Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da UFRRJ |
Texto Completo: | https://tede.ufrrj.br/jspui/handle/jspui/4776 |
Resumo: | Metarhizium spp. is a fungus widely used in the biological control of arthropods. Hemocytes, cells present in the hemolymph, are involved in the immune response of these ectoparasites, especially when challenged by pathogens. Tick methods for cells obtainment face difficulties and the knowledge about morphology of Rhipicephalus microplus hemocytes are very scarce, as well as the cell?s responses to infections. The present study had as objectives the establishment of a better protocol for obtaining hemocytes from Rhipicephalus microplus engorged females, morphological analysis of hemocytes and their conditions when infected by arthropodopathogenic fungi GFP. Different spin speeds were tested, and the method with higher cell viability and high cell concentration was applied after fungi physical inoculation into R. microplus engorged females. In the biological assay, the parameters of egg production, the incubation period of the larvae, the nutrition index and the percentage of control were evaluated followed by immersion of tick females using fungal suspension. After inoculation, hemolymph collection was performed and the hemocytes separated from plasma, resuspended in PBS and quantified in a Neubauer chamber. Hemocytes were submitted to hematoxylin and eosin and PAS staining and analyses of transmission electron microscopy were performed. For standardization, a centrifugation at 500 ? g for three minutes at 4 ? C was considered more efficient and applied after inoculation of fungi. Bioassay results of the two fungal isolates yielded very similar data, demonstrating that GFP molecular marking did not influence on fungi virulence against R. microplus. Hemocyte?s quantification after fungal inoculation showed that all isolates were able to decrease the concentration by approximately 80% in comparison to hemocytes from uninfected females. Under physiological conditions, five types of hemocytes (prohemocytes, granulocytes, spherulocytes, plasmatocytes and oenocytes) were identified and when females were infected by Metarhizium, these cells were presented in a cell death process and some cells presented fungus internalized in cytoplasm, suggesting a presence phagocytosis process. It was suggested that infection by entomopathogenic fungi can generate changes in the R. microplus hemocytes and directly influence on the concentration of these cells. The present study was the first to perform transmission electron microscopy on hemocytes of this species of tick and was the first to describe the cellular alterations due to fungal infection. |
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Bittencourt, V?nia Rita Elias Pinheiro505.198.676-53Angelo, Isabele da Costa090.347.797-18G?lo, Patr?cia SilvaMorena, Diva Denelle Spadacci132.654037-85http://lattes.cnpq.br/4606539860834297Paulo, J?ssica Fiorotti de2021-06-22T18:25:02Z2018-02-26PAULO, J?ssica Fiorotti de. Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP. 2018. 68 f. Disserta??o (Mestrado em Ci?ncias Veterin?rias) - Instituto de Veterin?ria, Universidade Federal Rural do Rio de Janeiro, Serop?dica - RJ, 2018.https://tede.ufrrj.br/jspui/handle/jspui/4776Metarhizium spp. is a fungus widely used in the biological control of arthropods. Hemocytes, cells present in the hemolymph, are involved in the immune response of these ectoparasites, especially when challenged by pathogens. Tick methods for cells obtainment face difficulties and the knowledge about morphology of Rhipicephalus microplus hemocytes are very scarce, as well as the cell?s responses to infections. The present study had as objectives the establishment of a better protocol for obtaining hemocytes from Rhipicephalus microplus engorged females, morphological analysis of hemocytes and their conditions when infected by arthropodopathogenic fungi GFP. Different spin speeds were tested, and the method with higher cell viability and high cell concentration was applied after fungi physical inoculation into R. microplus engorged females. In the biological assay, the parameters of egg production, the incubation period of the larvae, the nutrition index and the percentage of control were evaluated followed by immersion of tick females using fungal suspension. After inoculation, hemolymph collection was performed and the hemocytes separated from plasma, resuspended in PBS and quantified in a Neubauer chamber. Hemocytes were submitted to hematoxylin and eosin and PAS staining and analyses of transmission electron microscopy were performed. For standardization, a centrifugation at 500 ? g for three minutes at 4 ? C was considered more efficient and applied after inoculation of fungi. Bioassay results of the two fungal isolates yielded very similar data, demonstrating that GFP molecular marking did not influence on fungi virulence against R. microplus. Hemocyte?s quantification after fungal inoculation showed that all isolates were able to decrease the concentration by approximately 80% in comparison to hemocytes from uninfected females. Under physiological conditions, five types of hemocytes (prohemocytes, granulocytes, spherulocytes, plasmatocytes and oenocytes) were identified and when females were infected by Metarhizium, these cells were presented in a cell death process and some cells presented fungus internalized in cytoplasm, suggesting a presence phagocytosis process. It was suggested that infection by entomopathogenic fungi can generate changes in the R. microplus hemocytes and directly influence on the concentration of these cells. The present study was the first to perform transmission electron microscopy on hemocytes of this species of tick and was the first to describe the cellular alterations due to fungal infection.Metarhizium spp. ? um fungo amplamente utilizado no controle biol?gico de artr?podes. Os hem?citos, as c?lulas presentes no hemolinfa, est?o envolvidos na resposta imune destes ectoparasitas, principalmente quando desafiados por pat?genos. A obten??o dessas c?lulas ? dificultosa e muito pouco se conhece sobre a morfologia dos hem?citos de Rhipicephalus microplus e sua resposta ?s infec??es. O presente estudo teve como objetivos o estabelescimento de um melhor protocolo para a obten??o de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus e a an?lise morfol?gica dessas c?lulas em condi??es fisiol?gicas e quando infectadas por fungos artropodopatog?nicos marcados ou n?o com GFP. Foram testados diferentes velocidades de centrifuga??o da hemolinfa, e o m?todo com maior viabilidade celular e alta concentra??o de c?lulas foi aplicado ap?s a inocula??o f?ngica em f?meas ingurgitadas de R. microplus. Ap?s a exposi??o por imers?o, os par?metros das f?mea ingurgitadas avaliados foram o peso da massa de ovos, ?ndice de produ??o de ovos, per?odo de incuba??o das larvas, ?ndice nutricional e o percentual de controle. Posteriormente, a coleta de hemolinfa foi realizada e os hem?citos foram separados do plasma, ressuspensos em PBS e quantificados na c?mara de Neubauer. Al?m da quantifica??o, os hem?citos foram submetidos ?s colora??es de hematoxilina e eosina e PAS, al?m de serem submetidos ? microscopia eletr?nica de transmiss?o. Para a padroniza??o, a centrifuga??o em 500 ? g durante tr?s min a 4 ? C foi considerada mais eficiente, e foi aplicada ap?s inocula??o de fungos. Posteriormente, no bioensaio por imers?o, os resultados dos dois isolados f?ngicos foram muito parecidos entre si, demonstrando que a marca??o molecular n?o influenciou na virul?ncia dos fungos. No entanto, para a quantifica??o de hem?citos, a inocula??o foi utilizada e todos os isolados foram capazes de diminuir a concentra??o em aproximadamente 80% em compara??o com a quantidade de hem?citos de f?meas n?o infectadas. Em condi??es fisiol?gicas foram identificados cinco tipos de hem?citos (pr?-hem?citos, granul?citos, esferul?citos, plasmat?citos e oenocit?ides) e quando as f?meas foram infectadas com Metarhizium, estas c?lulas apresentavam em processo de morte celular e algumas c?lulas tamb?m possuiam a internaliza??o do fungo em quest?o, sugerindo a presen?a de um processo de fagocitose. Desta forma, sugere-se que a infec??o por fungos entomopatog?nicos pode gerar altera??es nos hem?citos de R. microplus e influenciar diretamente na concentra??o dessas c?lulas. Este trabalho foi o primeiro a realizar microscopia eletr?nica de transmiss?o em hem?citos desta esp?cie de carrapato e foi o primeiro a descrever altera??es celulares decorrentes de infec??o f?ngica atrav?s de microscopia eletr?nica de transmiss?o.Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2021-06-22T18:25:02Z No. of bitstreams: 1 2018 - J?ssica Fiorotti de Paulo.pdf: 2141637 bytes, checksum: 03c6a5c6bff3e7beeb888cecd36c7ed6 (MD5)Made available in DSpace on 2021-06-22T18:25:02Z (GMT). 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dc.title.por.fl_str_mv |
Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP |
dc.title.alternative.eng.fl_str_mv |
Morpho-physiological study of hemocytes Rhipicephalus microplus engorged females infected by GFP Metarhizium spp |
title |
Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP |
spellingShingle |
Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP Paulo, J?ssica Fiorotti de Resposta imune Fungos artropodopatog?nicos Morte celular Carrapato bovino Immune response Arthropodopathogenic fungi Cell death Bovine tick. Medicina Veterin?ria |
title_short |
Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP |
title_full |
Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP |
title_fullStr |
Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP |
title_full_unstemmed |
Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP |
title_sort |
Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP |
author |
Paulo, J?ssica Fiorotti de |
author_facet |
Paulo, J?ssica Fiorotti de |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Bittencourt, V?nia Rita Elias Pinheiro |
dc.contributor.advisor1ID.fl_str_mv |
505.198.676-53 |
dc.contributor.advisor-co1.fl_str_mv |
Angelo, Isabele da Costa |
dc.contributor.advisor-co1ID.fl_str_mv |
090.347.797-18 |
dc.contributor.referee1.fl_str_mv |
G?lo, Patr?cia Silva |
dc.contributor.referee2.fl_str_mv |
Morena, Diva Denelle Spadacci |
dc.contributor.authorID.fl_str_mv |
132.654037-85 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/4606539860834297 |
dc.contributor.author.fl_str_mv |
Paulo, J?ssica Fiorotti de |
contributor_str_mv |
Bittencourt, V?nia Rita Elias Pinheiro Angelo, Isabele da Costa G?lo, Patr?cia Silva Morena, Diva Denelle Spadacci |
dc.subject.por.fl_str_mv |
Resposta imune Fungos artropodopatog?nicos Morte celular Carrapato bovino |
topic |
Resposta imune Fungos artropodopatog?nicos Morte celular Carrapato bovino Immune response Arthropodopathogenic fungi Cell death Bovine tick. Medicina Veterin?ria |
dc.subject.eng.fl_str_mv |
Immune response Arthropodopathogenic fungi Cell death Bovine tick. |
dc.subject.cnpq.fl_str_mv |
Medicina Veterin?ria |
description |
Metarhizium spp. is a fungus widely used in the biological control of arthropods. Hemocytes, cells present in the hemolymph, are involved in the immune response of these ectoparasites, especially when challenged by pathogens. Tick methods for cells obtainment face difficulties and the knowledge about morphology of Rhipicephalus microplus hemocytes are very scarce, as well as the cell?s responses to infections. The present study had as objectives the establishment of a better protocol for obtaining hemocytes from Rhipicephalus microplus engorged females, morphological analysis of hemocytes and their conditions when infected by arthropodopathogenic fungi GFP. Different spin speeds were tested, and the method with higher cell viability and high cell concentration was applied after fungi physical inoculation into R. microplus engorged females. In the biological assay, the parameters of egg production, the incubation period of the larvae, the nutrition index and the percentage of control were evaluated followed by immersion of tick females using fungal suspension. After inoculation, hemolymph collection was performed and the hemocytes separated from plasma, resuspended in PBS and quantified in a Neubauer chamber. Hemocytes were submitted to hematoxylin and eosin and PAS staining and analyses of transmission electron microscopy were performed. For standardization, a centrifugation at 500 ? g for three minutes at 4 ? C was considered more efficient and applied after inoculation of fungi. Bioassay results of the two fungal isolates yielded very similar data, demonstrating that GFP molecular marking did not influence on fungi virulence against R. microplus. Hemocyte?s quantification after fungal inoculation showed that all isolates were able to decrease the concentration by approximately 80% in comparison to hemocytes from uninfected females. Under physiological conditions, five types of hemocytes (prohemocytes, granulocytes, spherulocytes, plasmatocytes and oenocytes) were identified and when females were infected by Metarhizium, these cells were presented in a cell death process and some cells presented fungus internalized in cytoplasm, suggesting a presence phagocytosis process. It was suggested that infection by entomopathogenic fungi can generate changes in the R. microplus hemocytes and directly influence on the concentration of these cells. The present study was the first to perform transmission electron microscopy on hemocytes of this species of tick and was the first to describe the cellular alterations due to fungal infection. |
publishDate |
2018 |
dc.date.issued.fl_str_mv |
2018-02-26 |
dc.date.accessioned.fl_str_mv |
2021-06-22T18:25:02Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
PAULO, J?ssica Fiorotti de. Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP. 2018. 68 f. Disserta??o (Mestrado em Ci?ncias Veterin?rias) - Instituto de Veterin?ria, Universidade Federal Rural do Rio de Janeiro, Serop?dica - RJ, 2018. |
dc.identifier.uri.fl_str_mv |
https://tede.ufrrj.br/jspui/handle/jspui/4776 |
identifier_str_mv |
PAULO, J?ssica Fiorotti de. Estudo morfo-fisiol?gico de hem?citos de f?meas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP. 2018. 68 f. Disserta??o (Mestrado em Ci?ncias Veterin?rias) - Instituto de Veterin?ria, Universidade Federal Rural do Rio de Janeiro, Serop?dica - RJ, 2018. |
url |
https://tede.ufrrj.br/jspui/handle/jspui/4776 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal Rural do Rio de Janeiro |
dc.publisher.program.fl_str_mv |
Programa de P?s-Gradua??o em Ci?ncias Veterin?rias |
dc.publisher.initials.fl_str_mv |
UFRRJ |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Instituto de Veterin?ria |
publisher.none.fl_str_mv |
Universidade Federal Rural do Rio de Janeiro |
dc.source.none.fl_str_mv |
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UFRRJ |
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UFRRJ |
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Biblioteca Digital de Teses e Dissertações da UFRRJ |
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