Immune defence reactions in freshwater crayfish, Cellular components

The freshwater signal crayfish, Pacifastacus leniusculus, is a well-established model for studying the immune system of invertebrates. Many important discoveries have been made in this species, among others related to the clotting reaction, hematopoiesis, the prophenoloxidase activating system, the functions of crustacean immune cells, and pathogen recogni-tion. In this thesis, a small contribution is made to this body of work, with the focus on the crayfish cellular defence reactions to the fungal pattern recog-nition protein, beta-1,3,-glucans, and to an oomycete, the type of pathogen that causes crayfish plague. Through mapping the proteomic reaction in the hemocytes to beta-1,3,-glucan, and then studying some of the identi-fied proteins in more detail, it brings us closer to understanding how these animals defend themselves against fungal infections without relying on adaptive immunity. A proteomic screening of hemocytes was conducted after an injection of laminarin, a beta-1,3,-glucan, and compared to the response to a saline injection and un-injected controls. When compared to both control groups, three proteins were specific to the laminarin group: a glycine-rich peptide, a Kazal-type proteinase inhibitor, and one putative chitin-binding protein; none of which had been previously described. Three other proteins were upregulated in both the saline and the laminarin group: one invertebrate-type (i-type) lysozyme, one crustin, and masquerade. The glycine-rich peptide and the i-type lysozyme were both investigated in de-tail for their potential functions in the immune- and wounding responses. The peptide was found to be expressed in several tissues, and have a specif-ic activity against the crayfish plague pathogen Aphanomyces astaci, with no effect against any other tested species of oomycete, fungi or bacteria. The i-type lysozyme (Pl-ilys), which was muramidase deficient and there-fore likely not involved in antibacterial defence, was found to be able to destabilise clots formed from the crayfish clotting protein and transglutam-inase. This result indicated a possible new function for muramidase-deficient i-type lysozymes in crustaceans. A single-cell RNA-sequencing study was also conducted to investigate the types of blood cells and hema-topoietic stem cells in P. leniusculus, the result of which shows that there are several potential subtypes among the of granular, semi-granular, hya-line and hematopoietic cells.