Evaluating the Antimicrobial Efficacy of a Designed Synthetic Peptide against Pathogenic Bacteria.

Recent research has focused on discovering peptides that effectively target multi-resistant bacteria while leaving healthy cells unharmed. In this work, we describe the antimicrobial properties of RK8, a peptide composed of eight amino acid residues. Its activity was tested against multidrug-resistant Gram-negative and Gram-positive bacteria. RK8's efficacy in eradicating mature biofilm and increasing membrane permeability was assessed using Sytox Green. Cytotoxicity assays were conducted both in vitro and in vivo models. Circular dichroism analysis revealed that RK8 adopted an extended structure in water and sodium dodecyl sulfate (SDS). RK8 exhibited MICs of 8-64 µM and MBCs of 4-64 µM against various bacteria, with higher effectiveness observed in Staphylococcus aureus (MRSA) and E. coli KPC+ strains than others. Ciprofloxacin and Vancomycin showed varying MIC and MBC values lower than RK8 for Gram-positive bacteria, but competitive for Gram-negative bacteria. The combination of RK8 and ciprofloxacin showed a synergistic effect. The RK8 peptides could reduce 38% of the mature A. baumannii biofilm. Sytox Green reagent achieved 100% membrane permeation of Gram-positive and Gram-negative bacteria. The RK8 peptide did not show cytotoxic effects against murine macrophages (64 µM), erythrocytes (100 µM) or Galleria mellanella larvae (960 µM). In the stability test against peptidases, the RK8 peptide was stable, maintaining around 60% of the molecule intact after 120 min of incubation. These results highlight the potential of RK8 to be a promising strategy for developing a new antimicrobial and antibiofilm agent, inspiring and motivating further research in antimicrobial peptides.

Chemical Composition, Antioxidant, and Cytotoxic Effects of Senna rugosa Leaf and Root Extracts on Human Leukemia Cell Lines.

Senna rugosa is a species found in the Cerrado and used in folk medicine as a vermifuge and in the treatment of poisonous snakebites accidents. In this work, we identified the main secondary metabolites present in ethanolic extracts of the leaves (ELSR) and roots (ERSR) of S. rugosa and evaluated the potential cytoprotective effect against cellular macromolecular damage, as well as the cytotoxic properties of the extracts on the K562 and Jurkat leukemic cell lines. The identification of metabolites was carried out by liquid chromatography coupled with mass spectrometry. The antioxidant activities were investigated by direct ABTS•+ and DPPH• radical scavenging methods, protection against oxidative damage in proteins, and DNA. Cytotoxic properties were investigated against healthy cells, isolated from human peripheral blood (PBMC) and leukemic cell lines. The leaf extracts contained catechin, rutin, epigallocatechin derivatives, kaempferol glycosides, luteolin, and dimeric and trimeric procyanidins, while the root extract profile showed obtusichromoneside derivatives, 2-methoxystypandrone, stilbene derivatives, naphthopyranones, and flavanone derivatives. The extracts showed antioxidant activity, with an IC50 of 4.86 ± 0.51 µg/mL and 8.33 ± 0.90 µg/mL in the ABTS assay for ELSR and ERSR, respectively. Furthermore, in the DPPH• assay, the IC50 was 19.98 ± 1.96 µg/mL for ELSR and 13.37 ± 1.05 µg/mL for ERSR. The extracts protected macromolecules against oxidative damage at concentrations of 5 µg/mL. The cytotoxicity test against leukemic strains was observed after 24 and 48 h of treatment. After 48 h, results against the K562 cell line demonstrate an IC50 of 242.54 ± 2.38 µg/mL and 223.00 ± 2.34 µg/mL for ELSR and ERSR, respectively. While against the Jurkat cell line, these extracts showed an IC50 of 171.45 ± 2.25 µg/mL and 189.30 ± 2.27 µg/mL, respectively. The results pertaining to PBMC viability demonstrated that the extracts showed selectivity for the leukemic cell lines. Together, our results reveal that the leaves and roots of S. rugosa have completely distinct and complex chemical compositions and expand their significant pharmacological potential in oxidative stress and leukemia conditions.

Boosting the antibacterial potential of a linear encrypted peptide in a Kunitz-type inhibitor (ApTI) through physicochemical-guided approaches.

Bacterial resistance has become a serious public health problem in recent years, thus encouraging the search for new antimicrobial agents. Here, we report an antimicrobial peptide (AMP), called PEPAD, which was designed based on an encrypted peptide from a Kunitz-type plant peptidase inhibitor. PEPAD was capable of rapidly inhibiting and eliminating numerous bacterial species at micromolar concentrations (from 4µM to 10 µM), with direct membrane activity. It was also observed that the peptide can act synergistically with ciprofloxacin and showed no toxicity in the G. mellonella in vivo assay. Circular dichroism assays revealed that the peptide's secondary structure adopts different scaffolds depending on the environment in which it is inserted. In lipids mimicking bacterial cell membranes, PEPAD adopts a more stable α-helical structure, which is consistent with its membrane-associated mechanism of action. When in contact with lipids mimicking mammalian cells, PEPAD adopts a disordered structure, losing its function and suggesting cellular selectivity. Therefore, these findings make PEPAD a promising candidate for future antimicrobial therapies with low toxicity to the host.

Computer-made peptide RQ18 acts as a dual antifungal and antibiofilm peptide though membrane-associated mechanisms of action.

The spread of fungi resistant to conventional drugs has become a threatening problem. In this context, antimicrobial peptides (AMPs) have been considered as one of the main alternatives for controlling fungal infections. Here, we report the antifungal and antibiofilm activity and some clues about peptide RQ18's mechanism of action against Candida and Cryptococcus. This peptide inhibited yeast growth from 2.5 µM and killed all Candida tropicalis cells within 2 h incubation. Moreover, it showed a synergistic effect with antifungal agent the amphotericin b. RQ18 reduced biofilm formation and promoted C. tropicalis mature biofilms eradication. RQ18's mechanism of action involves fungal cell membrane damage, which was confirmed by the results of RQ18 in the presence of free ergosterol in the medium and fluorescence microscopy by Sytox green. No toxic effects were observed in murine macrophage cell lines and Galleria mellonella larvae, suggesting fungal target selectivity. Therefore, peptide RQ18 represents a promising strategy as a dual antifungal and antibiofilm agent that contributes to infection control without damaging mammalian cells.

Investigating Effects of IR-780 in Animal Models of B16-F10 Melanoma: New Approach in Lung Metastasis.

IR-780 is a fluorescent marker, photostable and non-toxic, and is widely used in tumor targeting; however, studies on the impact of IR-780 in animal models of B16-F10 melanoma are scarce in the literature. Therefore, this study aims to analyze behavior of this marker in melanoma cells using in vitro and in vivo analyses with fluorescence microscopy to conduct an analysis of cell culture, and an in vivo imaging system for an analysis of cell culture, tumor targeting on animals, and organ examination. In vitro analysis showed that B16-F10 cells at a concentration of 2 × 105 cells.plate-1 allowed a better visualization using 20 µM of IR-780. Furthermore, the location of IR-780 accumulation was confirmed by its fluorescence microscopy. Through in vivo studies, fluorescence was not observed in subcutaneous nodules, and it was found that animals that received intraperitoneal injection of B16-F10 cells presented ascites and did not absorb IR-780. Additionally, animals exhibiting lung metastasis showed fluorescence in ex vivo lung images. Therefore, use of the IR-780 marker for evaluating the progression of tumor growth did not demonstrate efficiency; however, it was effective in diagnosing pulmonary metastatic tumors. Although this marker presented limitations, results of evaluating pulmonary involvement through ex vivo fluorescence imaging were determined based on intensity of fluorescence.

Evaluation of the Synthetic Multifunctional Peptide Hp-MAP3 Derivative of Temporin-PTa.

In recent years, antimicrobial peptides isolated from amphibian toxins have gained attention as new multifunctional drugs interacting with different molecular targets. We aimed to rationally design a new peptide from temporin-PTa. Hp-MAP3 (NH2-LLKKVLALLKKVL-COOH), net charge (+4), hydrophobicity (0.69), the content of hydrophobic residues (69%), and hydrophobic moment (0.73). For the construction of the analog peptide, the physicochemical characteristics were reorganized into hydrophilic and hydrophobic residues with the addition of lysines and leucines. The minimum inhibitory concentration was 2.7 to 43 µM against the growth of Gram-negative and positive bacteria, and the potential for biofilm eradication was 173.2 µM. Within 20 min, the peptide Hp-MAP3 (10.8 µM) prompted 100% of the damage to E. coli cells. At 43.3 µM, eliminated 100% of S. aureus within 5 min. The effects against yeast species of the Candida genus ranged from 5.4 to 86.6 µM. Hp-MAP3 presents cytotoxic activity against tumor HeLa at a concentration of 21.6 µM with an IC50 of 10.4 µM. Furthermore, the peptide showed hemolytic activity against murine erythrocytes. Structural studies carried out by circular dichroism showed that Hp-MAP3, while in the presence of 50% trifluoroethanol or SDS, an α-helix secondary structure. Finally, Amphipathic Hp-MAP3 building an important model for the design of new multifunctional molecules.

The synthetic antimicrobial peptide IKR18 displays anti-infectious properties in Galleria mellonella in vivo model.

Antimicrobial peptides (AMPs) are promising tools for developing new antibiotics. We described the design of IKR18, an AMP designed with the aid of computational tools. IKR18 showed antimicrobial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). CD studies revealed that IKR18 assumes an alpha-helical structure in the membrane-mimetic environment. The action mechanism IKR18 involves damage to the bacteria membrane, as demonstrated by Sytox green uptake. Furthermore, IKR18 displayed synergic and additive effects in combination with antibiotics ciprofloxacin and vancomycin. The peptide showed anti-biofilm activity in concentration and efficiency compared with commercial antibiotics, involving the direct death of bacteria, as confirmed by scanning electron microscopy. The anti-infective activity of IKR18 was demonstrated in the Galleria mellonella model infected with S. aureus, MRSA, and Acinetobacter baumannii. The novel bioinspired peptide, IKR18, proved to be effective in the control of bacterial infection, opening opportunities for the development of further assays, including preclinical models.

Purification, Characterization and Evaluation of the Antitumoral Activity of a Phospholipase A2 from the Snake Bothrops moojeni.

Nature presents a wide range of biomolecules with pharmacological potential, including venomous animal proteins. Among the protein components from snake venoms, phospholipases (PLA2) are of great importance for the development of new anticancer compounds. Thus, we aimed to evaluate the PLA2 anticancer properties from Bothrops moojeni venom. The crude venom was purified through three chromatographic steps, monitored by enzymatic activity and SDS-PAGE (12%). The purified PLA2 denominated BmPLA2 had its molecular mass and N-terminal sequence identified by mass spectrometry and Edman degradation, respectively. BmPLA2 was assayed against human epithelial colorectal adenocarcinoma cells (Caco-2), human rhabdomyosarcoma cells (RD) and mucoepidermoid carcinoma of the lung (NCI-H292), using human fibroblast cells (MRC-5) and microglia cells (BV-2) as a cytotoxicity control. BmPLA2 presented 13,836 Da and a 24 amino acid-residue homologue with snake PLA2, which showed a 90% similarity with other Bothrops moojeni PLA2. BmPLA2 displayed an IC50 of 0.6 µM against Caco-2, and demonstrated a selectivity index of 1.85 (compared to MRC-5) and 6.33 (compared to BV-2), supporting its selectivity for cancer cells. In conclusion, we describe a new acidic phospholipase, which showed antitumor activity and is a potential candidate in the development of new biotechnological tools.

Development of a novel anti-biofilm peptide derived from profilin of Spodoptera frugiperda.

Candida yeast infections are the fourth leading cause of death worldwide. Peptides with antimicrobial activity are a promising alternative treatment for such infections. Here, the antifungal activity of a new antimicrobial peptide-PEP-IA18-was evaluated against Candida species. PEP-IA18 was designed from the primary sequence of profilin, a protein from Spodoptera frugiperda, and displayed potent activity against Candida albicans and Candida tropicalis, showing a minimum inhibitory concentration (MIC) of 2.5 µM. Furthermore, the mechanism of action of PEP-IA18 involved interaction with the cell membrane (ergosterol complexation). Treatment at MIC and/or 10 × MIC significantly reduced biofilm formation and viability. PEP-IA18 showed low toxicity toward human fibroblasts and only revealed hemolytic activity at high concentrations. Thus, PEP-IA18 exhibited antifungal and anti-biofilm properties with potential applicability in the treatment of infections caused by Candida species.

Acrocomia aculeata (Jacq.) Lodd. ex Mart. Leaves Increase SIRT1 Levels and Improve Stress Resistance.

Oxidative stress is a metabolic disorder linked with several chronic diseases, and this condition can be improved by natural antioxidants. The fruit pulp of the palm Acrocomia aculeata (Jacq.) Lodd. ex Mart. is widely used in the treatment of various illnesses, but as far as we know, there are no reports regarding the properties of its leaves. Thus, we aimed to evaluate the antioxidant activity of A. aculeata leaf extracts obtained with water (EA-Aa), ethanol (EE-Aa), and methanol (EM-Aa) solvents. The extracts were chemically characterized, and their antioxidant activity was assessed through the scavenging of the free radicals DPPH and ABTS. EE-Aa and EM-Aa showed the highest amounts of phenolic compounds and free radical scavenging activity. However, EA-Aa was more efficient to protect human erythrocytes against AAPH-induced hemolysis and lipid peroxidation. Thus, we further show the antioxidant effect of EA-Aa in preventing AAPH-induced protein oxidation, H2O2-induced DNA fragmentation, and ROS generation in Cos-7 cells. Increased levels of Sirt1, catalase, and activation of ERK and Nrf2 were observed in Cos-7 treated with EA-Aa. We also verify increased survival in nematodes C. elegans, when induced to the oxidative condition by Juglone. Therefore, our results showed a typical chemical composition of plants for all extracts, but the diversity of compounds presented in EA-Aa is involved in the lower toxicity and antioxidant properties provided to the macromolecules tested, proteins, DNA, and lipids. This protective effect also proven in Cos-7 and in C. elegans was probably due to the activation of the Sirt1/Nrf2 pathway. Altogether, the low toxicity and the antioxidant properties of EA-Aa showed in all the experimental models support its further use in the treatment of oxidative stress-related diseases.

Dual Insecticidal Effects of Adenanthera pavonina Kunitz-Type Inhibitor on Plodia interpunctella is Mediated by Digestive Enzymes Inhibition and Chitin-Binding Properties.

The Indianmeal moth, Plodia interpunctella, is one of the most damaging pests of stored products. We investigated the insecticidal properties of ApKTI, a Kunitz trypsin inhibitor from Adenanthera pavonina seeds, against P. interpunctella larvae through bioassays with artificial diet. ApKTI-fed larvae showed reduction of up to 88% on larval weight and 75% in survival. Trypsin enzymes extracted from P. interpunctella larvae were inhibited by ApKTI, which also demonstrated capacity to bind to chitin. Kinetic studies revealed a non-competitive inhibition mechanism of ApKTI for trypsin, which were further corroborated by molecular docking studies. Furthermore, we have demonstrated that ApKTI exhibits a hydrophobic pocket near the reactive site loop probably involved in chitin interactions. Taken together, these data suggested that the insecticidal activity of ApKTI for P. interpunctella larvae involves a dual and promiscuous mechanisms biding to two completely different targets. Both processes might impair the P. interpunctella larval digestive process, leading to larvae death before reaching the pupal stage. Further studies are encouraged using ApKTI as a biotechnological tool to control insect pests in field conditions.

Biochemical characterization of a Kunitz inhibitor from Inga edulis seeds with antifungal activity against Candida spp.

We describe the characterization of IETI, the first trypsin inhibitor purified from Inga edulis, a tree widely distributed in Brazil. Two-step chromatography was used to purify IETI, a protein composed of a single peptide chain of 19,685.10 Da. Amino-terminal sequencing revealed that IETI shows homology with the Kunitz family, as substantiated by its physical-chemical features, such as its thermal (up to 70 °C) and wide-range pH stability (from 2 to 10), and the value of its dissociation constant (6.2 nM). IETI contains a single reactive site for trypsin, maintained by a disulfide bridge; in the presence of DTT, its inhibitory activity was reduced in a time- and concentration-dependent manner. IETI presented activity against Candida ssp., including C. buinensis and C. tropicalis. IETI inhibitory activity triggered yeast membrane permeability, affecting cell viability, thus providing support for the use of IETI in further studies for the control of fungal infections.

Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors.

Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI-induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI-induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs.

Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with insecticide activity against the Mediterranean flour moth.

This paper describes the characterization of a trypsin inhibitor from Poincianella pyramidalis seeds (PpyTI). The partial sequencing of PpyTI revealed homology to Kunitz inhibitors, clustered as a member of Family I03 in MEROPS database. PpyTI has a single polypeptide chain of 19,042 Da and presents stability at high temperatures (up to 70 °C) and a wide range of pH. In vitro assays showed that disulfide bridges have an important stabilization role of reactive site in PpyTI, a characteristic shared among several Kunitz inhibitors. Bioassays carried out with the Mediterranean flour moth (Anagasta kuehniella) revealed a significant decrease in both larval weight and survival of PpyTI-fed larvae, besides a larval stage extension. Through biochemical analysis, we demonstrated that the PpyTI insecticide effects were triggered by digestion process commitment, through the inhibition of trypsin and chymotrypsin activities, the major digestive enzymes in this species. The insecticide effects and biochemical characterization of PpyTI encourage further studies using this inhibitor for insect pest control.

The trypsin inhibitor from Entada acaciifolia seeds affects negatively the development of Mediterranean flour moth, Anagasta kuehniella.

The Mediterranean flour moth (Anagasta kuehniella) is a pest insect that attacks stored foods. The difficulty in controlling this kind of pest promotes the development of alternatives for pest control, among them the use of proteins with insecticide effect. In this work, we evaluated the role of a trypsin inhibitor purified from Entada acaciifolia seeds (EATI) on the A. kuehniella development. Different concentrations of inhibitor were added to a diet to determine its effects on insect performance. At 0.4%, the EATI decreases the larval weight and survival rates by 54.6% and 15%, respectively; in addition to the extension of the life cycle of insect. The biochemical analysis showed that the inhibitor is refractory to the digestion by midgut proteases, and led to a reduction of 32% in general proteolytic activity. A detailed analysis of the enzymatic activity revealed a decrease of 50% in trypsin activity as the chymotrypsin activity increased by 12%; possibly to compensate the commitment of the digestive process. The trypsins from the EATI-fed group stayed sensitive to the inhibition by EATI, and based on kinetic assays no new trypsin enzymes were produced as adaptation attempt. The insecticides effects observed for the EATI against this pest encourage a more in depth study of its possible long-term use as a biotechnological tool.

Insensitive trypsins are differentially transcribed during Spodoptera frugiperda adaptation against plant protease inhibitors.

0The fall armyworm (Spodoptera frugiperda) is an important pest insect due to high degree of polyphagia. In order to better understand its adaptation mechanism against plant protease inhibitors, bioassays were carried supplementing diet with the Kunitz trypsin inhibitor from Entada acaciifolia seeds (EATI). In vitro assays showed an increase of proteolytic activity in EATI-fed larvae midgut. Moreover, the trypsin enzymes showed insensitivity to inhibition with EATI. In order to understand what genes were overexpressed after chronic exposition to EATI, quantitative RT-PCR analyses were performed and revealed an increase in transcription of two trypsin genes, suggesting its participation in insensitivity of midgut trypsins. Another important result was the expression of one chymotrypsin gene, which is not expressed in control fed-larvae but induced in EATI-fed larvae. New regions of higher molecular weight showing proteolytic activity were visualized in inhibitor-fed larvae by zymography gel electrophoresis, proposing that the new enzymes expressed in response of inhibitor dietary would be formatting oligomers. This is a characteristic also observed in other pest insects that adapt to feed in plant protease inhibitors diet. Additional assays revealed that trypsins from EATI-fed larvae also became insensitive against Kunitz and Bowman-Birk inhibitors from soybean. This result suggests a possible involvement of the same S. frugiperda genes in adaptation against Kunitz and Bowman-Birk inhibitors in their host plants.

Purification of a Kunitz-type inhibitor from Acacia polyphyllaDC seeds: characterization and insecticidal properties against Anagasta kuehniella Zeller (Lepidoptera: Pyralidae).

Anagasta kuehniella is a polyphagous pest that causes economic losses worldwide. This species produces serine proteases as its major enzymes for protein digestion. In this study, a new serine-protease inhibitor was isolated from Acacia polyphylla seeds (AcKI).Further analysis revealed that AcKI is formed by two polypeptide chains with a relative molecular mass of ∼20 kDa. The effects of AcKI on the development, survival, and enzymatic activity of Anagasta kuehniella larvae were evaluated, by incorporating AcKI in an artificial diet. Bioassays revealed a reduction in larval weight of ∼50% with the lower concentration of AcKI used in the study (0.5%). Although additionalassays showed an increase in endogenous trypsin and chymotrypsin activities, with a degree of AcKI-insensivity, AcKI produces an anti nutritional effect on A. kuehniella, indicating AcKI as a promising bioinsecticide protein for engineering plants that are resistant to insect pests.

Structural insights regarding an insecticidal Talisia esculenta protein and its biotechnological potential for Diatraea saccharalis larval control.

Talisin is a seed-storage protein from Talisia esculenta that presents lectin-like activities, as well as proteinase-inhibitor properties. The present study aims to provide new in vitro and in silico biochemical information about this protein, shedding some light on its mechanistic inhibitory strategies. A theoretical three-dimensional structure of Talisin bound to trypsin was constructed in order to determine the relative interaction mode. Since the structure of non-competitive inhibition has not been elucidated, Talisin-trypsin docking was carried out using Hex v5.1, since the structure of non-competitive inhibition has not been elucidated. The predicted non-coincidence of the trypsin binding site is completely different from that previously proposed for Kunitz-type inhibitors, which demonstrate a substitution of an Arg(64) for the Glu(64) residue. Data, therefore, provide more information regarding the mechanisms of non-competitive plant proteinase inhibitors. Bioassays with Talisin also presented a strong insecticide effect on the larval development of Diatraea saccharalis, demonstrating LD50 and ED50 of ca. 2.0% and 1.5%, respectively.

Practical and theoretical characterization of Inga laurina Kunitz inhibitor on the control of Homalinotus coriaceus.

Digestive endoprotease activities of the coconut palm weevil, Homalinotus coriaceus (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Trypsin-like proteinases were major enzymes for H. coriaceus, with minor activity by chymotrypsin proteinases. More importantly, gut proteinases of H. coriaceus were inhibited by trypsin inhibitor from Inga laurina seeds. In addition, a serine proteinase inhibitor from I. laurina seeds demonstrated significant reduction of growth of H. coriaceus larvae after feeding on inhibitor incorporated artificial diets. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. We have constructed a three-dimensional model of the trypsin inhibitor complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor. Trypsin inhibitor of I. laurina shows structural features characteristic of the Kunitz type trypsin inhibitor. In summary, these findings contribute to the development of biotechnological tools such as transgenic plants with enhanced resistance to insect pests.