Padronização do cultivo in vitro de hemócitos de abelha Apis mellifera africanizada
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Data
2020-02-27
Autores
Deboni, Nicole Pavan Butolo
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Universidade Estadual Paulista (Unesp)
Resumo
Apesar da importância das abelhas como polinizadores, o declínio de suas populações tem sido alarmante e, constantemente, associado ao contato desses insetos com agrotóxicos. No caso de intoxicação, um dos primeiros sistemas afetados é o sistema imune, já que ele constitui a primeira barreira de defesa e manutenção do organismo. Contudo, avaliações dos impactos causados por agentes externos nesse sistema ainda são escassas e mensuradas de maneira indireta. Assim, buscando testes mais específicos a nível celular, o presente trabalho propôs a padronização do cultivo in vitro de hemócitos de abelha Apis mellifera africanizada, para posteriores estudos ecotoxicológicos. Tal padronização demandou o estabelecimento de diversos parâmetros, dentre eles a quantidade de células para o início do cultivo, a melhor temperatura e o estágio de desenvolvimento para coleta da hemolinfa, o método adequado para extração de hemolinfa, a determinação do meio de cultura ideal, das condições laboratoriais, etc. Primeiramente observou-se que as larvas de abelha A. mellifera africanizada em quinto instar possuíam uma quantidade significativamente maior de hemócitos circulantes (p<0,001) do que os outros estágios avaliados (recém emergidas e forrageiras), sendo assim, foi estabelecida a coleta da hemolinfa de larvas cultivadas in vitro, a fim de minimizar as chances de contaminações da cultura celular. Além disso, a temperatura também se mostrou um fator importante na variação da quantidade de hemócitos, estabelecendo-se a faixa de 32±2 oC como ideal para a coleta. Diversas técnicas de extração de hemolinfa das larvas também foram testadas. A técnica de extração, a partir de uma pequena incisão na região da cabeça da larva com o auxílio de uma tesoura oftalmológica, foi padronizada. A qualidade da amostra foi estabelecida por meio da leitura em turbidímetro, obtendo-se um valor de referência (22.432,35 - 24.504,87) dado em unidades nefelométricas de turbidez (NTUs) para as amostras de hemolinfa. Após as padronizações iniciais estabeleceu-se o cultivo dos hemócitos de larvas de abelha A. mellifera africanizada em quinto instar criadas in vitro, em que o meio de cultura Grace’s demonstrou o melhor desempenho para manutenção e proliferação celular, havendo uma média de 669 mil células após 24 horas de incubação e 1.141mil após 96 horas de incubação. As fotomicrografias em microscópio invertido com contraste de fase também permitiram observar a proliferação celular no meio Grace’s. A caracterização celular foi obtida por análise morfológica, através de uma característica dos hemócitos em que as células tornam-se alongadas devido ao reconhecimento de um agente estranho (placa de cultura); após as medições, essas células foram comparadas com outras encontradas em demais trabalhos com hemócitos de abelha no mesmo estágio de desenvolvimento, permitindo inferir que as células cultivadas eram hemócitos de larvas de abelha A. mellifera africanizada.
Despite the importance of bees as pollinators, the decline in their populations has been alarming and, constantly, associated with the contact of these insects with pesticides. In the case of intoxication, one of the first systems affected is the immune system, since it constitutes the body's first defense and maintenance barrier. However, assessments of the impacts caused by external agents in this system are still scarce and indirectly measured. Thus, looking for more specific tests at the cellular level, the present work proposed the standardization of in vitro cultivation of Africanized bee hemocytes Apis mellifera, for further ecotoxicological studies. Such standardization demanded the establishment of several parameters, among them the quantity of cells for the beginning of the culture, the best temperature and the development stage for the collection of hemolymph, the appropriate method for extraction of hemolymph, the determination of the ideal culture medium, laboratory conditions, etc. Firstly, it was observed that the larvae of Africanized A. mellifera bees in the fifth instar had a significantly greater amount of circulating hemocytes (p <0.001) than the other evaluated stages (newly emerged and forage), thus, collection of the hemolymph of larvae grown in vitro, in order to minimize the chances of contamination of cell culture. In addition, temperature also proved to be an important factor in the variation in the amount of hemocytes, establishing the range of 32 ± 2 oC as ideal for collection. Several techniques for extracting hemolymph from the larvae were also tested. The extraction technique, from a small incision in the larvae head region with the aid of ophthalmic scissors, was standardized. The quality of the sample was established by reading it in a turbidimeter, obtaining a reference value (22,432.35 - 24,504.87) given in nephelometric turbidity units (NTUs) for the hemolymph samples. After initial standardization, the cultivation of hemocytes of Africanized bee larvae A. mellifera in fifth instar created in vitro was established, in which Grace's culture medium demonstrated the best performance for cell maintenance and proliferation, with an average of 669 thousand cells after 24 hours of incubation and 1,141 thousand after 96 hours of incubation. The photomicrographs in an inverted microscope with phase contrast also made it possible to observe cell proliferation in Grace's medium. The cellular characterization was obtained by morphological analysis, through a characteristic of the hemocytes in which the cells become elongated due to the recognition of a foreign agent (culture plate); after the measurements, these cells were compared with others found in other studies with bee hemocytes at the same stage of development, allowing to infer that the cultured cells were hemocytes of A. mellifera Africanized bee larvae.
Despite the importance of bees as pollinators, the decline in their populations has been alarming and, constantly, associated with the contact of these insects with pesticides. In the case of intoxication, one of the first systems affected is the immune system, since it constitutes the body's first defense and maintenance barrier. However, assessments of the impacts caused by external agents in this system are still scarce and indirectly measured. Thus, looking for more specific tests at the cellular level, the present work proposed the standardization of in vitro cultivation of Africanized bee hemocytes Apis mellifera, for further ecotoxicological studies. Such standardization demanded the establishment of several parameters, among them the quantity of cells for the beginning of the culture, the best temperature and the development stage for the collection of hemolymph, the appropriate method for extraction of hemolymph, the determination of the ideal culture medium, laboratory conditions, etc. Firstly, it was observed that the larvae of Africanized A. mellifera bees in the fifth instar had a significantly greater amount of circulating hemocytes (p <0.001) than the other evaluated stages (newly emerged and forage), thus, collection of the hemolymph of larvae grown in vitro, in order to minimize the chances of contamination of cell culture. In addition, temperature also proved to be an important factor in the variation in the amount of hemocytes, establishing the range of 32 ± 2 oC as ideal for collection. Several techniques for extracting hemolymph from the larvae were also tested. The extraction technique, from a small incision in the larvae head region with the aid of ophthalmic scissors, was standardized. The quality of the sample was established by reading it in a turbidimeter, obtaining a reference value (22,432.35 - 24,504.87) given in nephelometric turbidity units (NTUs) for the hemolymph samples. After initial standardization, the cultivation of hemocytes of Africanized bee larvae A. mellifera in fifth instar created in vitro was established, in which Grace's culture medium demonstrated the best performance for cell maintenance and proliferation, with an average of 669 thousand cells after 24 hours of incubation and 1,141 thousand after 96 hours of incubation. The photomicrographs in an inverted microscope with phase contrast also made it possible to observe cell proliferation in Grace's medium. The cellular characterization was obtained by morphological analysis, through a characteristic of the hemocytes in which the cells become elongated due to the recognition of a foreign agent (culture plate); after the measurements, these cells were compared with others found in other studies with bee hemocytes at the same stage of development, allowing to infer that the cultured cells were hemocytes of A. mellifera Africanized bee larvae.
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Palavras-chave
Sistema imune, Hemolinfa, Contagem total de hemócitos, Cultura primária de hemócitos, Immune system, Hemolymph, Total hemocyte count, Primary culture of hemocytes