Neilier Rodrigues da Silva Júnior

Bio: Neilier Rodrigues da Silva Júnior is an academic researcher from Universidade Federal de Viçosa. The author has contributed to research in topics: Anticarsia gemmatalis & Protease. The author has an hindex of 3, co-authored 10 publications receiving 24 citations.

Intestinal proteolytic profile changes during larval development of Anticarsia gemmatalis caterpillars.

Abstract: Soybean is one of most consumed and produced grains in the world, and Anticarsia gemmatalis is a pest that causes great damage to this crop due to severe defoliation during its larval phase. Plants have mechanisms that lead to the inhibition of proteases in the intestine of these herbivores, hampering their development. Understanding this complex protease inhibitor is important for pest control. The objective of this study was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars. For this, the proteolytic profile of the gut in the third, fourth, and fifth instars were analyzed. Irreversible inhibitors of proteases were separately incubated with A. gemmatalis enzyme extracts at the third, fourth, and fifth instar to assess the contribution of these proteases to total proteolytic activity. The enzymatic extracts were also evaluated with specific substrates to confirm changes in the specific activities of trypsin-like, chymotrypsin-like, and cysteine proteases at different instars. The results showed that the protease profile of A. gemmatalis gut changes throughout its larval development. The activity of cysteine proteases was more intense in the first instar. On the contrary, the serine proteases showed major activities in the late stages of the larval phase. Zymogram analysis and protein identification by liquid chromatography-mass spectrometry indicated serine protease as the main protease class expressed in the fifth instar. These results may shift the focus from the rational development of the protease inhibitor to A. gemmatalis and other Lepidoptera, as the expression of major proteases is not constant.

Purification and characterization of trypsin produced by gut bacteria from Anticarsia gemmatalis.

Abstract: Purification of active trypsin in the digestive process of insects is essential for the development of potent protease inhibitors (PIs) as an emerging pest control technology and research into insect adaptations to dietary PIs. An important aspect is the presence of proteolytic microorganisms, which contribute to host nutrition. Here, we purified trypsins produced by bacteria Bacillus cereus, Enterococcus mundtii, Enterococcus gallinarum, and Staphylococcus xylosus isolated from the midgut of Anticarsia gemmatalis. The trypsins had a molecular mass of approximately 25 kDa. The enzymes showed increased activity at 40°C, and they were active at pH values 7.5–10. Aprotinin, bis-benzamidine, and soybean Kunitz inhibitor (SKTI) significantly inhibited trypsin activity. The l-1-tosyl-amido-2-phenylethylchloromethyl ketone (TPCK), pepstatin A, E-64, ethylenediamine tetraacetic acid, and calcium ions did not affect the enzyme activity at the concentrations tested. We infer the purified trypsins do not require calcium ions, by which they differ from the trypsins of other microorganisms and the soluble and insoluble trypsins characterized from A. gemmatalis. These data suggest the existence of different isoforms of trypsin in the velvetbean caterpillar midguts.

Small peptides inhibit gut trypsin‐like proteases and impair Anticarsia gemmatalis (Lepidoptera: Noctuidae) survival and development

Abstract: Background Anticarsia gemmatalis larvae are key defoliating pests of soybean plants. Inorganic insecticides, harmful to the environment and human health, are the main molecules used in the control of this pest. To apply more sustainable management methods, organic molecules with high specificities, such as proteinaceous protease inhibitors, have been sought. Thus, molecular docking studies, kinetics assays, and biological tests were performed to evaluate the inhibitory activity of two peptides (i.e., GORE1 and GORE2) rationally designed to inhibit trypsin-like enzymes, which are the main proteases of A. gemmatalis midgut. Results The molecular docking simulations revealed critical hydrogen bonding patterns of the peptides with key active site residues of trypsin-like proteases of A. gemmatalis and other Lepidopteran insects. The negative values of binding energy indicate that hydrogen bonds potentiate the tight binding of the peptides with trypsin-like proteases, predicting an effective inhibition. The inhibition's rate constants (𝐾𝑖) were 0.49 mM and 0.10 mM for GORE1 and GORE2, resulting in effective inhibition of the activity trypsin on the L-BApNA substrate in the in vitro tests, pointing at the peptide GORE2 has higher inhibitory capacity on the A. gemmatalis trypsins. In addition, the two peptides were determined to be reversible competitive inhibitors. The in vivo test demonstrated that the peptides harm the survival and development of A. gemmatalis larvae. Conclusion This results suggest that the peptides are potential candidates in the management of A. gemmatalis larvae and provide baseline information for the design of new trypsin-like inhibitors based on peptidomimetic tools. This article is protected by copyright. All rights reserved.

Analysis of the diversity of endosymbiotic microorganisms in two spider mite species

Abstract: The symbiosis relationship between arthropods and microorganisms is common in nature and may aid herbivores to counteract plant defences and increase fitness. The spider mite Tetranychus evansi is ...

Inhibitory effects of tripeptides to enzymatic activity and life cycle parameters of Anticarsia gemmatalis

Abstract: Chemical pesticides are the main tool used to handle lepidopteran insects in agriculture. However, in addition to causing environmental problems, the insects have acquired resistance to these molecules. Thus, the development of environmentally friendly biopesticides with higher specificity, such as organic protease inhibitors, has been encouraged. This work evaluated the inhibitory effects of two tripeptides, Pep1 and Pep2, obtained by removing the cleavage site of tripeptidyl substrates for trypsin-like proteases. Anticarsia gemmatalis were used as biochemical model and the trypsin-like activity of larvae exposed to different dosages of the tripeptides and protease inhibitors merged in an artificial diet was evaluated. Further, we selected one dose of each tripeptide to set a biological assay, where the total-proteolytic, cysteine-proteases, chymotrypsin-like and trypsin-like activities were determined. The tripeptides shown to have potential to inhibit trypsin-like serine proteases of A. gemmatalis midgut, being the lowest activity detected at doses of 170.4 μM and 251.2 μM for Pep1 and Pep2, respectively. The survival curves obtained using Kaplan-Meier estimators indicated that Pep2 and Pep1 affected survival when comparing to the control group. However, the effect of Pep1 on survival was more pronounced indicating the lowest percent of survival at the end of the larval phase (20%). In addition, larvae exposed to Pep1 maintained normal total proteolytic activity at the expense of the activities of chymotrypsin-like and cysteine proteases. Collectively, our findings revealed that the tripeptides, especially Pep1, exhibit toxic effects on A. gemmatalis and should be tested against other lepidopteran insects.

The winds of (evolutionary) change: Breathing new life into microbiology

Abstract: To date, over 1500 prokaryotes have been characterized by small subunit rRNA sequencing and molecular phylogeny has had an equally profound effect on our understanding of relationship among eukaryotic microorganisms. The universal phylogenetic tree readily shows however how artificial the strong distinction between the eukaryote and prokaryotes has become. The split between the Archaea and the Bacteria is now recognized as the primary phylogenetic division and that the Eucarya have branched from the same side of the tree as the Archaea. Both prokaryotic domains would seem to be of thermophilic origin suggesting that life arose in a very warm environment. Among the Archaea, all of the Crenarchaeota cultured to date are thermophiles, and the deepest euryarchaeal branchings are represented exclusively by thermophiles. Among the Bacteria, the deepest known branchings are again represented exclusively by thermophiles, and thermophilia is widely scattered throughout the domain. The Archaea comprise a small number of quite disparate phenotypes that grow in unusual niches. All are obligate or facultative anaerobes. All cultured crenarchaeotes are thermophilic, some even growing optimally above the normal boiling temperature of water. The Archaeoglobales are sulfate reducers growing at high temperatures. The extreme halophiles grow only in highly saline environments. The methanogens are confined to a variety of anaerobic niches, often thermophilic. The Bacteria, on the other hand, are notable as being the source of life`s photosynthetic capacity. Five kingdoms of bacteria contain photosynthetic species; and each of the five manifests a distinct type of (chlorophyll-based) photosynthesis.

Most dominant roles of insect gut bacteria: digestion, detoxification, or essential nutrient provision?

Abstract: The insect gut microbiota has been shown to contribute to the host’s digestion, detoxification, development, pathogen resistance, and physiology. However, there is poor information about the ranking of these roles. Most of these results were obtained with cultivable bacteria, whereas the bacterial physiology may be different between free-living and midgut-colonizing bacteria. In this study, we provided both proteomic and genomic evidence on the ranking of the roles of gut bacteria by investigating the anal droplets from a weevil, Cryptorhynchus lapathi. The gut lumen and the anal droplets showed qualitatively and quantitatively different subsets of bacterial communities. The results of 16S rRNA sequencing showed that the gut lumen is dominated by Proteobacteria and Bacteroidetes, whereas the anal droplets are dominated by Proteobacteria. From the anal droplets, enzymes involved in 31 basic roles that belong to 7 super roles were identified by Q-TOF MS. The cooperation between the weevil and its gut bacteria was determined by reconstructing community pathway maps, which are defined in this study. A score was used to rank the gut bacterial roles. The results from the proteomic data indicate that the most dominant role of gut bacteria is amino acid biosynthesis, followed by protein digestion, energy metabolism, vitamin biosynthesis, lipid digestion, plant secondary metabolite (PSM) degradation, and carbohydrate digestion, while the order from the genomic data is amino acid biosynthesis, vitamin biosynthesis, lipid digestion, energy metabolism, protein digestion, PSM degradation, and carbohydrate digestion. The PCA results showed that the gut bacteria form functional groups from the point of view of either the basic role or super role, and the MFA results showed that there are functional variations among gut bacteria. In addition, the variations between the proteomic and genomic data, analyzed with the HMFA method from the point of view of either the bacterial community or individual bacterial species, are presented. The most dominant role of gut bacteria is essential nutrient provisioning, followed by digestion and detoxification. The weevil plays a pioneering role in diet digestion and mainly digests macromolecules into smaller molecules which are then mainly digested by gut bacteria.

Genome of Tetranychus urticae reveals herbivorous pest adaptations

Abstract: The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant–herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

Propedia: a database for protein-peptide identification based on a hybrid clustering algorithm.

Abstract: Protein–peptide interactions play a fundamental role in a wide variety of biological processes, such as cell signaling, regulatory networks, immune responses, and enzyme inhibition. Peptides are characterized by low toxicity and small interface areas; therefore, they are good targets for therapeutic strategies, rational drug planning and protein inhibition. Approximately 10% of the ethical pharmaceutical market is protein/peptide-based. Furthermore, it is estimated that 40% of protein interactions are mediated by peptides. Despite the fast increase in the volume of biological data, particularly on sequences and structures, there remains a lack of broad and comprehensive protein–peptide databases and tools that allow the retrieval, characterization and understanding of protein–peptide recognition and consequently support peptide design. We introduce Propedia, a comprehensive and up-to-date database with a web interface that permits clustering, searching and visualizing of protein–peptide complexes according to varied criteria. Propedia comprises over 19,000 high-resolution structures from the Protein Data Bank including structural and sequence information from protein–peptide complexes. The main advantage of Propedia over other peptide databases is that it allows a more comprehensive analysis of similarity and redundancy. It was constructed based on a hybrid clustering algorithm that compares and groups peptides by sequences, interface structures and binding sites. Propedia is available through a graphical, user-friendly and functional interface where users can retrieve, and analyze complexes and download each search data set. We performed case studies and verified that the utility of Propedia scores to rank promissing interacting peptides. In a study involving predicting peptides to inhibit SARS-CoV-2 main protease, we showed that Propedia scores related to similarity between different peptide complexes with SARS-CoV-2 main protease are in agreement with molecular dynamics free energy calculation. Propedia is a database and tool to support structure-based rational design of peptides for special purposes. Protein–peptide interactions can be useful to predict, classifying and scoring complexes or for designing new molecules as well. Propedia is up-to-date as a ready-to-use webserver with a friendly and resourceful interface and is available at: https://bioinfo.dcc.ufmg.br/propedia

Complex Relationships at the Intersection of Insect Gut Microbiomes and Plant Defenses

Abstract: Insect herbivores have ubiquitous associations with microorganisms that have major effects on how host insects may interact in their environment. Recently, increased attention has been given to how insect gut microbiomes mediate interactions with plants. In this paper, I discuss the ecology and physiology of gut bacteria associated with insect herbivores and how they may shape interactions between insects and their various host plants. I first establish how microbial associations vary between insects with different feeding styles, and how the insect host physiology and ecology can shape stable or transient relationships with gut bacteria. Then, I describe how these relationships factor in with plant nutrition and plant defenses. Within this framework, I suggest that many of the interactions between plants, insects, and the gut microbiome are context-dependent and shaped by the type of defense and the isolates present in the environment. Relationships between insects and plants are not pairwise, but instead highly multipartite, and the interweaving of complex microbial interactions is needed to fully explore the context-dependent aspects of the gut microbiome in many of these systems. I conclude the review by suggesting studies that would help reduce the unsureness of microbial interactions with less-defined herbivore systems and identify how each could provide a path to more robust roles and traits.