Identification and molecular cloning of insecticidal toxins from the venom of the brown spider Loxosceles intermedia.

The venom of Loxosceles intermedia was investigated for the presence of insecticidal toxins active against Spodoptera frugiperda (Lepdoptera: Noctuidade), an insect that has caused great reductions in corn production in Brazil. A combination of gel filtration (Sephadex G-100) and ion-exchange chromatography (Carboxymethyl Cellulose, CM 52) resulted in four major fractions that were submitted to biological assay. Fraction 4 was further purified by a reverse phase HPLC (C18 Column) resulting in peptides active against Spodoptera frugiperda. Three new potential insecticidal toxins named LiTx x 1, LiTx x 2 and LiTx x 3 were identified. The partial amino terminal sequences of these peptides were obtained and used to clone the corresponding cDNAs with the help of degenerate oligonucleotides. The amino acid sequence deduced from the cDNA of LiTx x 1, LiTx x 2 and LiTx x 3 revealed mature proteins of approximately 7.4, 7.9 and 5.6 kDa.

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue.

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.

Hematological cell findings in bone marrow and peripheral blood of rabbits after experimental acute exposure to Loxosceles intermedia (brown spider) venom.

The purpose of this work was to find out the cellular changes occurring in bone marrow and peripheral blood after acute exposure to the venom of Loxosceles intermedia. Doses of 40 microg of venom were injected intradermally into five rabbits, and five rabbits receiving only phosphate-buffered saline (PBS) were used as controls. Bone marrow and peripheral blood samples were obtained before the envenomation and 4, 8, 12, 24 and 48 h, and 5, 10, 15, 20 and 30 days after envenomation. In bone marrow samples we assessed cellularity, nucleated red cells, megakaryocytes and neutrophils, and in peripheral blood we assessed red cells (red cell concentration, hemoglobin and hematocrit), leukocytes, neutrophils and platelets. Our objective was to find out if the venom has a direct effect on bone marrow and peripheral blood or if changes in both of them are secondary to the needs of tissues, and if there is a good correlation between histopathological and hematological findings. We found that the red cell parameters were not affected by the venom, except for nucleated red cells which decreased after venom exposure. The depression of megakaryocyte numbers and thrombocytopenia showed a strong correlation with the histopathologic changes observed in skin biopsies obtained from the rabbits. The changes in cellularity and neutrophils of bone marrow were strongly correlated with those in peripheral blood and skin. The thrombocytopenia and neutropenia in peripheral blood are due to marrow depression, which may be a consequence of an extensive migration of platelets and neutrophils to the necrotic lesion or the marrow depression may be a transitory effect of evenoming by L. intermedia.