ABSTRACT
ABSTRACT In this study, we investigated the chromosomes of three species of Sicarius spiders from the Brazilian Caatinga, using classical and molecular cytogenetic techniques. Based on the phylogenetic approach, we also discussed about the variation of diploid number, types of sex chromosome system and changes in the localization of ribosomal genes of Scytodoidea. Sicarius are Synspermiata spiders that together with the genera Loxosceles and Hexophthalma constitute the family Sicariidae. In this group, the available cytogenetic data showed a low diploid number range (2n♂=18 to 2n♂=23) and the presence of only multiple sex chromosome systems (X1X2Y and X1X20). Mitotic metaphase cells exhibited 2n♂=16+X1X2Y for Sicarius cariri and S. ornatus, and 2n♂=18+XY for S. tropicus. In these species, silver impregnation revealed nucleolar organizer region (Ag-NOR) on the terminal region of pair 1. In S. ornatus and S. tropicus, the results obtained with fluorescent in situ hybridization (FISH) using 18S rDNA probe were similar to Ag-NOR, however in S. cariri, the ribosomal sites were localized in the terminal region of the X1 sex chromosome. In this work, we presented the first description of a simple sex chromosome system for Sicariidae, helping to understand how the XY sex chromosome system evolved from the X1X2Y system. Additionally, FISH data incongruous with Ag-NOR indicate that the cytogenetic studies in Sicariidae allow investigating the relation between the karyotype evolution and the distribution and the activity of rDNA genes.
RESUMEN En este estudio, investigamos los cromosomas de tres especies de arañas Sicarius de la Caatinga brasileña, utilizando técnicas de citogenética clásica y molecular. Usando un enfoque filogenético, también discutimos la variación del número diploide, los tipos de sistema cromosómico sexual y los cambios en la localización de los genes ribosómicos en Scytodoidea. Los Sicarius son arañas Synspermiata que, junto con los géneros Loxosceles y Hexophthalma, constituyen a la familia Sicariidae. En este grupo, los datos citogenéticos disponibles mostraron un rango de número diploide bajo (2n♂=18 a 2n♂=23) y únicamente la presencia de sistemas de cromosomas sexuales múltiples (X1X2Y y X1X20). Las células mitóticas en metafase mostraron 2n♂=16+X1X2Y para Sicarius cariri y S. ornatus, y 2n♂=18+XY para S. tropicus. En estas especies, la impregnación de plata reveló la región organizadora nucleolar (Ag-NOR) en la región terminal del par 1. En S. ornatus y S. tropicus, los resultados obtenidos con la hibridación in situ fluorescente (FISH) utilizando la sonda de ADNr 18S fueron similares a los de Ag-NOR, sin embargo, en S. cariri los sitios ribosomales se localizaron en la región terminal del cromosoma sexual X1. En este trabajo, presentamos la primera descripción de un sistema cromosómico sexual simple para Sicariidae, ayudando a entender cómo el sistema cromosómico sexual XY evolucionó a partir del sistema X1X2Y. Además, los datos de FISH incongruentes con Ag-NOR indican que los estudios citogenéticos en Sicariidae permiten investigar la relación entre la evolución del cariotipo y la distribución y la actividad de los genes de ADNr.
ABSTRACT
The spider family Sicariidae includes three genera, Hexophthalma, Sicarius and Loxosceles. The three genera share a common characteristic in their venoms: the presence of Sphingomyelinases D (SMase D). SMases D are considered the toxins that cause the main pathological effects of the Loxosceles venom, that is, those responsible for the development of loxoscelism. Some studies have shown that Sicarius spiders have less or undetectable SMase D activity in their venoms, when compared to Hexophthalma. In contrast, our group has shown that Sicarius ornatus, a Brazilian species, has active SMase D and toxic potential to envenomation. However, few species of Sicarius have been characterized for their toxic potential. In order to contribute to a better understanding about the toxicity of Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from Sicarius tropicus and compare them with that from Loxosceles laeta, one of the most toxic Loxosceles venoms. We show here that S. tropicus venom presents active SMases D. However, regarding hemolysis development, it seems that these toxins in this species present different molecular mechanisms of action than that described for Loxosceles venoms, whereas it is similar to those present in bacteria containing SMase D. Besides, our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms' composition may play a role in the toxic potential of venoms from Sicarius species.
Subject(s)
Evolution, Molecular , Hemolysis/drug effects , Phosphoric Diester Hydrolases/toxicity , Spider Venoms/toxicity , Spiders/enzymology , Animals , Cell Survival/drug effects , Female , HaCaT Cells , Humans , Keratinocytes/drug effects , Keratinocytes/pathology , Male , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Sex Factors , Species Specificity , Spider Venoms/enzymology , Spider Venoms/genetics , Spiders/classification , Spiders/geneticsABSTRACT
The spider family Sicariidae includes three genera, Hexophthalma, Sicarius and Loxosceles. The three genera share a common characteristic in their venoms: the presence of Sphingomyelinases D (SMase D). SMases D are considered the toxins that cause the main pathological effects of the Loxosceles venom, that is, those responsible for the development of loxoscelism. Some studies have shown that Sicarius spiders have less or undetectable SMase D activity in their venoms, when compared to Hexophthalma. In contrast, our group has shown that Sicarius ornatus, a Brazilian species, has active SMase D and toxic potential to envenomation. However, few species of Sicarius have been characterized for their toxic potential. In order to contribute to a better understanding about the toxicity of Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from Sicarius tropicus and compare them with that from Loxosceles laeta, one of the most toxic Loxosceles venoms. We show here that S. tropicus venom presents active SMases D. However, regarding hemolysis development, it seems that these toxins in this species present different molecular mechanisms of action than that described for Loxosceles venoms, whereas it is similar to those present in bacteria containing SMase D. Besides, our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms’ composition may play a role in the toxic potential of venoms from Sicarius species.
ABSTRACT
Envenomation by Loxosceles spiders (Sicariidae family) has been thoroughly documented. However, little is known about the potential toxicity of members from the Sicarius genus. Only the venom of the Brazilian Sicarius ornatus spider has been toxicologically characterized. In Chile, the Sicarius thomisoides species is widely distributed in desert and semidesert environments, and it is not considered a dangerous spider for humans. This study aimed to characterize the potential toxicity of the Chilean S. thomisoides spider. To do so, specimens of S. thomisoides were captured in the Atacama Desert, the venom was extracted, and the protein concentration was determined. Additionally, the venoms were analyzed by electrophoresis and Western blotting using anti-recombinant L. laeta PLD1 serum. Phospholipase D enzymatic activity was assessed, and the hemolytic and cytotoxic effects were evaluated and compared with those of the L. laeta venom. The S. thomisoides venom was able to hydrolyze sphingomyelin as well as induce complement-dependent hemolysis and the loss of viability of skin fibroblasts with a dermonecrotic effect of the venom in rabbits. The venom of S. thomisoides showed intraspecific variations, with a similar protein pattern as that of L. laeta venom at 32-35 kDa, recognized by serum anti-LlPLD1. In this context, we can conclude that the venom of Sicarius thomisoides is similar to Loxosceles laeta in many aspects, and the dermonecrotic toxin present in their venom could cause severe harm to humans; thus, precautions are necessary to avoid exposure to their bite.
Subject(s)
Arthropod Proteins/toxicity , Fibroblasts/drug effects , Hemolysis/drug effects , Phospholipase D/toxicity , Phosphoric Diester Hydrolases/toxicity , Skin/drug effects , Spider Bites/enzymology , Spider Venoms/toxicity , Spiders , Animals , Arthropod Proteins/metabolism , Cell Line , Cell Survival/drug effects , Female , Fibroblasts/pathology , Humans , Hydrolysis , Male , Necrosis , Phospholipase D/metabolism , Rabbits , Skin/pathology , Sphingomyelins/metabolism , Spider Venoms/enzymologyABSTRACT
BACKGROUND: Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. METHODS: The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. RESULTS: We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. CONCLUSIONS: People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.
ABSTRACT
Background Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.
Subject(s)
Antibodies, Heterophile/analysis , Phospholipase D/immunology , Spider Venoms/immunology , Spider Bites/complicationsABSTRACT
Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.(AU)
Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Phospholipase D/isolation & purification , Spider Venoms/toxicity , Antibodies, Heterophile/blood , Antivenins/therapeutic use , Enzyme-Linked Immunosorbent Assay/methods , Immunoblotting/methodsABSTRACT
Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used (31)P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.
Subject(s)
Arthropod Proteins/chemistry , Phospholipase D/chemistry , Spider Venoms/chemistry , Spiders/enzymology , Animals , Arthropod Proteins/metabolism , Crystallography, X-Ray , Lipids , Nuclear Magnetic Resonance, Biomolecular , Phospholipase D/metabolism , Spider Venoms/metabolism , Substrate Specificity/physiologyABSTRACT
The present study details the pathological and parasitological findings of parasitic ventriculitis and nematode infections in the large intestines of two female Rhea americana americana birds. The birds were housed in captivity, and both exhibited poor body condition and lethargy. The rheas were sent to the Veterinary Hospital of the Veterinary School, Universidade Federal de Minas Gerais (UFMG) and, despite medical care, the clinical condition of the birds did not improve. The birds died two days after admission, and were submitted to necropsy. Gross, histopathology and parasitological analysis resulted in the identification of Sicarius uncinipenis, which is associated with parasitic ventriculitis, while Deletrocephalus cesarpintoi was identified in the large intestine of both rheas. The apparent clinical indications, including loss of appetite and death, combined with the discovery of numerous parasites and other pathology changes, supported the conclusion that the death of the birds was caused by the parasitic infection. Further investigations of these infections in free-living and captive rheas are required, such that accurate data on the incidence and pathogenicity of these parasites can be obtained.
O presente estudo relata os achados patológicos e parasitológicos de ventriculite parasitária e da infecção por nematódeo no intestino grosso em duas fêmeas Rhea americana americana. As aves eram mantidas em cativeiro e ambas apresentaram condição corporal ruim e inapetência. As emas foram encaminhadas para o Hospital Veterinário da Escola de Veterinária da UFMG e, apesar dos cuidados médicos, não houve melhora na condição clínica. As aves morreram dois dias após a internação e foram encaminhadas para a necropsia. Ao exame macroscópico, histopatológico e parasitológico, Sicarius uncinipenis foi identificado e associado com ventriculite parasitária, enquanto Deletrocephalus cesarpintoi foi identificado no intestino grosso. Possivelmente, o quadro de inapetência e morte foi causado pela infecção parasitária, pois os parasitos eram numerosos. Mais investigações dessa infecção são necessárias em emas de vida livre e cativeiro, para a obtenção de informações mais precisas da incidência e patogenicidade desses parasitos.
