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The envenomation from the Bothrops genus is characterized by systemic and local effects caused by the main toxin families in the venom. In Bothrops pubescens venom we were able to identify 89 protein groups belonging to 13 toxin families with the bottom-up proteomics approach and 40 unique proteoforms belonging to 6 toxin families with the top-down proteomics approach. We also identified multi-proteoform complexes of dimeric L-amino acid oxidase using native top-down mass spectrometry.

Jaburetox (Jbtx) is an insecticidal peptide derived from Canavalia ensiformis urease, whose mechanism of action is not completely elucidated. We employed behavioral, electromyographical and electrophysiological protocols to identify the cellular and molecular targets involved in the Jbtx entomotoxicity in cockroaches and locusts. In Nauphoeta cinerea, Jbtx (32 μg/g) altered the locomotory behaviour inducing a significative decrease in the distance travelled followed by a significant increase in stopped time (52 ± 85 cm and 2573 ± 89 s, p .05, n = 40). Jbtx (8 to 32 μg/g body weight, respectively) also increased the leg and antennae grooming activities (p .05, n = 40, respectively). Jbtx (8 to 16 μg/g) induced a maximum neuromuscular blockade of 80.72% (n = 6, p .05) and was cardiotoxic, decreasing the cockroach heart rate. The electrophysiological profiles of both muscle and nerve of L. migratoria showed that Jbtx (2.5 × 10

Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect's digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 μM). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.

The distribution of corazonin in the central nervous system of the heteropteran insect Triatoma infestans was studied by immunohistochemistry. The presence of corazonin isoforms was investigated using MALDI-TOF mass spectrometry in samples containing the brain, the subesophageal ganglion, the corpora cardiaca-corpus allatum complex and the anterior part of the aorta. Several groups of immunopositive perikarya were detected in the brain, the subesophageal ganglion and the thoracic ganglia. Regarding the brain, three clusters were observed in the protocerebrum. One of these clusters was formed by somata located near the entrance of the ocellar nerves whose fibers supplied the aorta and the corpora cardiaca. The remaining groups of the protocerebrum were located in the lateral soma cortex and at the boundary of the protocerebrum with the optic lobe. The optic lobe housed immunoreactive somata in the medial soma layer of the lobula and at the level of the first optic chiasma. The neuropils of the deutocerebrum and the tritocerebrum were immunostained, but no immunoreactive perikarya were detected. In the subesophageal ganglion, immunostained somata were found in the soma layers of the mandibular and labial neuromeres, whereas in the mesothoracic ganglionic mass, they were observed in the mesothoracic, metathoracic and abdominal neuromeres. Immunostained neurites were also found in the esophageal wall. The distribution pattern of corazonin like immunoreactivity in the central nervous system of this species suggests that corazonin may act as a neurohormone. Mass spectrometric analysis revealed that [Arg 7]-corazonin was the only isoform of the neuropeptide present in T. infestans tissue samples. Fil: Settembrini, Beatriz Patricia. Universidad Austral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; Argentina Fil: De Pasquale, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; Argentina Fil: Postal, Melissa. Universidade Federal do Rio Grande do Sul; Brasil Fil: Pinto, Paulo M.. Universidade de Caxias do Sul; Brasil Fil: Carlini, Célia Regina R S. Universidade Federal do Rio Grande do Sul; Brasil Fil: Villar, Marcelo Jose. Universidad Austral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

There are several anti-oxidant enzyme families that play pivotal roles in facilitating the survival of parasites. Glutathione transferases (GSTs) are members of the anti-oxidant family that can detoxify a broad range of exogenous or endogenous compounds including reactive oxidative species. GSTs have been studied as vaccine candidates, immunodiagnostic markers and as treatment targets. Helminths of the genus Angiostrongylus live inside arteries of vertebrates and two main species are associated with accidental human infections: Angiostrongylus costaricensis adult worms live inside the mesenteric arteries and larvae of Angiostrongylus cantonensis become trapped in the central nervous system vasculature. Since the interactions between angiostrongylid nematodes and their vertebrate hosts are poorly understood, this study characterized the anti-oxidant enzymatic activities of A. cantonensis from female worms by collecting excreted and secreted (ES) and total extract (TE) molecules. Catalase (CAT) and superoxide dismutase (SOD) activities were found both in the ES and TE while glutathione peroxidase (GPX) and GST were found only in the TE. GSTs were purified by glutathione agarose affinity column (AcGST) and the pool of eluted GSTs was analyzed by mass spectrometry (LC-MS/MS) and de novo sequencing (Masslynx software). Sequences from two peptides (AcGSTpep1 and AcGSTpep2) present high identity to the N-terminal and C-terminal from sigma class GSTs of nematodes. It is known that these GST enzymes are associated with host immune regulation. Furthermore, understanding the role of parasite-derived anti-oxidant molecules is important in understanding host-parasite interactions.

Metarhizium anisopliae is a filamentous fungus used in the biological control of arthropods and produces several chitinases in order to break the host cuticle chitin fibers. Chitinase function during fungal cell development and/or infection processes is also an important aspect when analyzing the life cycle of entomopathogens. The expression profile analysis of the endochitinase chi2 gene acquired by RT-PCR experiments indicated the presence of two different transcripts, suggesting the occurrence of alternative splicing in the chi2 gene. The presence of two transcripts, characterized by the removal or retention of the second 72 bp intron, was further confirmed by DNA sequencing, Northern blot and qRT-PCR. Furthermore, we detected the synthesis of two different proteins from the transcripts by two-dimensional Western blot and mass spectrometry analyses. This is the first reported occurrence of alternative splicing in M. anisopliae.