Metais como o chumbo (Pb) podem constituir uma fonte de poluição ambiental difusa de difícil controle e remediação. Espécies cosmopolitas como os siris-azuis do gênero Callinectes sp. podem ser empregados para indicar a disponibilidade de contaminantes no ambiente. Diferentes autores verificaram a presença de metais em Callinectes sp., todavia, biomarcadores moleculares e imunológicos referentes à exposição destes metais tem recebido pouca atenção, principalmente para espécie-alvo deste estudo, o Callinectes danae . Diante disso, o presente trabalho teve por objetivo avaliar os efeitos deletérios do Pb na resposta imune celular e molecular em Callinectes danae. Para tanto, foi realizada caracterização dos hemócitos circulantes presentes na hemolinfa como parâmetro imunológico inicial; experimentos de exposição in vivo a 2 µ g/mL de Pb por 24h, 48h e 96h em indivíduos coletados em Cananéia, para avaliar parâmetros como: Quantificação total de Pb no hepatopâncreas, brânquias e músculos, por espectrômetro de absorção atômica; contagem diferencial de hemócitos por citometria de fluxo; contagem total de hemócitos em câmara de neubauer; viabilidade celular dos hemócitos circulantes e avaliação da expressão gênica de metalotioneínas (MTCu-II; MTCd-I e MTCd-II) por RT-qPCR no hepatopâncreas. Além disso, foram realizados experimentos in vitro e avaliado a atividade fagocítica após exposições a 0; 50; 100; 250; 500; 1000 ng/mL de Pb, por 1h, em siris-azuis coletados no complexo estuarino de Cananéia e na Baía do Araçá em São Sebastião. Os resultados demonstraram que a espécie alvo do presente estudo possui três populações de hemócitos, sendo estas: Granulócitos, semigranulócitos e hialinócitos. A contagem total e diferencial dos hemócitos variou consideravelmente entre os indivíduos, o que inviabilizou a utilização deste parametro como um biomarcador. Os ensaios de viabilidade celular apresentaram redução dos hemócitos vivos apenas em 96h de exposição. A bioacumulação de Pb foi estatisticamente significativa em todos tecidos amostrados, principalmente nas brânquias, onde houve aumento em 48h e redução em 96h. A expressão gênica de MTCu-II e MTCd-I não apresentaram diferenças nos períodos avaliados. Apenas MTCd-II foi regulada positivamente em 48h seguido de redução em 96h, indicando que a isoforma de metalotioneína a ser escolhida é uma variável a ser considerada e que possivelmente apenas esta isoforma consiga interagir com uma possível contaminação com Pb. Os experimentos de fagocitose in vitro apresentaram um curva decrescente com relação ao aumento das concentrações de Pb, em ambos locais de coleta, sugerindo uma resposta dose-dependente e demonstrando que a atividade fagocítica é uma forte candidata para posteriores estudos de monitoramento ambiental frente contaminações de Pb. Para tanto, mais estudos de aprofundamento nestes biomarcadores, diretamente no ambiente, devem ser conduzidos.

Metals such as lead (Pb) can be a source of diffuse environmental pollution that is difficult to control and remediate. Cosmopolitan species such as blue crabs of the genus Callinectes sp. can be used to indicate the availability of contaminants in the environment. Different authors have verified the presence of metals in Callinectes sp. however, molecular and immunological biomarkers regarding the exposure of these metals have received little attention, especially for the target species of this study, Callinectes danae. Therefore, the present work aimed to evaluate the deleterious effects of Pb on cellular and molecular immune responses in Callinectes danae, to find new tools for environmental biomonitoring of estuaries. For this, a characterization of circulating hemocytes present in hemolymph was performed as the initial immunological parameter; In vivo exposure experiments using 2 µ g/mL Pb for 24h, 48h and 96h in individuals collected in Cananéia, to evaluate parameters such as: total quantification of Pb in hepatopancreas, gills and muscles by atomic absorption spectrometer; Differential hemocyte count by flow cytometry; Total hemocyte count in neubauer chamber; Cell viability of circulating hemocytes and evaluation of metallothionein gene expression (MTCu-II; MTCd-I and MTCd-II) in hepatopancreas by RT-qPCR. In addition, in vitro experiments were performed and phagocytic activity was evaluated after exposure to 0; 50; 100; 250; 500; 1000 ng/mL Pb for 1h in blue crabs collected in Cananéia and Araçá Bay. The results showed that the target species of the present study has three populations of hemocytes: Granulocytes, semigranulocytes and hyalinocytes. The total and differential hemocyte count varied considerably among individuals, which made the use of this parameter as a biomarker unfeasible. Cell viability assays showed reduction of living hemocytes only at 96h of exposure. Pb bioaccumulation was statistically significant in all tissues sampled, especially in the gills, where there was a increase at 48h and a decrease at 96h. The gene expression of TCM-II and TCM-I showed no differences in the evaluated periods. Only MTCd-II was up-regulated at 48h followed by a down-regulation at 96h, indicating that the metallothionein isoform to be chosen is a variable to be considered and that possibly only this isoform can interact with a possible Pb contamination. In vitro phagocytosis experiments showed a decreasing curve regarding the increase of Pb concentrations in both collection sites, suggesting a dose dependent response and demonstrating that phagocytic activity is a strong candidate for environmental monitoring work against Pb contamination. Metals such as lead (Pb) can be a source of diffuse environmental pollution that is difficult to control and remediate. Cosmopolitan species such as blue crabs of the genus Callinectes sp. can be used to indicate the availability of contaminants in the environment. Different authors have verified the presence of metals in Callinectes sp. however, molecular and immunological biomarkers regarding the exposure of these metals have received little attention, especially for the target species of this study, Callinectes danae. Therefore, the present work aimed to evaluate the deleterious effects of Pb on cellular and molecular immune responses in Callinectes danae, to find new tools for environmental biomonitoring of estuaries. For this, a characterization of circulating hemocytes present in hemolymph was performed as the initial immunological parameter; In vivo exposure experiments using 2 g/mL Pb for 24h, 48h and 96h in individuals collected in Cananéia, to evaluate parameters such as: total quantification of Pb in hepatopancreas, gills and muscles by atomic absorption spectrometer; Differential hemocyte count by flow cytometry; Total hemocyte count in neubauer chamber; Cell viability of circulating hemocytes and evaluation of metallothionein gene expression (MTCu-II; MTCd-I and MTCd-II) in hepatopancreas by RT-qPCR. In addition, in vitro experiments were performed and phagocytic activity was evaluated after exposure to 0; 50; 100; 250; 500; 1000 ng/mL Pb for 1h in blue crabs collected in Cananéia and Araçá Bay. The results showed that the target species of the present study has three populations of hemocytes: Granulocytes, semigranulocytes and hyalinocytes. The total and differential hemocyte count varied considerably among individuals, which made the use of this parameter as a biomarker unfeasible. Cell viability assays showed reduction of living hemocytes only at 96h of exposure. Pb bioaccumulation was statistically significant in all tissues sampled, especially in the gills, where there was a increase at 48h and a decrease at 96h. The gene expression of TCM-II and TCM-I showed no differences in the evaluated periods. Only MTCd-II was up-regulated at 48h followed by a down-regulation at 96h, indicating that the metallothionein isoform to be chosen is a variable to be considered and that possibly only this isoform can interact with a possible Pb contamination. In vitro phagocytosis experiments showed a decreasing curve regarding the increase of Pb concentrations in both collection sites, suggesting a dose dependent response and demonstrating that phagocytic activity is a strong candidate for environmental monitoring work against Pb contamination.