by 10 Gy. CONCLUSION: The present study suggests that Lepidopteran insect cells carry a stronger antioxidant system that protects against radiation-induced macromolecular damage, growth inhibition and cell death.

Effect of gamma radiation on Phenoloxidase pathway, antioxidant defense mechanism in Helicoverpa armigera (Lepidoptera: Noctuidae) and its implication in inherited sterility towards pest suppression.

PURPOSE: To investigate age-correlated radiosensitivity in highly radioresistant lepidopteran pest, Helicoverpa armigera, upon exposure to ionizing radiation and to examine the irradiation impact on stress-molecular responses in F1 (first-filial) progeny of irradiated (100 Gy) male moths in relation to its reproductive behavior. MATERIALS AND METHODS: Efficacy of sub-lethal gamma radiation was evaluated on two markedly apart ontogenic stages, neonates and adult moths. Differential growth, reproductive behavior and stress-indicating molecular responses were examined upto F1 progeny of sub-sterilized moths. Free-radical scavenging enzymes, superoxide dismutase (SOD), catalase (CAT) and Phenoloxidase cascade enzymes, pro-phenoloxidase (PPO), its activating enzyme (PPAE) were studied in irradiated and irradiated plus microbial challenge regimen (dual-stress) by Real-time RT-PCR (reverse-transcription-polymerase-chain-reaction). RESULTS: An inverse correlation of radiosensitivity with developmental age of insect was observed. F1 sterility was higher than parent sterility. F1 progeny exhibited protraction in development and decreased survival upon irradiation. Sex ratio in F1 progeny was skewed towards males. PPO, PPAE, SOD and CAT transcripts were downregulated upon neonate irradiation resulting in enhanced vulnerability of larvae to incidental microbial challenge. These transcripts were upregulated in F1 progeny of sub-sterilized male moths (100 Gy) upon dual-stress. CONCLUSIONS: Irradiation impact on stress-indicating molecular responses in F1 progeny is correlated with its reproductive performance. These observations will permit defining regimen having pragmatic viability of 'F1 sterility technique' for pest suppression. Gamma dose of 100 Gy would ensure balance between induced sterility of males and their field competitiveness. These parameters would facilitate integration of biocontrol strategy with parabiological 'Sterile Insect Release Technique'.

Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of γ-radiation.

PURPOSE: To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells. MATERIALS AND METHODS: Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500-2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit. RESULTS: A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism. CONCLUSIONS: Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50-200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.

A calcium-insensitive attenuated nitrosative stress response contributes significantly in the radioresistance of Sf9 insect cells.

Lepidopteran insects/insect cells display 50-100 times higher radioresistance than humans, and are evolutionarily closest to mammals amongst all radioresistant organisms known. Compared to mammalian cells, Lepidopteran cells (TN-368, Sf9) display more efficient antioxidant system and DNA repair and suffer considerably less radiation-induced DNA/cytogenetic damage and apoptosis. Recent studies indicate that a considerably lower radiation-induced oxidative stress may significantly reduce macromolecular damage in Lepidopteran cells. Since nitrosative stress contributes in radiation-induced cellular damage, we investigated its nature in the γ-irradiated Sf9 cells (derived from Spodoptera frugiperda; order Lepidoptera; family Noctuidae) and compared with BMG-1 human cell line having significant NOS expression. Radiation induced considerably less ROS/RNS in Sf9 cells, which remained unchanged on treatment with NOS inhibitor l-NMMA. Surprisingly, growth of Sf9 cultures or irradiation could not induce NO or its metabolites, indicating negligible basal/radiation-induced NOS activity that remained unchanged even after supplementation with arginine. Cytosolic calcium release following high-dose (1000-2000Gy at 61.1cGys(-1)) γ-irradiation or H(2)O(2) (250µM) treatment also failed to generate NO in Sf9 cells having high constitutive levels of calmodulin, whereas BMG-1 cells displayed considerable calcium-dependent NO generation even following 10Gy dose. These results strongly imply the lack of calcium-mediated NOS activity in Sf9 cells. Addition of exogenous NO from GSH-NO caused considerable increase in radiation-induced apoptosis, indicating significant contribution of constitutively attenuated nitrosative stress response into the radioresistance of Lepidopteran cells. Our study demonstrates for the first time that a calcium-insensitive, attenuated nitrosative stress response may contribute significantly in the unusual radioresistance displayed by Lepidopteran insect cells.

Interaction of Bacillus thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal activity in Spodoptera frugiperda.

Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.

Mitochondrial antioxidant defence in radio-resistant Lepidopteran insect cells.

Cells isolated from Lepidopteran insects (butterfly and moths) display very high radioresistance as compared to mammals and other insect species. Since free radical induced mitochondrial damage under stress conditions is very crucial for cellular fate determination, antioxidant system is the major protective modality required to minimize stress-induced damage and to modulate cellular sensitivity. In this study, we predict the mitochondrial localization potential and co-existence of important antioxidant enzymes in insect cells and compare with other radiosensitive (mammals, Dipteran insects) and radioresistant (nematodes) species. Our study clearly demonstrates the inter-species variation in then localization potential of various antioxidant enzymes. A higher mitochondrial localization potential as a function of mitoprot score was evident for all important antioxidant enzymes in the lepidopteran insect Bombyx mori (Mn-SOD, 0.694; GPx, 0.862; TRPx, 0.997; TR, 0.9), besides an unusual mitochondrial localization prediction for catalase (0.453). We further found coexistence of glutathione and thioredoxin system in the mitochondria of lepidopteran insects as also reported in various plant species. On the basis of above observations, we hypothesize that a strong mitochondrial antioxidant enzyme system including the unusual coexistence of catalase, glutathione and thioredoxin system may help minimize the free radical mediated damage to mitochondria and can contribute to the intrinsic radioresistance of lepidopteran insects.