Novel Inhibitor of Glutamate-Cysteine Ligase Catalytic Subunit against Tribolium castaneum: High-Throughout Virtual Screening, Molecular Docking and Dynamics Simulation, and Bioassay.

The enzyme glutamate-cysteine ligase catalytic subunit (Gclc) is a rate-limiting enzyme in the biosynthesis of glutathione that is involved in antioxidant defense, detoxification of xenobiotics, and/or its metabolites and regulates the cell cycle and immune function. Therefore, Gclc presents an appealing target for the development of novel insecticides. In this study, we conducted high-throughput virtual screening from the ZINC20 database and identified three compounds with high binding affinity to the Tribolium castaneum Gclc (TcGclc). Ultimately, we selected ZINC000032992384 due to its superior stability and lowest binding energy, as determined through molecular dynamics simulations. Bioassay results revealed that the IC50 value of ZINC000032992384 was 19.70 µM lower than that of BSO (49.67 µM). Furthermore, the larval mortality in the ZINC000032992384 treated group was 63.8%, significantly higher than that of the controls (29.1% in the dichlorvos group and 6.4% in the acetone group). This study provides novel insights for the development of a Gclc-targeted inhibitor as a potent insecticide based on the interaction between receptors and ligands.

Increasing protein identifications in bottom-up proteomics of T. castaneum - Exploiting synergies of protein biochemistry and bioinformatics.

Depending on the respective research question, LC-MS/MS based bottom-up proteomics poses challenges from the initial biological sample all the way to data evaluation. The focus of this study was to investigate the influence of sample preparation techniques and data analysis parameters on protein identification in Tribolium castaneum by applying free software proteomics platform Max Quant. Multidimensional protein extraction strategies in combination with electrophoretic or chromatographic off-line protein pre-fractionation were applied to enhance the spectrum of isolated proteins from T. castaneum and reduce the effect of co-elution and ion suppression effects during nano-LC-MS/MS measurements of peptides. For comprehensive data analysis, MaxQuant was used for protein identification and R for data evaluation. A wide range of parameters were evaluated to gain reproducible, reliable, and significant protein identifications. A simple phosphate buffer, pH 8, containing protease and phosphatase inhibitor cocktail and application of gentle extraction conditions were used as a first extraction step for T.castaneum proteins. Furthermore, a two-dimensional extraction procedure in combination with electrophoretic pre-fractionation of extracted proteins and subsequent in-gel digest resulted in almost 100% increase of identified proteins when compared to chromatographic fractionation as well as one-pot-analysis. The additionally identified proteins could be assigned to new molecular functions or cell compartments, emphasizing the positive effect of extended sample preparation in bottom-up proteomics. Besides the number of peptides during post-processing, MaxQuant's Match between Runs exhibited a crucial effect on the number of identified proteins. A maximum relative standard deviation of 2% must be considered for the data analysis. Our work with Tribolium castaneum larvae demonstrates that sometimes - depending on matrix and research question - more complex and time-consuming sample preparation can be advantageous for isolation and identification of additional proteins in bottom-up proteomics.

Effects of defect action level of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) fragments on quality of wheat flour.

BACKGROUND: Tribolium castaneum (Herbst) is one of the most important secondary storage pests of all types of flour and flour-based products. The present study focuses on the fragment producing behaviour of T. castaneum in wheat flour during storage and its effect on the quality parameters and defect action level (DAL) of fragments. The US Food and Drug Administration has set a DAL of 75 insect fragments in 50 g of flour. Box-Behnken design was used to optimize the storage conditions (storage period in days and temperature in degrees Celsius) and insect density (numbers) to keep insect fragments below the DAL. RESULTS: Optimization results indicated that the presence of single number of adult of T. castaneum is enough to cross the DAL of insect fragments within a storage period of 21 days at a storage temperature of 30 °C. Insect fragments cause perceptible changes in the quality of wheat flour. When sample attained DAL of T. castaneum fragments in wheat flour,the various quality parameters were analysed in that moisture content of wheat flour was 10.8 ± 0.26%, total colour change was 2.052 (ΔE value), T. castaneum progeny emergence was 19.66 ± 1, uric acid was 1.8 ± 0.16 g kg-1 and microbial count was 7.34 ± 0.5 cfu g-1 . CONCLUSIONS: Results from the present study indicate that the presence of even a single adult of stored pest in wheat flour should not be ignored. It is mandatory to determine the threshold level and frequent sampling is required to achieve zero tolerance of stored product insects in food commodities. © 2021 Society of Chemical Industry.

Cuticular Hydrocarbon Trails Released by Host Larvae Lose their Kairomonal Activity for Parasitoids by Solidification.

Successful host search by parasitic wasps is often mediated by host-associated chemical cues. The ectoparasitoid Holepyris sylvanidis is known to follow chemical trails released by host larvae of the confused flour beetle, Tribolium confusum, for short-range host location. Although the hexane-extractable trails consist of stable, long-chain cuticular hydrocarbons (CHCs) with low volatility, the kairomonal activity of a trail is lost two days after release. Here, we studied whether this loss of kairomonal activity is due to changes in the chemical trail composition induced by microbial activity. We chemically analyzed trails consisting of hexane extracts of T. confusum larvae after different time intervals past deposition under sterile and non-sterile conditions. GC-MS analyses revealed that the qualitative and quantitative pattern of the long-chain CHCs of larval trails did not significantly change over time, neither under non-sterile nor sterile conditions. Hence, our results show that the loss of kairomonal activity of host trails is not due to microbially induced changes of the CHC pattern of a trail. Interestingly, the kairomonal activity of trails consisting of host larval CHC extracts was recoverable after two days by applying hexane to them. After hexane evaporation, the parasitoids followed the reactivated host trails as they followed freshly laid ones. Cryo-scanning electron microscopy showed that the trails gradually formed filament-shaped microstructures within two days. This self-assemblage of CHCs was reversible by hexane application. Our study suggests that the long-chain CHCs of a host trail slowly undergo solidification by a self-assembling process, which reduces the accessibility of CHCs to the parasitoid's receptors as such that the trail is no longer eliciting trail-following behavior.

Synthesis, Quantification, and Characterization of Fatty Acid Amides from In Vitro and In Vivo Sources.

Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented.

Insecticidal and acetylcholine esterase inhibition activity of Rhododendron thymifolium essential oil and its main constituent against two stored product insects.

In this work, we investigated the bioactivities of the essential oil (EO) extracted from the Rhododendron thymifolium and its principal germacrone against Lasioderma serricorne and Tribolium castaneum. The EO was obtained by steam distillation. Germacrone was obtained by cryogenic crystallization. The bioactivity of EO and germacrone was tested via contact and repellent activity assays. The results showed that EO and germacrone possessed contact and repellent activities against two species of insects. EO exhibited obvious contact activity against the L. serricorn adults, larvae and T. castaneum larvae with LD50 values of 29.15 µg/adult, 42.73 µg/larva, 19.65 µg/larva respectively. Germacrone exhibited excellent contact activity against the L. serricorne adults, larvae and the T. castaneum larvae with LD50 values of 17.18 µg/adult, 20.94 µg/larva, 20.93 µg/larva respectively. And at the highest testing concentrations (78.63 and 15.73 nL/cm2), the repellent activity of EO and germacrone on two target insects was comparable to that of the positive control (DEET) after 30 h exposure. In especially, in the treatment of the 120 h after the repellent activity of EO and germacrone against T.castaneum adults and larvae were still very significant and showed the same level percentage repellency as DEET. Meanwhile, germacrone exhibited inhibition of acetylcholinesterase activity with IC50 values of 3%. The results indicated that the EO of R. thymifolium and germacrone had the potential to be developed as natural insecticides and repellents for the control of T. castaneum and L. serricorne.

Volatile chemical composition of Piper sancti-felicis Trel essential oil and its biocidal action against Tribolium castaneum (Herbst)

This research assessed the fumigant activity of the essential oil from Piper sancti-felicis Trel and five of its components on the Tribolium castaneum (Herbst) biological model. Hydrodistillation was used for extraction of the essential oil, with separation and identification of the compounds through gas chromatography coupled to mass spectrometry (GC-MS). The fumigant was evaluated through gas dispersion on the T. castaneum. The majority compounds found in the EO were b-nerolidol (15.4%), 3-carene (14.9%), p-cymene (9.1%), spathulenol (8.2%), a-cubebene (6.2%) and calamenene (5.2%). Piper sancti-felicis displayed fumigant activity with a LC50 = 108.5 & 956;g L-1 air, and other individual monoterpenes tested such as & 945;-terpinolene (LC50 = 110.1 & 956;g L-1 air), p-cymene (LC50 = 120.3 & 956;g L-1 air), 3-carene (LC50 = 130.6 & 956;g L-1 air), (R) -limonene (CL50 = 189.6 & 956;g L-1 air), and a-pinene (LC50 = 213.1 & 956;g L-1 air), were significantly less toxic than methyl pyrimiphos used as a positive control, CL50 = 87.4 & 956;g L-1 air. The essential oil of P. sancti-felicis can be considered as a natural source of biocides.

Preliminary Study on the Differences in Hydrocarbons Between Phosphine-Susceptible and -Resistant Strains of Rhyzopertha dominica (Fabricius) and Tribolium castaneum (Herbst) Using Direct Immersion Solid-Phase Microextraction Coupled with GC-MS.

Phosphine resistance is a worldwide issue threatening the grain industry. The cuticles of insects are covered with a layer of lipids, which protect insect bodies from the harmful effects of pesticides. The main components of the cuticular lipids are hydrocarbon compounds. In this research, phosphine-resistant and -susceptible strains of two main stored-grain insects, T. castaneum and R. dominica, were tested to determine the possible role of their cuticular hydrocarbons in phosphine resistance. Direct immersion solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) was applied to extract and analyze the cuticular hydrocarbons. The results showed significant differences between the resistant and susceptible strains regarding the cuticular hydrocarbons that were investigated. The resistant insects of both species contained higher amounts than the susceptible insects for the majority of the hydrocarbons, sixteen from cuticular extraction and nineteen from the homogenized body extraction for T. castaneum and eighteen from cuticular extraction and twenty-one from the homogenized body extraction for R. dominica. 3-methylnonacosane and 2-methylheptacosane had the highest significant difference between the susceptible and resistant strains of T. castaneum from the cuticle and the homogenized body, respectively. Unknown5 from the cuticle and 3-methylhentriacontane from the homogenized body recorded the highest significant differences in R. dominica. The higher hydrocarbon content is a key factor in eliminating phosphine from entering resistant insect bodies, acting as a barrier between insects and the surrounding phosphine environment.

Antibacterial activity of a Tribolium castaneum defensin in an in vitro infection model of Streptococcus pneumoniae.

Streptococcus pneumoniae (S. pneumoniae) is the most common bacterial cause of community-acquired pneumonia. Increasing rates of antibiotic-resistant S. pneumoniae strains impair therapy and necessitate alternative treatment options. In this study, we analysed insect-derived antimicrobial peptides (AMPs) for antibacterial effects on S. pneumoniae in a human in vitro infection model.AMP effects on bacterial growth were examined by colony forming unit (CFU)-assays, and growth curve measurements. Furthermore, cytotoxicity to primary human macrophages was detected by measuring lactate-dehydrogenase release to the supernatant. One AMP (Defensin 1) was tested in a model of primary human monocyte-derived macrophages infected with S. pneumoniae strain D39 and a multi-resistant clinical isolate. Inflammatory reactions were characterised by qPCR and multiplex-ELISA.In total, the antibacterial effects of 23 AMPs were characterized. Only Tribolium castaneum Defensin 1 showed significant antibacterial effects against S. pneumoniae strain D39 and a multi-resistant clinical isolate. During in vitro infection of primary human macrophages with S. pneumoniae D39, Defensin 1 displayed strong antibacterial effects, and consequently reduced bacteria-induced cytokine expression and release.In summary, Tribolium castaneum Defensin 1 showed profound antibacterial effectivity against Streptococcus pneumoniae D39 and a multi-resistant clinical isolate without unwanted cytotoxic or inflammatory side effects on human blood-derived macrophages.

Chemical composition of essential oils from four Rhododendron species and their repellent activity against three stored-product insects.

The objective of this work was to analyze four essential oils (EOs) from Rhododendron species in China and evaluate their repellent activity against Tribolium castaneum, Lasioderma serricorne, and Liposcelis bostrychophila adults. These four EOs from Rhododendron species, including R. capitatum, R. przewalskii, R. mucronulatum, and R. micranthum, were obtained by hydrodistillation. Major components of four EOs were identified as sesquiterpenoids by GC-MS. The relatively high components included cedrene (22.20%), borneol (36.64%), 4-(2,3,4,6-tetramethylphenyl)-3-buten-2-one (27.74%), and germacrene D (27.60%). Repellent activity of EOs from Rhododendron species was investigated against T. castaneum, L. serricorne, and L. bostrychophila adults for the first time. In this study, EOs had demonstrated their repellent activities against three stored-product insects in 2- and 4-h exposure. The above results can not only provide comprehensive utilization of plant resources of Rhododendron genus but also establish a very good perspective of novel application to control stored-product insects.

Insecticidal and repellent efficacy against stored-product insects of oxygenated monoterpenes and 2-dodecanone of the essential oil from Zanthoxylum planispinum var. dintanensis.

Essential oils (EOs) extracted from leaves (EL) and fruit pericarp (EFP) of Zanthoxylum planispinum var. dintanensis were analyzed for their chemical composition by GC-MS technique and evaluated for their fumigant, contact toxicity and repellency against three stored-product insects, namely Tribolium castaneum, Lasioderma serricorne, and Liposcelis bostrychophila adults. Results of GC-MS analysis manifested that EL and EFP of Z. planispinum var. dintanensis were mainly composed of oxygenated monoterpenes. Major components included linalool, sylvestrene and terpinen-4-ol. The obvious variation observed between two oil samples was that EL contained 2-dodecanone (11.52%) in addition to the above mentioned components, while this constituent was not detected in EFP. Bioassays of insecticidal and repellent activities were performed for EL, EFP as well as some of their individual compounds (linalool, terpinen-4-ol and 2-dodecanone). Testing results indicated that EL, EFP, linalool, terpinen-4-ol and 2-dodecanone exhibited potent insecticidal and repellent activities against the three target insects selected. Among the three individual compounds, 2-dodecanone was significantly toxic to T. castaneum (LD50 = 5.21 µg/adult), L. serricorne (LD50 = 2.54 µg/adult) and L. bostrychophila (LD50 = 23.41 µg/cm2) in contact assays and had beneficial repellent effects on L. serricorne at 2 and 4 h post-exposure. The anti-insect efficacy of Z. planispinum var. dintanensis EO suggests it has potential to be used as botanical insecticide or repellent to control pest damage in warehouses and grain stores.

Context-dependent effects of cold stress on behavioral, physiological, and life-history traits of the red flour beetle.

Animals are exposed in nature to a variety of stressors. While stress is generally harmful, mild stress can also be beneficial and contribute to reproduction and survival. We studied the effect of five cold shock events versus a single cold shock and a control group, representing three levels of stress (harsh, mild, and no stress), on behavioral, physiological, and life-history traits of the red flour beetle (Tribolium castaneum, Herbst 1797). Beetles exposed to harsh cold stress were less active than a control group: they moved less and failed more frequently to detect a food patch. Their probability to mate was also lower. Beetle pairs exposed to harsh cold stress frequently failed to reproduce at all, and if reproducing, females laid fewer eggs, which were, as larvae in mid-development, smaller than those in the control group. However, harsh cold stress led to improved female starvation tolerance, probably due to enhanced lipid accumulation. Harsh cold shock also improved tolerance to an additional cold shock compared to the control. Finally, a single cold shock event negatively affected fewer measured response variables than the harsh cold stress, but also enhanced neither starvation tolerance nor tolerance to an additional cold shock. The consequences of a harsher cold stress are thus not solely detrimental but might even enhance survival under stressful conditions. Under benign conditions, nevertheless, harsh stress impedes beetle performance. The harsh stress probably shifted the balance point of the survival-reproduction trade-off, a shift that did not take place following exposure to mild stress.

Isolation and antiproliferation of tumor cells by a novel peptide (TC22) from the beetle Tribolium castaneum.

Anticancer peptides (ACPs) are biologically anticancer active molecules that are produced by mammals, plants, insects and microorganisms. Here, a new peptide (TC22) with the amino acid sequence MTVVLLLIVLPLLGGVHSSGIL was identified and characterized from the beetle Tribolium castaneum. We found it inhibited the growth and viability of HeLa and MCF-7 cells. Flow cytometry analysis demonstrated the TC22 induced HeLa cell apoptosis, and activated caspase-9 and caspase-3. Furthermore, TC22 led to ROS generation, and triggered p53 transcription and expression. Taken together, our results indicated that TC22 exhibited high anticancer capacity via activating p53, inducing ROS generation and through a mitochondrial pathway. This research provided a novel natural source peptide with strong anticancer capacity. These findings provide some novel insights on the potential candidate reagent in cancer treatment.

Response of Tribolium castaneum to dietary mannitol, with remarks on its possible nutritive effects.

Mannitol, one of the sugar alcohols, is often used as a low-calorific carbohydrate by animals. In some insects, mannitol acts as a cryoprotectant to endure coldness, but also become a poisonous agent. Adults of the red flour beetle Tribolium castaneum were shown to recognize mannitol as a factor stimulating their feeding behavior, but it remains unclear whether T. castaneum can utilize mannitol as a source of nutrition, because the enzymes needed to metabolize mannitol are unknown in this species. This study shows that T. castaneum utilizes mannitol as a nutrient in a dietary assay based on a sole carbon source added to artificial gypsum diet. The amount of mannitol excreted was less than that ingested, suggesting that it is absorbed in the insect body. The hemolymph of T. castaneum contained no mannitol but contained trehalose, a known blood sugar in insects, even after being fed mannitol. This study also revealed that dietary mannitol was metabolized to triglyceride, the main component of the fat body, forming lipid droplets. It was found that metabolites of a mannitol-supplemented diet extend the lifespan of T. castaneum, compared with those obtained by metabolizing a mannitol-free diet. Given that the insects presented transcriptional changes upon being fed carbohydrates, it might be possible to identify specific genes related to mannitol-specific metabolism by their upregulation upon mannitol intake in T. castaneum. The present study investigated mannitol-responsive gene expression using RNA-Seq. Twenty-eight genes, including those encoding trehalose-6-phosphate synthase and fatty acid synthase, were differentially expressed between beetles that were fed or not fed mannitol. The identification of upregulated genes provides us with important insights into the molecular events following mannitol intake.

Telomerase Inhibition by a New Synthetic Derivative of the Aporphine Alkaloid Boldine.

Telomerase, the enzyme responsible for cell immortality, is an important target in anti-cancer drug discovery. Boldine, an abundant aporphine alkaloid of Peumus boldus, is known to inhibit telomerase at non-toxic concentrations. Cytotoxicity of N-benzylsecoboldine hydrochloride (BSB), a synthetic derivative of boldine, was determined using the MTT method in MCF7 and MDA-MB231 cells. Aliquots of cell lysates were incubated with various concentrations of BSB in qTRAP (quantitative telomere repeat amplification protocol)-ligand experiments before substrate elongation by telomerase or amplification by hot-start Taq polymerase. The crystal structure of TERT, the catalytic subunit of telomerase from Tribolium castaneum, was used for docking and molecular dynamics analysis. The qTRAP-ligand data gave an IC50 value of about 0.17 ± 0.1 µM for BSB, roughly 400 times stronger than boldine, while the LD50 in the cytotoxicity assays were 12.5 and 21.88 µM, respectively, in cells treated for 48 h. Although both compounds interacted well with the active site, MD analysis suggests a second binding site with which BSB interacts via two hydrogen bonds, much more strongly than boldine. Theoretical analyses also evaluated the IC50 for BSB as submicromolar. BSB, with greater hydrophobicity and flexibility than boldine, represents a promising structure to inhibit telomerase at non-toxic concentrations.

Self-Assembled Coacervates of Chitosan and an Insect Cuticle Protein Containing a Rebers-Riddiford Motif.

The interactions among biomacromolecules within insect cuticle may offer new motifs for biomimetic material design. CPR27 is an abundant protein in the rigid cuticle of the elytron from Tribolium castaneum. CPR27 contains the Rebers-Riddiford (RR) motif, which is hypothesized to bind chitin. In this study, active magnetic microrheology coupled with microscopy and protein particle analysis techniques were used to correlate alterations in the viscosity of chitosan solutions with changes in solution microstructure. Addition of CPR27 to chitosan solutions led to a 3-fold drop in viscosity. This change was accompanied by the presence of micrometer-sized coacervate particles in solution. Coacervate formation had a strong dependence on chitosan concentration. Analysis showed the existence of a critical CPR27 concentration beyond which a significant increase in particle count was observed. These effects were not observed when a non-RR cuticular protein, CP30, was tested, providing evidence of a structure-function relationship related to the RR motif.

Cuticular Hydrocarbons of Tribolium confusum Larvae Mediate Trail Following and Host Recognition in the Ectoparasitoid Holepyris sylvanidis.

Parasitic wasps which attack insects infesting processed stored food need to locate their hosts hidden inside these products. Their host search is well-known to be guided by host kairomones, perceived via olfaction or contact. Among contact kairomones, host cuticular hydrocarbons (CHCs) may provide reliable information for a parasitoid. However, the chemistry of CHC profiles of hosts living in processed stored food products is largely unknown. Here we showed that the ectoparasitoid Holepyris sylvanidis uses CHCs of its host Tribolium confusum, a worldwide stored product pest, as kairomones for host location and recognition at short range. Chemical analysis of T. confusum larval extracts by gas chromatography coupled with mass spectrometry revealed a rich blend of long-chain (C25-C30) hydrocarbons, including n-alkanes, mono-, and dimethylalkanes. We further studied whether host larvae leave sufficient CHCs on a substrate where they walk along, thus allowing parasitoids to perceive a CHC trail and follow it to their host larvae. We detected 18 CHCs on a substrate that had been exposed to host larvae. These compounds were also found in crude extracts of host larvae and made up about a fifth of the CHC amount extracted. Behavioral assays showed that trails of host CHCs were followed by the parasitoids and reduced their searching time until successful host recognition. Host CHC trails deposited on different substrates were persistent for about a day. Hence, the parasitoid H. sylvanidis exploits CHCs of T. confusum larvae for host finding by following host CHC trails and for host recognition by direct contact with host larvae.

Crystallographic Studies of Telomerase.

Telomeres are nucleoprotein complexes that maintain the ends of our chromosomes thus providing genomic stability. Telomerase is a ribonucleoprotein reverse transcriptase that replicates the short tandem repeats of DNA known as telomeres. The telomeric DNA is specifically associated with two major complexes, the shelterin and CST complexes both of which are involved in telomere length regulation and maintenance along with telomerase. Obtaining structural information on these nucleoprotein complexes has been a major bottleneck in fully understanding the mechanism of action of telomeric nucleoproteins for over two decades. The recent advances in molecular and structural biology have enabled us to obtain atomic resolution structures of telomeric proteins alone and in complex with their nucleic acid substrates transforming the field and our understanding and interpretation of this unique biological pathway. Here we report our approach to obtain the structure of the Triobolium castaneum catalytic subunit of telomerase TERT (tcTERT) in its apo- and substrate-bound states.

The influence of the feeding Flour Beetle Tribolium confusum-infested fodder on the selected indices of the health status of rats.

The present experiment was designed to demonstrate differences in the degree of fodder contamination with benzoquinones at various Tribolium confusum levels, the impact of infested feed on the beetle population and the impact of infested feed on the health status of rats. The feeding studies were done on female rats divided into 3 groups: a control group and two experimental groups. Experimental groups were fed with a fodder infested by 150 individuals of T. confusum per kg (group D1) and 300 individuals of T. confusum per kg (group D2). The insects were grown in the fodder for 5 months and the contaminated fodder was given to the laboratory animals for 8 weeks. After that period the rats were sacrificed, blood was drawn for morphological, biochemical and immunological analyses, as well as the samples of internal organs were taken for histopathology. Regardless of initial degree of infestation, after 5 months incubation period the content of benzoquinones in fodder reached the maximum level that reduced beetle population. The resulting concentration to benzoquinones had no effect upon feed intake nor growth of rate, whereas caused the presence of these substances in feces, urine and also in tissues which was indicated by pathological lesions observed in the study. The results obtained point to the possibility of the benzoquinones accumulation in the organisms of farm animals fed fodder containing pests.

Flexibility and extracellular opening determine the interaction between ligands and insect sulfakinin receptors.

Despite their fundamental importance for growth, the mechanisms that regulate food intake are poorly understood. Our previous work demonstrated that insect sulfakinin (SK) signaling is involved in inhibiting feeding in an important model and pest insect, the red flour beetle Tribolium castaneum. Because the interaction of SK peptide and SK receptors (SKR) initiates the SK signaling, we have special interest on the structural factors that influence the SK-SKR interaction. First, the three-dimensional structures of the two T. castaneum SKRs (TcSKR1 and TcSKR2) were generated from molecular modeling and they displayed significance in terms of the outer opening of the cavity and protein flexibility. TcSKR1 contained a larger outer opening of the cavity than that in TcSKR2, which allows ligands a deep access into the cavity through cell membrane. Second, normal mode analysis revealed that TcSKR1 was more flexible than TcSKR2 during receptor-ligand interaction. Third, the sulfated SK (sSK) and sSK-related peptides were more potent than the nonsulfated SK, suggesting the importance of the sulfate moiety.