Flow cytometric characterization of hemocytes of the flat oyster, Ostrea chilensis.

The flat oyster, Ostrea chilensis, native to New Zealand (NZ) and Chile is considered an important ecological, cultural and fisheries resource. Currently, commercial landings of this species in NZ are restricted due to low population numbers caused by ongoing mortalities resulting from the presence of the haplosporidian parasite, Bonamia exitiosa. More recently, the arrival of B. ostreae in NZ led to major mortalities in farmed stocks. To understand how diseases caused by Bonamia spp. affect this oyster species, a more complete understanding of its biology, physiology and immune system is needed. The present study characterized, for the first time, hemocytes of adult O. chilensis, from the Foveaux Strait, NZ, using flow cytometry (FCM) and histology. Based on the internal complexity of the hemocytes, two main circulating hemocyte populations were identified: granulocytes and hyalinocytes (accounting for ~30% and ~70% of the total circulating hemocyte population, respectively). These were further divided into two sub-populations of each cell type using FCM. A third sub-population of granulocytes was identified using histology. Using FCM, functional and metabolic characteristics were investigated for the two main hemocyte types. Granulocytes showed higher phagocytic capabilities, lysosomal content, neutral lipid content and reactive oxygen species production compared to hyalinocytes, indicating their important role in cellular immune defence in this species. Methods of hemocyte sampling and storage were also investigated and flow cytometric protocols were detailed and verified to allow effective future investigations into the health status of this important species.

Hemócitos de Bradybaena similaris e Megalobulimus abbreviatus (Gastropoda, Stylommatophora) / Hemocytes of Bradybaena similaris and Megalobulimus abbreviatus (Gastropoda, Stylommatophora)

Hemocytes act in the defense system against invading organisms, foreign particles aiding the recognition of what is own to the body of gastropods and what is not. Information and studies on the hemocytes in species of mollusks healthy (no infections), especially in Bradybaena similaris (Fèrussac, 1821) and Megalobulimus abbreviatus (Bequaert, 1948) are scarce. Therefore, this work aims at characterization and quantification of hemocytes present in the hemolymph of these two species. In this work three cell types were identified in the hemolymph of both species: round cells, hyalinocytes and granulocytes. The three types of hemocytes were measured, and the average of total diameter and the nucleus for each was calculated. On B. similaris, the average diameter of round cells was 10.7 µm, of hyalinocytes was 20 µm and of granulocytes was 25.4 µm. On M. abbreviatus, the average diameter of round cells was 11.7 µm, of hyalinocytes was 21.5 µm and of granulocytes was 30.5 µm. Although the hyalinocytes have similar averages between B. similaris and M. abbreviatus, the cells were demonstrated significant differences in their total diameter and size of the nucleus (p<0.0001). The average density of cells per ml without distinction of cellular type was 197,813 cells/ml for M. abbreviatus, and 416,333 cells/ml for B. similaris. The most frequent hemocytes in M. abbreviatus and B. similaris were hyalinocytes, unlike other gastropods.(AU)

Os hemócitos atuam no sistema de defesa contra organismos invasores e partículas estranhas, auxiliando o reconhecimento do que é próprio do corpo dos grastrópodes e o que não é. São escassas as informações e estudos sobre os hemócitos em espécies de moluscos saudáveis (sem infecções), principalmente em Bradybaena similaris (Fèrussac, 1821) and Megalobulimus abbreviatus (Bequaert, 1948). Portanto, este trabalho tem como objetivos a caracterização e quantificação dos hemócitos presentes na hemolinfa destas duas espécies. Neste trabalho, foram identificados três tipos celulares na hemolinfa de ambas espécies: as células redondas, hialinócitos e granulócitos. Os três tipos de hemócitos foram medidos e foi calculada a média do diâmetro total e do núcleo para cada um deles. Para B. similaris, o diâmetro médio das células redondas foi de 10,7 µm, dos hialinócitos foi de 20 µm e dos granulócitos de 25,4 µm. Para M. abbreviatus, o diâmetro médio foi de 11,7 µm para as células redondas, de 21,5 µm para os hialinócitos e de 30,5 µm para os granulócitos. Embora os hialinócitos possuam médias parecidas entre B. similaris e M. abbreviatus, foram detectadas diferenças significativas do diâmetro celular total e diâmetro do núcleo (p<0,0001) dessas células entre as espécies estudadas. A densidade média de células por ml, sem distinção de tipo celular foi de 197.813 células/ml para M. abbreviatus, e de 416.333 células/ml para B. similaris. Diferentemente de outros gastrópodes, os hemócitos mais frequentes em M. abbreviatus e em B. similaris foram os hialinócitos.(AU)

Hemócitos de Bradybaena similaris e Megalobulimus abbreviatus (Gastropoda, Stylommatophora) / Hemocytes of Bradybaena similaris and Megalobulimus abbreviatus (Gastropoda, Stylommatophora)

Hemocytes act in the defense system against invading organisms, foreign particles aiding the recognition of what is own to the body of gastropods and what is not. Information and studies on the hemocytes in species of mollusks healthy (no infections), especially in Bradybaena similaris (Fèrussac, 1821) and Megalobulimus abbreviatus (Bequaert, 1948) are scarce. Therefore, this work aims at characterization and quantification of hemocytes present in the hemolymph of these two species. In this work three cell types were identified in the hemolymph of both species: round cells, hyalinocytes and granulocytes. The three types of hemocytes were measured, and the average of total diameter and the nucleus for each was calculated. On B. similaris, the average diameter of round cells was 10.7 µm, of hyalinocytes was 20 µm and of granulocytes was 25.4 µm. On M. abbreviatus, the average diameter of round cells was 11.7 µm, of hyalinocytes was 21.5 µm and of granulocytes was 30.5 µm. Although the hyalinocytes have similar averages between B. similaris and M. abbreviatus, the cells were demonstrated significant differences in their total diameter and size of the nucleus (p<0.0001). The average density of cells per ml without distinction of cellular type was 197,813 cells/ml for M. abbreviatus, and 416,333 cells/ml for B. similaris. The most frequent hemocytes in M. abbreviatus and B. similaris were hyalinocytes, unlike other gastropods.

Os hemócitos atuam no sistema de defesa contra organismos invasores e partículas estranhas, auxiliando o reconhecimento do que é próprio do corpo dos grastrópodes e o que não é. São escassas as informações e estudos sobre os hemócitos em espécies de moluscos saudáveis (sem infecções), principalmente em Bradybaena similaris (Fèrussac, 1821) and Megalobulimus abbreviatus (Bequaert, 1948). Portanto, este trabalho tem como objetivos a caracterização e quantificação dos hemócitos presentes na hemolinfa destas duas espécies. Neste trabalho, foram identificados três tipos celulares na hemolinfa de ambas espécies: as células redondas, hialinócitos e granulócitos. Os três tipos de hemócitos foram medidos e foi calculada a média do diâmetro total e do núcleo para cada um deles. Para B. similaris, o diâmetro médio das células redondas foi de 10,7 µm, dos hialinócitos foi de 20 µm e dos granulócitos de 25,4 µm. Para M. abbreviatus, o diâmetro médio foi de 11,7 µm para as células redondas, de 21,5 µm para os hialinócitos e de 30,5 µm para os granulócitos. Embora os hialinócitos possuam médias parecidas entre B. similaris e M. abbreviatus, foram detectadas diferenças significativas do diâmetro celular total e diâmetro do núcleo (p<0,0001) dessas células entre as espécies estudadas. A densidade média de células por ml, sem distinção de tipo celular foi de 197.813 células/ml para M. abbreviatus, e de 416.333 células/ml para B. similaris. Diferentemente de outros gastrópodes, os hemócitos mais frequentes em M. abbreviatus e em B. similaris foram os hialinócitos.