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19 Jun 2017

Characterisation of the Innate Immune Responses of Marron

Bambang Widyo Prastowo, 2015
Curtin University


Cherax cainii (marron) is an indigenous species to Western Australia, and a prominent commercial aquaculture species. The immunological defense of C. cainii depends on the innate immune system however, little is known about the specifics of the C. cainii defense mechanisms. Therefore, the work herein aims at gaining deeper insights into the C. cainii immunological response, with particular emphasis on the cellular component, the haemocyte. Haemocyte responses including morphologic characteristics, nitric oxide (NO) production, population changes and phagocytosis were evaluated in response to inflammatory stimulus from Gram negative bacteria and their endotoxin and the environmental effect of temperature.

Immunological challenge by live and heat-killed V. mimucus and lipopolysaccharide (LPS) was conducted on whole C. cainii and in vitro, through haemocyte culture, at various temperatures. Techniques and technologies to assess responses included light and transmission electron microscopy (TEM), flow cytometry (FCM) and the Griess assay for NO production.

Major findings include the identification of three morphologically distinct haemocyte cell types in keeping with findings in related species: called hyaline cells (HC), small granular cells (SGC) and large granular cells (LGC). Total and differential haemocyte counts changed when marron were cultivated in different water temperatures: total haemocyte counts increased from 1.9 to 4.9 ×106 cells ml−1 when environment temperature increased from 20 to 30 °C. Hyaline cells represented the most abundant cell type (42.5%), followed by SGCs (35%) and LGCs (22.5%): data from 25 °C. Haemocyte activation in response to live and heat-killed V. mimucus and LPS, both in vivo and in vitro, resulted in a change in ratio of haemocyte type, with HC increasing in number and both SGC and LGC decreasing. HCs in vitro tends to dominate and the SGCs/LGCs fraction is diminished. HCs rise in proportion because granulocytes are extinguished (or convert to HCs) in the course of the immune response. These changes were affected by both incubation time and temperature. However, the in vivo’s DHCs, show relatively more granularity than the in vitro studies. This is consistent with the idea that the host response is based around the granular fraction. However, phagocytic activity assessed through TEM and FCM revealed that all three haemocyte cell types are involved in phagocytosis of Gram negative bacteria, with HC and SGC demonstrating the XIII highest activity. Furthermore, haemocyte activation with bacteria resulted in NO production, with highest levels produced by SGCs and LGCs.

These findings are the first in C. cainii and demonstrate that circulating haemocyte are a dynamic population of cells involved in the elimination of pathogens. In fact there are heaps of HCs present in vitro but these cannot granulate in vitro to restore the balance and also the hematopoietic tissue (Hpt) appears to modulate the cell types in vivo. The recommended lineages of C. cainii haemocytes have been established upon morphological characteristics, since currently only a little information know about the cellular and biochemical signals involved in regulating C. cainii haemocyte type in vivo. The data is suggestive that there is one cell line forming three distinct haemocyte subtypes of a contiguous nature. The information about the innate immune repertoires of C. cainii in response to the invasion of diseases is very important to help further our understanding about the mechanism of freshwater crayfish immunity to disease infection and developing strategies for management of the disease.

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