Extraction, Chemical Composition and Insecticidal Activities of Lantana camara Linn. Leaf Essential Oils against Tribolium castaneum, Lasioderma serricorne and Callosobruchus chinensis.

Storage pests and the food spoilage they cause are problems of great concern. Using essential oil obtained from different plants as an insecticide against these storage pests can be considered an environmentally friendly pest management option. Lantana camara Linn. (family Verbenaceae) is a flowering species, and is also a noxious weed that can proliferate well in nearly all geographical habitats. A biopesticide derived from the essential oil extracted from this plant can offer an effective solution for controlling storage pests. The goal of this study is to extract and analyse the chemical composition of essential oil obtained from L. camara leaves, and assess its effectiveness as a bioactive substance against three storage pests: Tribolium castaneum, Lasioderma serricorne, and Callosobruchus chinensis. The yield of essential oil extracted from L. camara leaves was about 0.24 ± 0.014%. By employing the GC-MS technique, the major phytochemicals contained in L. camara leaf essential oil were identified as caryophyllene (69.96%), isoledene (12%), and ɑ-copaene (4.11%). The essential oil exhibited excellent fumigant toxicity (LC50 of 16.70 mg/L air for T. castaneum, 4.141 mg/L air for L. serricorne and 6.245 mg/L air for C. chinensis at 24 h), contact toxicity (LC50 of 8.93 mg/cm2 for T. castaneum, 4.82 mg/cm2 for L. serricorne and 6.24 mg/cm2 for C. chinensis after 24 h) along with effective repellent activity towards the test insects. In addition, the oil showed no significant phytotoxicity on the germination of paddy seeds. This presents the potential to utilize a weed in developing a biopesticide for effectively managing stored product insects because of its strong bioactivity.

Ligand-binding properties of chemosensory protein 1 in Callosobruchus chinensis to mung bean volatiles.

Callosobruchus chinensis (Coleoptera: Fabaceae) is a worldwide pest that feeds exclusively on legumes, and is the most serious pest affecting mung beans. Usually, the insect olfactory system plays a predominant role in searching for host plants and egg-laying locations. Chemosensory proteins (CSPs), are mainly responsible for transporting specific odour molecules from the environment. In this study, we found that the CSP1 gene of adult C. chinensis displayed antennae-biased expression using quantitative real-time PCR (qRT-PCR) analysis. The binding properties of 23 mung bean volatiles were then determined through several analyses of in vitro recombinant CSP1 protein, including fluorescence competitive binding assay, homology modelling, molecular docking, and site-directed mutagenesis. Fluorescence competitive binding assays showed that CchiCSP1 protein could bind to four mung bean volatiles and was most stable at pH 7.4. After site-directed mutation of three key amino acid bases (L39, V25, and Y35), their binding affinities to each ligand were significantly decreased or lost. This indicated that these three amino acid residues may be involved in the binding of CchiCSP1 to different ligands. We further used Y-tube behavioural bioassays to find that the four mung bean volatiles had a significant attraction or repulsion response in adult C. chinensis. The above findings confirm that the CchiCSP1 protein may be involved in the response of C. chinensis to mung bean volatiles and plays an important role in olfactory-related behaviours. The four active volatiles are expected to develop into new behavioural attractants or repellents in the future.

Comparative transcriptome analysis of Callosobruchus chinensis (L.) (Coleoptera: Chrysomelidae-Bruchinae) after heat and cold stress exposure.

Callosobruchus chinensis is regarded as one of the cosmopolitan pests of legume crops and can cause tremendous losses to a variety of beans. This study focused on comparative transcriptome analyses of C. chinensis exposed to 45 °C (heat stress), 27 °C (ambient temperature) and -3 °C (cold stress) for 3 h to investigate the gene differences and underlying molecular mechanisms. There were 402 and 111 differentially expressed genes (DEGs) identified in the heat and cold stress treatments, respectively. "cell process", "cell" and "binding" were the main enriched functions and biological processes revealed by gene ontology (GO) analysis. The clusters of orthologous genes (COG) showed that DEGs were assigned to the categories: "posttranslational modification, protein turnover, chaperones", "lipid transport and metabolism", and "general function prediction only". With respect to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the "longevity regulating pathway-multiple species", "carbon metabolism", "peroxisome", "protein processing in endoplasmic", "glyoxylate and dicarboxylate metabolism" pathways were significantly enriched. The annotation and enrichment analysis revealed that genes encoding heat shock proteins (Hsps) and cuticular proteins were significantly upregulated under high and low-temperature stresses, respectively. In addition, some DEGs encoding "Protein lethal essential for life", "Reverse transcriptase", "DnaJ domain", "Cytochrome" and "Zinc finger protein" were also upregulated to varying degrees. Transcriptomic data were validated using qRT‒PCR, which confirmed that they were consistent. In this paper, the temperature tolerance of C. chinensis adults was evaluated and the results showed that female adults were more sensitive to heat and cold stress than males, and the upregulation of heat shock protein and epidermal protein was the largest in DEGs after heat and cold stress, respectively. These findings provide a reference for further understanding the biological characteristics of C. chinensis adults and the molecular mechanisms underlying the response to low and high temperatures.

Identify immune-related genes of adzuki bean weevil (Callosobruchus chinensis) in response to bacteria challenge by transcriptome analysis.

BACKGROUND: Callosobruchus chinensis is one of the important postharvest pests in legume growing areas. Bacterial pesticide is a potential alternative method to control storage pests. However, the effect of these pathogen bacteria on storage pests, and the molecular mechanisms of insect response remain to be to investigated. RESULTS: Using the next generation sequencing technology, we established a transcriptomic library for C. chinensis larvae in response to Escherichia coli. Total of 355 differential expressed genes (DEGs) were identified, which 178 DEGs were upregulated, and 177 DEGs were downregulated compared to control group. To validate the RNA-seq analysis, 20 DEGs and 14 immune-related genes were selected to perform quantitative polymerase chain reaction (RT-qPCR). These immune-related genes were involved in recognition (peptidoglycan recognition proteins), signal transduction (fibrinogen-related proteins, serine proteinases and NF-κB), and execution effectors (phenoloxidase, defensin, attacin, and antimicrobial peptide). In addition, genes that encode digestive and respiratory enzymes were altered in C. chinensis larvae in response to infection. Some genes that involved in juvenile hormone and insulin pathway appeared to express differentially, suggesting that pathogen infection might lead to developmental arrest. Furthermore, iron homeostasis and chitin metabolism appeared significantly altered after infection. CONCLUSION: In this study, we characterized the immune response of C. chinensis larvae in response to E. coli using RNA-seq, from pathogen recognition, signal transduction, to execution. Some other identified genes were involved in iron homeostasis, respiration, and digestion. A better understanding of molecular response of beetle to pathogen will facilitate us to develop an available strategy to control storage pests.

Pesticidal efficacy, mode of action and safety limits profile of essential oils based nanoformulation against Callosobruchus chinensis and Aspergillus flavus.

The study explores the pesticidal efficacy, mode of action, and safety limit profile of essential oils-based formulation using the combination of Myristica fragrans (M), Bunium persicum (B), and Zanthoxylum alatum (Z) (1:1:1 v/v/v) and their nanoformulation (Ne-MBZ) against the Callosobruchus chinensis, Aspergillus flavus and aflatoxin B1 production. Linalool, γ-terpinene, and cuminaldehyde were identified as the major compounds of the formulation (MBZ) by Gas chromatography-mass spectrometry (GC-MS). Nanoencapsulation of developed formulation (Ne-MBZ) was prepared using chitosan and characterized by scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), and Fourier transform infrared spectroscopy (FTIR). The pesticidal efficacy of nanoformulation (Ne-MBZ) against C. chinensis IC50 (0.14 µl/ml), A. flavus (0.8 µl/ml) and AFB1 (0.8 µl/ml) was significantly higher in both in-vitro and in-situ conditions than the sum of their individual revealing a notable synergistic effect. Besides, the detailed mode of pesticidal action and safety limit profile were explored using biochemical, in-silico and ADMET (absorption, distribution, metabolism, excretion, and toxicity) approaches.

Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species.

The smallest 32 amino acid α-amylase inhibitor from Amaranthus hypochondriacus (AAI) is reported. The complete gene of pre-protein (AhAI) encoding a 26 amino acid (aa) signal peptide followed by the 43 aa region and the previously identified 32 aa peptide was cloned successfully. Three cysteine residues and one disulfide bond conserved within known α-amylase inhibitors were present in AhAI. Identical genomic and open reading frame was found to be present in close relatives of A. hypochondriacus namely Amaranthus paniculatus, Achyranthes aspera and Celosia argentea. Interestingly, the 3'UTR of AhAI varied in these species. The highest expression of AhAI was observed in A. hypochondriacus inflorescence; however, it was not detected in the seed. We hypothesized that the inhibitor expressed in leaves and inflorescence might be transported to the seeds. Sub-cellular localization studies clearly indicated the involvement of AhAI signal peptide in extracellular secretion. Full length rAhAI showed differential inhibition against α-amylases from human, insects, fungi and bacteria. Particularly, α-amylases from Helicoverpa armigera (Lepidoptera) were not inhibited by AhAI while Tribolium castaneum and Callosobruchus chinensis (Coleoptera) α-amylases were completely inhibited. Molecular docking of AhAI revealed tighter interactions with active site residues of T. castaneum α-amylase compared to C. chinensis α-amylase, which could be the rationale behind the disparity in their IC50. Normal growth, development and adult emergence of C. chinensis were hampered after feeding on rAhAI. Altogether, the ability of AhAI to affect the growth of C. chinensis demonstrated its potential as an efficient bio-control agent, especially against stored grain pests.

Genetic differentiation and diversity of Callosobruchus chinensis collections from China.

Callosobruchus chinensis (Linnaeus) is one of the most destructive pests of leguminous seeds. Genetic differentiation and diversity analysis of 345 C. chinensis individuals from 23 geographic populations using 20 polymorphic simple sequence repeats revealed a total of 149 alleles with an average of 7.45 alleles per locus. The average Shannon's information index was 1.015. The gene flow and genetic differentiation rate values at the 20 loci ranged from 0.201 to 1.841 and 11.0-47.2%, with averages of 0.849 and 24.4%, respectively. In the 23 geographic populations, the effective number of alleles and observed heterozygosity ranged from 1.441 to 2.218 and 0.191-0.410, respectively. Shannon's information index ranged from 0.357 to 0.949, with the highest value in Hohhot and the lowest in Rudong. In all comparisons, the fixation index (F ST ) values ranged from 0.049 to 0.441 with a total F ST value of 0.254 among the 23 C. chinensis populations, indicating a moderate level of genetic differentiation and gene flow among these populations. Analysis of molecular variance revealed that the genetic variation within populations accounted for 76.7% of the total genetic variation. The genetic similarity values between populations varied from 0.617 to 0.969, whereas genetic distances varied from 0.032 to 0.483. Using unweighted pair-group method using arithmetical averages cluster analysis, the 23 geographic collections were classified into four distinct genetic groups but most of them were clustered into a single group. The pattern of the three concentrated groups from polymerase chain reactions analysis showed a somewhat different result with cluster.

Resistance of Faba Bean and Pea Germplasm to Callosobruchus chinensis (Coleoptera: Bruchidae) and Its Relationship With Quality Components.

In total, 339 faba bean (Vicia faba L.) and 100 pea (Pisum sativum L.) accessions were screened for their ability to resist Callosobruchus chinensis L. in free choice laboratory tests. Four, 15, and 43 faba bean varieties were highly resistant, resistant, and moderately resistant to C. chinensis, respectively. Three immune, three highly resistant, and six resistant accessions were discovered among the pea germplasm. The faba bean and pea varieties presented a hundred-kernel weight reduction varied from 0.18 to 35.36% for faba bean varieties and 0 to 56.53% for pea varieties. Varieties with brown and black seed color had significantly fewer wormholes and higher C. chinensis resistance than varieties with light-color seeds. Resistance to C. chinensis showed a significant, positive correlation with catechin, total polyphenol, and γ-aminobutyric acid contents, but a significant, negative correlation with oligosaccharide content. Correlation coefficients (r) between infestation rate of faba bean and total phenol, catechin, and oligosaccharide contents were -0.9723, -0.8071, and 0.7631, respectively. The values of r for pea resistance and total phenol, catechin, and oligosaccharide content were -0.8846, -0.7666, and 0.8308, respectively. The results suggest that quality components in faba bean and pea have a great role in resistance against C. chinensis.

Leaves of Lantana camara Linn. (Verbenaceae) as a potential insecticide for the management of three species of stored grain insect pests.

Insects cause extensive damage to stored grains and their value added products. Among the stored grain pests Sitophilus oryzae (L.) Callosobruchus chinensis (Fab.) and Tribolium castaneum (Herbst.) are considered as destructive pests in India. Plants may provide alternatives to currently used insect control agents as they constitute rich source in bioactive molecules. Lantana camara, an erect shrub, which grows widely in the tropics, exhibits insecticidal activity against several insects. The methanol extract from leaves of L. camara has fumigant and contact toxicity against S. oryzae, C. chinesis and T. castaneum. In fumigant assays, The LC50 for S. oryzae was 128 µl/L(1), C. chinensis 130.3 µl/L(1), and T. castaneum 178.7 µl/L(1). The LD50 values for S. oryzae C. chinensis and T. castaneum in contact toxicity were 0.158, 0.140 and 0.208 mg/cm(2), respectively. For grain treatment, a concentration of 500 mg/L(1) and 7 days exposure were needed to obtain 90 - 100 % population extinction in all three insects. Probit analysis showed that C. chinensis were more susceptible than S. oryzae and T. castaneum. Gaschromatography-Mass Spectrometer (GCMS) studies for extracts indicated the presence of potent fumigant molecules in L. camara. The prospect of utilizing L. camara as potent fumigant insecticide is discussed.

Purification of a novel α-amylase inhibitor from local Himalayan bean (Phaseolus vulgaris) seeds with activity towards bruchid pests and human salivary amylase.

Six bean (Phaseolus vulgaris L.) cultivars of Himalayan region were analysed for α- amylase inhibitor activity. The α-amylase inhibitor from seeds of screened bean cultivar KR-9, showing maximum inhibitory activity was purified using ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-100) and ion exchange chromatography (DEAE-Sephadex). The inhibitor was purified to homogeneity as judged by native-PAGE with 14.22 fold purification and 71.66% recovery. Purified inhibitor consisted of three subunits of molecular weight 15,488, 18,620 and 26,302 daltons, respectively as determined by SDS-PAGE. It was found to be heat stable up to 30 °C-40 °C and had two pH optima of 5.0 and 6.9. Nature of inhibition was found to be of non-competitive type. The purified inhibitor was found to be effective against α-amylases extracted from larvae of Callosobruchus chinensis, Tribolium castaneum and gut enzyme of Spodoptera littoralis. Larvae of Tribolium castaneum fed on flour mixed with purified inhibitor for 5 days showed 100% larval mortality. Purified α-amylase inhibitor was also found to inhibit human salivary α-amylase, suggesting its potential in prevention and therapy of obesity and use as drug design targets for treatment of diabetes. The gene encoding the inhibitor may be used to develop transgenic plants resistant against insect pests.

Insecticidal property of Ocimum essential oil embedded polylactic acid packaging films for control of Sitophilus oryzae and Callosobruchus chinensis.

Naturally derived essential oils (EOs) from Ocimum basilicum (OB) and Ocimum gratissimum (OG) were incorporated in polylactic acid (PLA) matrix with concentration of 2.5 % and 5 % respectively by solvent casting method, with an aim to develop insecticidal polymer pouches (insect proof polymer pouches). The major components following GC-MS analysis revealed the presence of EOs as estragole (69.998 %) in OB and thymol (29.843 %) in OG. The developed films with EOs showed remarkable insecticidal activity with 100 % and 80 % mortality rate for OB and OG respectively at 2.5 % concentration against rice weevil (Sitophilus oryzae). While, both OB and OG EOs observed 100 % mortality against pulse beetle (Callosobruchus chinensis) after 96 h exposure in presence of grain conditions and the insecticidal efficacy was equivalent to the commercial hermetic bags (CSB). The physical property of film showed increase in the film thickness in EO-PLA films whereas the mechanical properties of EO-PLA films matrix have increased the polymer flexibility with decreasing tensile strength (TS) and Youngs modulus (EM) as TS OB 5 %, OG 5 % - 15.64 MPa, 17.66 MPa respectively. While, water vapour transmission rate (WVTR) was reduced to 0.015 g/m2.hr, optical characteristics showed slight significant change in colour and FTIR spectra exhibited a change in -OH stretching compared to blank PLA matrix. The surface morphology of the blank and EO embedded PLA films visualized by SEM depicted a complete homogenization of essential oils in the PLA matrix.

Screening of Comprehensive Panel of Cultivated and Wild Vigna Species for Resistance to Pulse Beetle, Callosobruchus chinensis L.

Pulses are a key source of dietary proteins in human nutrition. Despite several efforts to increase the production, various constraints, such as biotic and abiotic factors, threaten pulse production by various means. Bruchids (Callosobruchus spp.) are the serious issue of concern, particularly in storage conditions. Understanding host-plant resistance at morphological, biochemical and molecular levels is the best way to minimize yield losses. The 117 mungbean (Vigna radiata L. Wilczek) genotypes, including endemic wild relatives, were screened for resistance against Callosobruchus chinensis; among them, two genotypes, PRR 2008-2 and PRR 2008-2-sel, which belong to V. umbellata (Thumb.), were identified as highly resistant. The expression of antioxidants in susceptible and resistant genotypes revealed that the activity of phenylalanine ammonia lyase (PAL) was upregulated in the highly resistant wild Vigna species and lower in the cultivated susceptible genotypes, along with other biomarkers. Further, the SCoT-based genotyping revealed SCoT-30 (200 bp), SCoT-31 (1200 bp) and SCoT-32 (300 bp) as unique amplicons, which might be useful for developing the novel ricebean-based SCAR markers to accelerate the molecular breeding programme.

Screening of behaviorally active compounds based on the interaction between two chemosensory proteins and mung bean volatiles in Callosobruchus chinensis.

Chemosensory proteins (CSPs) are involved in the earliest steps of the olfactory process by binding and transporting odorants and play a crucial role in the insect's search for food and egg-laying sites. In the present study, the tissue expression profiles showed that both CchiCSP3 and CchiCSP5 of Callosobruchus chinensis were highly expressed in the adult antennae. Subsequently, the recombinant CchiCSP3 and CchiCSP5 proteins were analysed using fluorescence competitive binding assays, and both showed binding affinities for the three mung bean volatiles. Molecular docking and site-directed mutagenesis revealed four key amino acid residues in CchiCSP3 (L47, W80, Y81, and L84) and CchiCSP5 (Y28, K46, L49, and I72). Electroantennogram (EAG) and dual-choice biobehavioral assays showed that the antennae of adult C. chinensis were electrophysiologically active in response to stimulation with all three behaviorally active compounds and that octyl 4-methoxycinnamate and ß-ionone had a significant luring effect on adult C. chinensis, whereas vanillin had a significant avoidance effect. Our study screened three effective behaviorally active compounds based on the involvement of two CchiCSPs in the recognition of mung bean volatiles, providing an opportunity to develop an alternative control strategy using behavioral disruptors to limit the impact of pests.

A Phylogeographical Analysis of the Beetle Pest Species Callosobruchus chinensis (Linnaeus, 1758) in China.

Callosobruchus chinensis (Coleoptera Bruchidae), is a pest of different varieties of legumes. In this paper, a phylogeographical analysis of C. chinensis was conducted to provide knowledge for the prevention and control of C. chinensis. A total of 224 concatenated mitochondrial sequences were obtained from 273 individuals. Suitable habitat shifts were predicted by the distribution modelling (SDM). Phylogeny, genetic structure and population demographic history were analyzed using multiple software. Finally, the least-cost path (LCP) method was used to identify possible dispersal corridors and genetic connectivity. The SDM results suggested that the distribution of C. chinensis experienced expansion and contraction with changing climate. Spatial distribution of mtDNA haplotypes showed there was partial continuity among different geographical populations of C. chinensis, except for the Hohhot (Inner Mongolia) population. Bayesian skyline plots showed that the population had a recent expansion during 0.0125 Ma and 0.025 Ma. The expansion and divergent events were traced back to Quaternary glaciations. The LCP method confirmed that there were no clear dispersal routes. Our findings indicated that climatic cycles of the Pleistocene glaciations, unsuitable climate and geographic isolation played important roles in the genetic differentiation of C. chinensis. Human activities weaken the genetic differentiation between populations. With the change in climate, the suitable areas of C. chinensis will disperse greatly in the future.

Experimental evolution of competing bean beetle species reveals long-term reversals of short-term evolution, but no consistent character displacement.

Interspecific competition for shared resources should select for evolutionary divergence in resource use between competing species, termed character displacement. Many purported examples of character displacement exist, but few completely rule out alternative explanations. We reared genetically diverse populations of two species of bean beetles, Callosobruchus maculatus and Callosobruchus chinensis, in allopatry and sympatry on a mixture of adzuki beans and lentils, and assayed oviposition preference and other phenotypic traits after four, eight, and twelve generations of (co)evolution. C. maculatus specializes on adzuki beans; the generalist C. chinensis uses both beans. C. chinensis growing in allopatry emerged equally from both bean species. In sympatry, the two species competing strongly and coexisted via strong realized resource partitioning, with C. chinensis emerging almost exclusively from lentils and C. maculatus emerging almost exclusively from adzuki beans. However, oviposition preferences, larval survival traits, and larval development rates in both beetle species did not vary consistently between allopatric versus sympatric treatments. Rather, traits evolved in treatment-independent fashion, with several traits exhibiting reversals in their evolutionary trajectories. For example, C. chinensis initially evolved a slower egg-to-adult development rate on adzuki beans in both allopatry and sympatry, then subsequently evolved back toward the faster ancestral development rate. Lack of character displacement is consistent with a previous similar experiment in bean beetles and may reflect lack of evolutionary trade-offs in resource use. However, evolutionary reversals were unexpected and remain unexplained. Together with other empirical and theoretical work, our results illustrate the stringency of the conditions for character displacement.

Laboratory evaluation of seven pakistani strains of entomopathogenic nematodes against a stored grain insect pest, Pulse beetle Callosobruchus chinensis (L.).

Seven Pakistani strains of entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis were tested against last instar and adult stages of the pulse beetle, Callosobruchus chinensis (L.). These nematodes included Steinernema pakistanense Shahina, Anis, Reid and Maqbool (Ham 10 strain); S. asiaticum Anis, Shahina, Reid and Rowe (211 strain); S. abbasi Elawad, Ahmad and Reid (507 strain); S. siamkayai Stock, Somsook and Reid (157 strain); S. feltiae Filipjev (A05 strains); Heterorhabditis bacteriophora Poinar (1743 strain); and H. indica Poinar, Karunakar and David (HAM-64 strain). Activity of all strains was determined at four different nematode densities in Petri dishes and in concrete containers. A significant nematode density effect was detected for all nematode species tested. Overall, Heterorhabditis bacteriophora, S. siamkayai, and S. pakistanense were among those that showed the highest virulence to pulse beetle larvae and adults. For all nematode species, the last larval stage of the pulse beetle seems to be more susceptible than the adult. LC(50) values in Petri dish and concrete containers were 14-340 IJs/larvae and 41-441 IJs/larvae, respectively, and 59-1376 IJs/adult and 170-684/adult, respectively.

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To clarify the effects of protease inhibitors on the activities of midgut proteases in Callosobruchus chinensis (L.) larvae, the inhibitory effects of four kinds of protease inhibitors on the activities of midgut proteases of C. chinensis larvae were examined in vitro and in feeding conditions with indoor artificial insect inoculation. The activities of total protease, trypsin-like enzyme and chymotrypsin-like enzyme in the midgut of C. chinensis larvae fed on artificial mung bean that contained different contents of mung bean types in inhibitor (MBTI) were examined. The results showed that those four protease inhibitors had significant inhibitory effects on the activities of total protease, trypsin-like enzyme and chymotrypsin-like enzyme in C. chinensis larvae. The inhibitory effect was more significant when the concentration was higher. Among those inhibitors, the inhibitory effect of 20 µg·mL-1 MBTI on three kinds of enzyme activities was the strongest, with reduction of 62.5%, 41.2% and 38.7%, respectively. Ovomucoid inhibitor (OI) had the lowest inhibitory effect. The activities of three enzymes in the midgut were also inhibited by C. chinensis larvae fed with artificial mung bean containing different inhibitors. The activities of three enzymes increased with the prolongation of the instar after feeding, but they were significantly lower than that in control. The inhibitory effect of MBTI was the strongest. The inhibitory effect on the activities of total protease and trypsin-like enzyme was gradually enhanced with the increases of MBTI contents when C. chinensis larvae were fed with artificial mung bean with different contents MBTI. The inhibitory effect on chymotrypsin-like enzyme activity was not significant. When the content of MBTI was up to 20%, the activity of chymotrypsin-like enzyme was obviously inhibited.

Coevolution of competing Callosobruchus species does not stabilize coexistence.

Interspecific resource competition is expected to select for divergence in resource use, weakening interspecific relative to intraspecific competition, thus promoting stable coexistence. More broadly, because interspecific competition reduces fitness, any mechanism of interspecific competition should generate selection favoring traits that weaken interspecific competition. However, species also can adapt to competition by increasing their competitive ability, potentially destabilizing coexistence. We reared two species of bean beetles, the specialist Callosobruchus maculatus and the generalist C. chinensis, in allopatry and sympatry on a mixture of adzuki beans and lentils, and assayed mutual invasibility after four, eight, and twelve generations of evolution. Contrary to the expectation that coevolution of competitors will weaken interspecific competition, the rate of mutual invasibility did not differ between sympatry and allopatry. Rather, the invasion rate of C. chinensis, but not C. maculatus, increased with duration of evolution, as C. chinensis adapted to lentils without experiencing reduced adaptation to adzuki beans, and regardless of the presence or absence of C. maculatus. Our results highlight that evolutionary responses to interspecific competition promote stable coexistence only under specific conditions that can be difficult to produce in practice.

Characterization of two coleopteran α-amylases and molecular insights into their differential inhibition by synthetic α-amylase inhibitor, acarbose.

Post-harvest insect infestation of stored grains makes them unfit for human consumption and leads to severe economic loss. Here, we report functional and structural characterization of two coleopteran α-amylases viz. Callosobruchus chinensis α-amylase (CcAmy) and Tribolium castaneum α-amylase (TcAmy) along with their interactions with proteinaceous and non-proteinaceous α-amylase inhibitors. Secondary structural alignment of CcAmy and TcAmy with other coleopteran α-amylases revealed conserved motifs, active sites, di-sulfide bonds and two point mutations at spatially conserved substrate or inhibitor-binding sites. Homology modeling and molecular docking showed structural differences between these two enzymes. Both the enzymes had similar optimum pH values but differed in their optimum temperature. Overall, pattern of enzyme stabilities were similar under various temperature and pH conditions. Further, CcAmy and TcAmy differed in their substrate affinity and catalytic efficiency towards starch and amylopectin. HPLC analysis detected common amylolytic products like maltose and malto-triose while glucose and malto-tetrose were unique in CcAmy and TcAmy catalyzed reactions respectively. At very low concentrations, wheat α-amylase inhibitor was found to be superior over the acarbose as far as complete inhibition of amylolytic activities of CcAmy and TcAmy was concerned. Mechanism underlying differential amylolytic reaction inhibition by acarbose was discussed.