Abstract:
Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus
zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS
Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as
compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by
flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was
gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety
(detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared
to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the
supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc
treated cells (P%0:05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in
cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish
haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the
polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by
LPS, thereby indicating common innate immune recognition mechanisms to bacterial antigens between cells from
mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of
invertebrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic
compounds.
