Evidence-based insecticide resistance in South American tomato leaf miner, Phthorimaea absoluta (Meyrick) under laboratory selection.

The South American tomato moth, Phthorimaea absoluta (Meyrick), is one of the key pests of tomato in India. Since its report in 2014, chemical control has been the main means of tackling this pest, both in the open field and protected cultivation. Despite regular insecticidal sprays, many outbreaks were reported from major tomato-growing regions of South India during 2019-2020. A study was conducted to investigate the effect of insecticide resistance on biology, biochemical enzymes, and gene expression in various P. absoluta field populations viz., Bangalore, Kolar, Madurai, Salem, and Anantapur to commonly used insecticides such as flubendiamide, cyantraniliprole, and indoxacarb. Increased levels of insecticide resistance ratios (RR) were recorded in P. absoluta populations of different locations. A significant increase in cytochrome P450 monooxygenase (CYP/MFO) and esterase levels was noticed in the resistant population compared to susceptible one. Through molecular studies, we identified four new CYP genes viz., CYP248f (flubendiamide), CYP272c, CYP724c (cyantraniliprole), and CYP648i (indoxacarb). The expression levels of these genes significantly increased as the folds of resistance increased from G1 to G20 (generation), indicating involvement of the identified genes in insecticide resistance development in P. absoluta. In addition, the resistant populations showed decreased fecundity, increased larval development period, and adult longevity, resulting in more crop damage. The information generated in the present study thus helps in understanding the development of insecticide resistance by P. absoluta and suggests the farmers and researchers to use insecticides wisely by adopting insecticide resistance management as a strategy under integrated pest management.

Modeling Commodity Flow in the Context of Invasive Species Spread: Study of Tuta absoluta in Nepal.

Trade and transport of goods is widely accepted as a primary pathway for the introduction and dispersal of invasive species. However, understanding commodity flows remains a challenge owing to its complex nature, unavailability of quality data, and lack of systematic modeling methods. A robust network-based approach is proposed to model seasonal flow of agricultural produce and examine its role in pest spread. It is applied to study the spread of Tuta absoluta, a devastating pest of tomato in Nepal. Further, the long-term establishment potential of the pest and its economic impact on the country are assessed. Our analysis indicates that regional trade plays an important role in the spread of T. absoluta. The economic impact of this invasion could range from USD 17-25 million. The proposed approach is generic and particularly suited for data-poor scenarios.

Development of a test grid using Eye Movement Perimetry for screening glaucomatous visual field defects.

BACKGROUND: Eye Movement Perimetry (EMP) uses Saccadic Eye Movement (SEM) responses for visual field evaluation. Previous studies have demonstrated significant delay in initiation of SEMs among glaucoma patients in comparison with healthy subjects. The aim of the current study was to develop an EMP-based screening grid to identify glaucomatous visual field defects. METHODS: An interactive test consisting of 36 locations and two stimulus contrasts (162 cd/m2 and 190 cd/m2 on a background of 140 cd/m2) was evaluated in 54 healthy subjects and 50 primary glaucoma patients. Each subject was presented a central fixation target combined with the random projection of Goldmann size III peripheral targets. Instructions were given to look at each peripheral target on detection and then re-fixate at the central fixation target while the saccades were assessed using an eye tracker. From each seen peripheral target, the Saccadic Reaction Time (SRT) was calculated for contrast level 162 cd/ m2. These values were used to plot Receiver Operating Characteristic (ROC) curves for each test locations and the Area Under the Curve (AUC) values were used to identify the locations with highest susceptibility to glaucomatous damage. Each stimulus location with an AUC less than 0.75 along with its mirrored test location around the horizontal axis were eliminated from the grid. RESULTS: The mean age was 48.1 ± 16.6 years and 50.0 ± 14.5 years for healthy subjects and glaucoma patients respectively. A significant increase of SRT values by 76.5% (p < 0.001) was found in glaucoma patients in comparison with the healthy subjects. From the ROC analysis, ten out of 36 locations meeting the cut-off criteria of AUC were eliminated resulting in a new grid containing 26 test locations. SRT values were significantly different (p < 0.05) between the healthy subjects and glaucoma irrespective of the grids used. CONCLUSIONS: The present study resulted in a screening grid consisting of 26 locations predominantly testing nasal, superior and inferior areas of the visual field. An internal validation of the modified grid showed 90.4% of screening accuracy which makes it a potential approach for population based glaucoma screening.

Heteroplasmy due to coexistence of mtCOI haplotypes from different lineages of the Thrips tabaci cryptic species group.

Heteroplasmy is the existence of multiple mitochondrial DNA haplotypes within the cell. Although the number of reports of heteroplasmy is increasing for arthropods, the occurrence, number of variants, and origins are not well studied. In this research, the occurrence of heteroplasmy was investigated in Thrips tabaci, a putative species complex whose lineages can be distinguished by their mitochondrial DNA haplotypes. The results from this study showed that heteroplasmy was due to the occurrence of mitochondrial cytochrome oxydase I (mtCOI) haplotypes from two different T. tabaci lineages. An assay using flow cytometry and quantitative real-time PCR was then used to quantify the per cell copy number of the two mtCOI haplotypes present in individuals exhibiting heteroplasmy from nine geographically distant populations in India. All of the T. tabaci individuals in this study were found to exhibit heteroplasmy, and in every individual the per cell copy number of mtCOI from lineage 3 comprised 75-98% of the haplotypes detected and was variable among individuals tested. There was no evidence to suggest that the presense of lineage-specific haplotypes was due to nuclear introgression; however, further studies are needed to investigate nuclear introgression and paternal leakage during rare interbreeding between individuals from lineages 2 and 3.

Sequence analysis of Indian iris yellow spot virus ambisense genome segments: evidence of interspecies RNA recombination.

The nucleotide sequence of M- and S-RNA segments of an Indian iris yellow spot virus (IYSV) were determined. Sequence comparisons showed that both of these sequences shared less than 95 % identity with those other known IYSV isolates. Phylogenetic analysis revealed that the S- and M-RNA sequences of known IYSV isolates clustered with those of the tospoviruses, tomato yellow ring virus, polygonum ringspot virus and hippeastrum chlorotic ringspot virus. Further, multiple recombination detection methods detected inter- and intra-species recombination events that clustered primarily within the intergenic regions of S- and M-RNA, suggesting that these are possibly recombination hotspots in IYSV and closely related tospoviruses.

Risk Factors of Diabetes among Obese Persons Attending Medical Outpatient Department in a Bangalore Hospital.

In this study, carried out in KC General Hospital, Bangalore, 90 obese persons were selected by non-probability convenience sampling technique using a descriptive corre- lational design. The data was analysed using descriptive and inferential statistics. Majority of obese persons had moderate knowledge (73.3%) and neutral attitude (82.2%) regarding risk factors of diabetes. There was a positive correlation (r=0.59) between the knowledge and attitude of obese persons; a statistically significant asso- ciation was found between level of knowledge and demographic variables such as (i) age, educational background and family income of the obese persons and (ii) grade of obesity and educational background of the obese persons. The study found that obese persons have moderate knowledge and neutral attitude regarding risk factors of diabetes. There is need for health education about risk factors of diabetes among obese persons through such means as a pamphlet.

[Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm, Helicoverna armigera].

Reverse-transcription quantitative real-time PCR (RT-qPCR), a sensitive technique is being extensively employed in quantification of gene expression. However this requires normalization with suitable reference gene (RG) which is crucial in minimizing inter sample variations. Information regarding suitable RG is scarce in general and more so in insects, including the cotton bollworm, Helicoverpa armigera, an economically important pest. In management of this pest RNA interference (RNAi), is perceived as a potential tool, which is achieved by double-stranded RNA (dsRNA) delivery. These studies demand accurate quantification of gene silencing. In this study we assessed the suitability of five RGs viz. ß-actin (ACTB), 18S rRNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB) and elongation fator-1-alfa (EF1-α) for gene expression studies in dsRNA treatment and across different developmental stages of H. armigera and ranked using geNorm, NormFinder and BestKeeper software programs. Data analysis revealed that best ranked RGs were varied in dsRNA treatment and in developmental stages. Under dsRNA treatment, 18S and GAPDH were more stable whereas, TUB and GAPDH were more stable across developmental stages. We also demonstrate that inappropriate selection of RG led to erroneous estimation of the target gene, chymotrypsin, expression. These results facilitate accurate quantification of gene expression in H. armigera.

Insilico Structural 3D Modelling of Novel Cry1Ib9 and Cry3A Toxins from Local Isolates of Bacillus thuringiensis.

Three-dimensional (3D) models for the 79.2 kDa activated Cry1Ib9 and 77.4 kDa activated Cry3A δ-endotoxins from Bacillus thuringiensis (Bt) native isolates that are specifically toxic to Coleopteran insect pests were constructed by utilizing homology modeling online tool. Evidences presented here, based on the identification of structural equivalent residues of Cry1Ib9 and Cry3A toxin through homology modelling indicate that, they share a common Bt toxin tridimensional structure. The main differences observed in Cry1I9 domain I at positions α2b (S56-I60), α4 (F78-l93) and additionally ß0 (Q10-L12), α8a (T280-V282) were observed, in domain II at positions α9b (P333-L339), ß6(T390-Q393), ß7(V398-W404), ß8 (V418-W425), ß9 (E453-N454), ß10 (S470-I479) where as in domain III the changes were observed at positions ß19 (R601-F607), ß20 (609-L613), ß21 (S618-F627) and α11a (K655-F664), α13, α14 components present at downstream sites, where as in Cry3A main differences observed in domain I is at the position of α4 (P105-I152), α5 (Q163-A185), ß1A(E190-L192), α6 (F193-Y217), Domain II is not consevered and main variations were observed at ß2 (E292-L295), ß3(V299-L308), ß4(I340-F347), ß5(D356-P368), ß6(I375-T377), ß7(V389-F394), ß8(K398-N405), ß9(Y416-Y427), ß10 (T436-Y439), ß12(G476-H495), ß12A (M503-I504) where as in domain III main variations observed at positions of ß18 (P583-I593), ß19(F604-S610), ß20(P611-L615), ß21(N619-G626). Cry1Ib9 and Cry3A contain the most variable regions in the loops of domain II, which determine the specificity of these toxins. These are the first models of Coleopteran-active protein from native isolates of Bt and its importance can be perceived since members of this group of toxins are potentially important candidates for coleoptera insect pest control programs.

One-step DNA fragment assembly for expressing intron-containing hairpin RNA in plants for gene silencing.

Double-stranded RNA-mediated RNA interference in plants involves generating a construct expressing intron-containing hairpin RNA (ihpRNA), which usually is a cumbersome, multistep process. Here, we describe a simplified method involving single steps of PCR, restriction, ligation, and transformation for assembling an ihpRNA construct for plant transformation. Our method has several advantages over the currently available ones, viz., wider choice of restriction sites and facility for rapid screening of positive clones, among others. We demonstrate the utility of this approach in assembling the tomato phytoene desaturase gene. This simplified DNA fragment assembly strategy for ihpRNA construction facilitates high-throughput gene silencing in plants.

Molecular characterization and genetic diversity of insecticidal crystal protein genes in native Bacillus thuringiensis isolates.

The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.

Bacillus thuringiensis isolates from Great Nicobar Islands.

Bacillus thuringiensis (Bt) strains were isolated from soil samples of Great Nicobar Islands, one of the "hottest biodiversity hotspots," where no collection has been characterized previously. The 36 new Bt isolates were obtained from 153 samples analyzed by crystal protein production with light/phase-contrast microscopy, determination of cry gene profile by SDS-PAGE, evaluation of toxicity against Coleopteran, and Lepidopteran insect pests, finally cloning and sequencing. Majority of the isolates showed the presence of 66-35 kDa protein bands on SDS-PAGE while the rest showed >130, 130, 73, and 18 kDa bands. The variations in crystal morphology and mass of crystal protein(s) purified from the isolates of Bt revealed genetic and molecular diversity. Based on the toxicity test, 50 % of isolates were toxic to Ash weevils, 16 % isolates were toxic to cotton bollworm, 38 % isolates were toxic both to ash weevil as well as cotton bollworm, while 11 % of the isolates did not exhibit any toxicity. PCR analysis unveiled prepotency of cry1B- and cry8b-like genes in these isolates. This study appoints the first isolation and characterization of local B. thuringiensis isolates in Great Nicobar Islands. Some of these isolates display toxic potential and, therefore, could be adopted for future applications to control some agriculturally important insect pests in the area of integrated pest management for sustainable agriculture.

Effect of diet delivered various concentrations of double-stranded RNA in silencing a midgut and a non-midgut gene of Helicoverpa armigera.

Ribonucleic acid interference (RNAi) is a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA). Recently, RNAi has gained popularity as a reverse genetics tool owing to its tremendous potential in insect pest management, which includes Helicoverpa armigera. However, its efficiency is mainly governed by dsRNA concentration, frequency of application, target gene, etc. Therefore, to obtain a robust RNAi response in H. armigera, we evaluated various concentrations of dsRNA and its frequency of applications delivered through diet in silencing a midgut gene, chymotrypsin and a non-midgut gene, juvenile hormone acid methyl transferase (jhamt) of H. armigera. The extent of target gene silencing was determined by employing reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Our study revealed four significant findings: (i) single application of dsRNA elicited a delayed and transient silencing, while multiple applications resulted in early and persistent silencing of the above genes; (ii) silencing of the non-midgut gene (jhamt) through diet delivered dsRNA revealed prevalence of systemic silencing probably due to communication of silencing signals in this pest; (iii) the extent of silencing of chymotrypsin was positively correlated with dsRNA concentration and was negatively correlated with jhamt; (iv) interestingly, over-expression (15­18 folds) of an upstream gene, farnesyl diphosphate synthase (fpps), in juvenile hormone (JH) biosynthetic pathway at higher concentrations of jhamt dsRNA was the plausible reason for lesser silencing of jhamt. This study provides an insight into RNAi response of target genes, which is essential for RNAi design and implementation as a pest management strategy.

DNA barcoding and elucidation of cryptic aphid species (Hemiptera: Aphididae) in India.

Rapid, precise and timely identification of invasive pest insects such as aphids is important and a challenge worldwide due to their complex life cycles, parthenogenetic reproduction, sex and colour morphs. In this respect, DNA barcoding employing a 658 bp fragment of 5' region of the mitochondrial cytochrome oxidase I (CO-I) gene is an effective tool in addressing the above. In the present study, we employed CO-I for discriminating 142 individuals representing 32 species of aphids from India. Sequence analyses revealed that the intraspecific and interspecific distances ranged from zero to 3.8% and 2.31 to 18.9%, respectively. In addition, the study also showed for the first time the prevalence of three cryptic species, namely Brevicoryne brassicae (Linnaeus), Hyperomyzus carduellinus (Theobald) and Brachycaudus helichrysi (Kaltenbach) from India. Our work has clearly demonstrated that DNA barcoding is an efficient and accurate method for identification of aphid species (including cryptic species), an approach that potentially could play an important role in formulating viable pest management strategies, more especially biocontrol.

Expression of cry3A gene and its toxicity against Asian Gray Weevil Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae).

Coleopterans are the most damaging pests of many agricultural and forestry crops; there is an urgent need to develop effective biopesticides against these insects. Enhancers of Bt toxicity typify an opportunity to improve currently available commercial products into more effective control agents against diverse pests. A 1.9 kb DNA fragment, PCR amplified from native isolates of Bt using cry3A gene specific primers was cloned in expression vector pQE-80L and then used for transformation of Escherichia coli M15 cells. The sequence of the cloned crystal protein gene showed almost complete homology with a Coleopteran active Cry3A toxin gene with 117 mutations scattered in different domain regions encoding a protein of 645 amino acid residues in length, with a predicted molecular mass of 77.4 kDa. Phylogenetic analysis could be compulsive for new/novel Bacillus thuringiensis strains, allowing them to be grouped with related Cry proteins. The toxicity of Bt protein was determined against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) LC50 152 ng cm(-2). Genes coding for Coleopteran active Cry3A proteins have been isolated and their efficient expression will provide the tools necessary to increase the efficacy of Cry-based biopesticide against economically important beetles.

Automated Brain Tumor Detection using Ideal Shallow Neural Network with Artificial Jellyfish Optimization.

INTRODUCTION: Brain tumors are predicted from Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) scan images. In recent years, image processing-based automated tools are developed to predict tumor areas with less human interference. However, such automated tools are suffering from computational complexity and reduced accuracy in certain critical images. In the proposed work, an Ideal Shallow Neural Network (ISNN) is utilized to improve the prediction accuracy, and the computational complexity is reduced by implementing an Artificial Jellyfish Optimization (AJO) algorithm for minimizing the feature dimensionality. METHOD: The proposed method utilizes MRI images for the verification process as they are more informative than the CT scan image. The BRATS and the Kaggle datasets are used in this work and a Gabor filtering technique is used for noise reduction and a histogram equalization is used for enhancing the tumor boundary regions. The classification results observed from the AJO-ISNN are further forwarded towards the segmentation process and which uses the Centroid Weighted Segmentation (WCS) along with a Grasshopper Optimization Algorithm (GOA) for improving the segmentation over the boundary regions of the brain tumor. RESULT: The experimental result indicates a classification accuracy of 95.14% on the proposed AJO-ISNN model and AJO-ISNN is comparatively better than the Convolutional Neural Network (CNN) model accuracy of 85.41% and VGG 19 model accuracy of 93.75% while implemented with the AJO optimization model. Similarly, the Dice Similarity Coefficient of the proposed CWS-GOA also reaches 93.15% when performed with both BRATS and Kaggle datasets. CONCLUSION: Apart from the accuracy attainments the proposed work classifies and segments the tumor region in around 65 seconds on average of 200 image verifications and that is comparatively better than the previous multi-cascaded CNN and the InceptionV3 models.

Molecular characterization of the Indian isolate (Ka-To) of tomato spotted wilt virus (TSWV) infecting tomato (Solanum lycopersicum L.).

Tomato spotted wilt virus (TSWV) infecting tomato has been identified as an emerging constraint for tomato cultivation in the southern Indian states of Karnataka and Tamil Nadu. Infection of TSWV produces circular necrotic ring spots on leaves, stem and floral necrosis and necrotic ringspots on fruits of tomato. In this study, we describe the characterization of TSWV isolate (Ka-To) infecting tomato from India based on biological, serological and molecular assay. Pathogenicity of TSWV (Ka-To) isolate was established by mechanical inoculation of sap from infected leaves on tomato, cowpea and datura which expressed necrotic or chlorotic local lesions. Samples were tested positive in the serological assay performed with TSWV-specific immunostrips. Further, reverse transcription polymerase chain reaction (RT-PCR) amplification of coat protein gene followed by sequencing, unequivocally confirmed the identity of TSWV. The obtained full-length nucleotide sequences of Ka-To isolate [L RNA-MK977648; M RNA-MK977649; and S RNA-MK977650] had greater similarity to the TSWV isolates of Spain and Hungary infecting tomato and pepper. The phylogenetic and recombination analysis showed the evidence for reassortment and recombination in the genome of Ka-To isolate. To the best of our knowledge, this is the first confirmed evidence for the occurrence of TSWV on tomato in India. Information obtained in this study issues a forewarning on the emergence of TSWV on vegetable ecosystem in the Indian subcontinent, requiring urgent management strategies to curtail its pestilence. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03579-y.

Modified competing polymerase chain reaction primer for single tube quantitative PCR.

We developed a modified polymerase chain reaction (PCR) primer with 3' phosphate instead of hydroxyl group for single-tube accurate transcript quantification. 18S ribosomal RNA (rRNA) reference gene-specific modified primer was used for precise single-tube quantification of two target transcripts, namely chymotrypsin and jhamt (juvenile hormone acid methyl transferase) of Helicoverpa armigera. A comparative study of 3' phosphorylated primers, 3' mismatched primers, and commercial Competimers revealed that 3' phosphorylation was more efficient than the 3' mismatch and was on par with Competimers in blocking the primer extension. Thus, the modified primers can be used in single-tube, economical, and accurate PCR quantification of the target gene using any assay-specific reference gene.

Screening, diversity and partial sequence comparison of vegetative insecticidal protein (vip3A) genes in the local isolates of Bacillus thuringiensis Berliner.

Characterization, direct sequencing of the PCR amplicon and phylogenetic relationship was done to discover a novel Vip protein genes of the Bt isolates, to improve the prospects for insect control, more Vip proteins should be sought out and researched to predict their insecticidal activity. Characterization was based on direct sequencing of PCR amplicon using primers specific to vip3A gene was presented here. 12 out of 18 isolates screened were positive for vip gene-specific primers. Homology search for the partial sequences using BLAST showed that 11 isolates had high similarity to vip3Aa gene and only one fragment with vip3Ae gene (25-100% at nucleotide and amino acid level). Phylogenetic analysis showed that the gene sequences were responsible for geographic separation for divergence within vip genes, consistent with the evaluation of distinct bacterial population. Despite the geographical distances, strains harbouring vip genes have originated from common ancestors may significantly contribute to control resistant insect pests. Some strains have evolved to be quite distinct and others remain as members of closely related groups. The reported method is a powerful tool to find novel Vip3A proteins from large-scale Bt strains which is effective in terms of time and cost. Further the Vip proteins produced by different strains of B. thuringiensis are unique in terms of the sequence divergence and hence may also differ in their insecticidal activities.

The evolutionary process of invasion in the fall armyworm (Spodoptera frugiperda).

The fall armyworm (FAW; Spodoptera frugiperda) is one of the major agricultural pest insects. FAW is native to the Americas, and its invasion was first reported in West Africa in 2016. Then it quickly spread through Africa, Asia, and Oceania, becoming one of the main threats to corn production. We analyzed whole genome sequences of 177 FAW individuals from 12 locations on four continents to infer evolutionary processes of invasion. Principal component analysis from the TPI gene and whole genome sequences shows that invasive FAW populations originated from the corn strain. Ancestry coefficient and phylogenetic analyses from the nuclear genome indicate that invasive populations are derived from a single ancestry, distinct from native populations, while the mitochondrial phylogenetic tree supports the hypothesis of multiple introductions. Adaptive evolution specific to invasive populations was observed in detoxification, chemosensory, and digestion genes. We concluded that extant invasive FAW populations originated from the corn strain with potential contributions of adaptive evolution.