Avaliação morfológica e bioquímica em abelhas recém emergidas da espécie Scaptotrigona postica, Latreille 1807 (Hymenoptera, Apidae), expostas a clotianidina durante a fase larval
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Data
2022-08-26
Autores
Camargo, Isabella Fernanda
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Universidade Estadual Paulista (Unesp)
Resumo
O Brasil é o país com maior biodiversidade de abelhas sem ferrão do mundo. Dentre as espécies, Scaptotrigona postica desempenha papel fundamental nos serviços de polinização de plantas nativas e cultivadas. No entanto, a saúde das populações desses polinizadores está sendo ameaçada devido a múltiplos fatores, dentre eles, o uso intensivo de agrotóxicos. Esses compostos químicos chegam à colmeia por meio de abelhas forrageiras e, uma vez lá, podem ser ingeridos pelas larvas durante a alimentação. No Brasil são utilizados diferentes grupos de agrotóxicos, entre eles a clotianidina, um ingrediente sistêmico, cujos efeitos são desconhecidos em larvas e adultos de abelhas S. postica. Dessa forma, o presente estudo teve como objetivo compreender os possíveis impactos da clotianidina por meio da: (I) investigação dos efeitos de concentrações realísticas de campo, na sobrevivência e desenvolvimento de larvas e adultos de abelhas S. postica; (II) avaliação da toxicidade deste inseticida na morfologia de tecidos de órgãos de metabolização de substâncias (intestino e túbulos de Malpighi) de recém emergidas; e (III) análise da atividade de enzimas relacionadas a desintoxicação (Carboxilesterase 3 – CaEs-3 e Glutationa-S-transferase - GST) e enzimas antioxidantes (Glicose-6-fosfato desidrogenase – G6PD e Glutationa peroxidase - GPx), nos órgãos supracitados de abelhas recém emergidas. Para que os objetivos fossem alcançados, foram realizadas sucessivas diluições no alimento para atingir as concentrações de clotianidina a serem oferecidas às larvas: 1 ng i.a./μL (concentração de campo) e 0,1 ng i.a./μL (concentração de campo/10), o alimento puro foi oferecido ao grupo controle. Após a distribuição do alimento (25 μL/larva) em placas de acrílico, larvas de primeiro instar foram coletadas e transferidas. Para os bioensaios in vitro, os efeitos foram observados de acordo com a progressão das fases de desenvolvimento: 5ª instar larval; defecação; taxa de mortalidade; taxa de pupação; taxa de emergência; mudança na coloração de olhos das pupas e; tempo em cada fase de desenvolvimento. Para as análises de alterações morfológicas, lâminas contendo secções dos órgãos sob coloração de hematoxilina e eosina foram analisadas. Para cada alteração observada foi levado em consideração o índice de relevância dos danos patológicos nos órgãos. Já para as análises de modulação da atividade das enzimas, os grupos expostos foram individualmente comparados com o grupo controle e diferenças significativas foram consideradas. Os dados obtidos foram submetidos a análises estatísticas no software RStudio. Os resultados demonstraram que a clotianidina afetou significativamente as taxas de sobrevivência, pupação e emergência das abelhas em ambos os grupos expostos, quando comparados ao controle. O tempo em cada fase de desenvolvimento apresentou significância estatística para o 5ª instar larval e defecação. Não houve diferença no tempo entre os grupos no período pupal, apenas em pupa de olho marrom, demonstrando que a clotianidina foi mais tóxica no período larval do que pupal. Os resultados das alterações morfológicas demonstraram que a clotianidina danificou severamente os órgãos analisados nos grupos expostos. Foram observadas alterações como perda de material citoplasmático; perda de borda em escova; aumento da eliminação de células para o lúmen; perda de ninhos de células regenerativas; inchaço celular e picnose. Já com relação aos resultados de atividade enzimática, a concentração de campo de clotianidina modulou diferencialmente a atividade das enzimas CaEs-3, GST e G6PD, com exceção da GPx. Os resultados obtidos são inéditos e contribuem para o desenvolvimento de medidas protetivas para a conservação da S. postica, uma vez que este estudo é o primeiro a avaliar os efeitos da exposição larval a clotianidina para essa espécie de abelha. Desse modo, esse trabalho fornece dados relevantes a serem incluídos nas investigações do IBAMA e nos esquemas de avaliação de risco ambiental de agrotóxicos para abelhas sem ferrão.
Brazil is the country with the greatest biodiversity of stingless bees in the world. Among the species, Scaptotrigona postica plays a fundamental role in the pollination services of native and cultivated plants. However, the health of populations of these pollinators is being threatened due to multiple factors, among them, the intensive use of pesticides. These chemical compounds reach the hive through forager bees and, once there, can be ingested by the larvae during feeding. In Brazil, different groups of pesticides are used, including clothianidin, a systemic ingredient whose effects are unknown in larvae and adults of S. postica bees. Thus, the present study aimed to understand the possible impacts of clothianidin through: (I) investigation of the effects of realistic field concentrations on the survival and development of larvae and adults of S. postica bees; (II) evaluation of the toxicity of this insecticide in the morphology of tissues of substances metabolizing organs (intestine and Malpighian tubules) of newly emerged; and (III) analysis of the activity of detoxification-related enzymes (Carboxylesterase 3 - CaEs-3 and Glutathione-S-transferase - GST) and antioxidant enzymes (Glucose-6-phosphate dehydrogenase - G6PD and Glutathione peroxidase - GPx) in aforementioned organs of newly emerged bees. To achieve the objectives, successive dilutions were carried out in the food to reach the concentrations of clothianidin to be offered to the larvae: 1 ng a.i./μL (field concentration) and 0.1 ng a.i./μL (field concentration/10), pure food was offered to the control group. After distributing food (25 μL/larva) on acrylic plates, first instar larvae were collected and transferred. For the in vitro bioassays, the effects were observed according to the progression of the developmental stages: 5th larval instar; defecation; mortality rate; pupation rate; emergency fee; change in pupae eye color and time at each stage of development. For the analysis of morphological changes, slides containing sections of the organs stained with hematoxylin and eosin were analyzed. For each observed alteration, the relevance index of pathological damage to the organs was considered. For the analysis of enzyme activity modulation, the exposed groups were individually compared with the control group and significant differences were considered. The data obtained were submitted to statistical analysis in the RStudio software. The results showed that clothianidin significantly affected the survival, pupation and emergence rates of bees in both exposed groups when compared to the control. The time in each developmental stage was statistically significant for the 5th larval instar and defecation. There was no difference in time between groups in the pupal period, only in browneyed p upae, demonstrating that clothianidin was more toxic in the larval period than in the pupal period. The results of the morphological alterations showed that clothianidin severely damaged the organs analyzed in the exposed groups. Changes such as loss of cy toplasmic material were observed; brush edge loss; increased clearance of cells into the lumen; loss of regenerative cell nests; cellular swelling and pyknosis. Regarding the enzymatic activity results, the clothianidin field concentration differentially m odulated the activity of the CaEs GST and G6PD enzymes, except for GPx3, . The results obtained are unprecedented and contribute to the development of protective measures for the conservation of postica S. , since this study is the first to assess the effects of larval exposure to clothianidin for this bee species. Thus, this wo rk provides relevant data to be included in IBAMA investigations and in the environmental risk assessment schemes of pesticides for stingless bees .
Brazil is the country with the greatest biodiversity of stingless bees in the world. Among the species, Scaptotrigona postica plays a fundamental role in the pollination services of native and cultivated plants. However, the health of populations of these pollinators is being threatened due to multiple factors, among them, the intensive use of pesticides. These chemical compounds reach the hive through forager bees and, once there, can be ingested by the larvae during feeding. In Brazil, different groups of pesticides are used, including clothianidin, a systemic ingredient whose effects are unknown in larvae and adults of S. postica bees. Thus, the present study aimed to understand the possible impacts of clothianidin through: (I) investigation of the effects of realistic field concentrations on the survival and development of larvae and adults of S. postica bees; (II) evaluation of the toxicity of this insecticide in the morphology of tissues of substances metabolizing organs (intestine and Malpighian tubules) of newly emerged; and (III) analysis of the activity of detoxification-related enzymes (Carboxylesterase 3 - CaEs-3 and Glutathione-S-transferase - GST) and antioxidant enzymes (Glucose-6-phosphate dehydrogenase - G6PD and Glutathione peroxidase - GPx) in aforementioned organs of newly emerged bees. To achieve the objectives, successive dilutions were carried out in the food to reach the concentrations of clothianidin to be offered to the larvae: 1 ng a.i./μL (field concentration) and 0.1 ng a.i./μL (field concentration/10), pure food was offered to the control group. After distributing food (25 μL/larva) on acrylic plates, first instar larvae were collected and transferred. For the in vitro bioassays, the effects were observed according to the progression of the developmental stages: 5th larval instar; defecation; mortality rate; pupation rate; emergency fee; change in pupae eye color and time at each stage of development. For the analysis of morphological changes, slides containing sections of the organs stained with hematoxylin and eosin were analyzed. For each observed alteration, the relevance index of pathological damage to the organs was considered. For the analysis of enzyme activity modulation, the exposed groups were individually compared with the control group and significant differences were considered. The data obtained were submitted to statistical analysis in the RStudio software. The results showed that clothianidin significantly affected the survival, pupation and emergence rates of bees in both exposed groups when compared to the control. The time in each developmental stage was statistically significant for the 5th larval instar and defecation. There was no difference in time between groups in the pupal period, only in browneyed p upae, demonstrating that clothianidin was more toxic in the larval period than in the pupal period. The results of the morphological alterations showed that clothianidin severely damaged the organs analyzed in the exposed groups. Changes such as loss of cy toplasmic material were observed; brush edge loss; increased clearance of cells into the lumen; loss of regenerative cell nests; cellular swelling and pyknosis. Regarding the enzymatic activity results, the clothianidin field concentration differentially m odulated the activity of the CaEs GST and G6PD enzymes, except for GPx3, . The results obtained are unprecedented and contribute to the development of protective measures for the conservation of postica S. , since this study is the first to assess the effects of larval exposure to clothianidin for this bee species. Thus, this wo rk provides relevant data to be included in IBAMA investigations and in the environmental risk assessment schemes of pesticides for stingless bees .
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Palavras-chave
Neonicotinoide, Meliponíneos, Criação in vitro, Danos morfológicos, Atividade enzimática, Neonicotinoid, Meliponines, In vitro creation, Morfological damage, Enzymatic activity