Endotoxin-induced coagulation reactions and phenoloxidase activity modulation in Sudanonautes africanus hemolymph...

Sudanonautes africanus is a freshwater crab local to Nigeria and West Africa that has no documentation of its innate immunity reactions. The objective of this study was to assess the effect of endotoxin (lipopolysaccharide [LPS]) on coagulation and on phenoloxidase (PO) activity in the hemolymph fractions of S. africanus. The hemolymph from each of 10 live crabs was obtained by carapace puncture and then fractionated into plasma and hemocytes. The hemocytes were then processed and then fractionated into hemocyte lysate (HL), hemocyte lysate supernatant (HLS), and hemocyte lysate debris (HLD). In one study, each fraction was then incubated with a fixed level of LPS in the presence or absence of exogenous calcium (Ca(2+)) ion. In another study, the LPS concentration was varied in order to study its effect on protein coagulation when an optimal ratio mixture of plasma:HLS was present as well as on PO activity in the plasma and HLS fractions. The results of the first set of studies demonstrated that a presence of Ca(2+) in the LPS-induced clotting reactions was essential. The next set of studies showed that a 7:1 plasma:HLS mixture yielded a higher level of coagulation than any other ratio tested in the presence of 1 EU LPS/ml. When this same plasma:HLS mixture ratio was used to ascertain the effect of varying LPS level on coagulation, the response trended higher up to a dose of 3.0 EU/ml., and decreased thereafter until 7 EU/ml. As expected based on the effect of LPS on PO activation, an increasing presence of LPS led to a general trend increase in activity of the enzyme in the plasma fraction; however, the effect was moreover inhibitory in the HLS fraction. From the results here, we conclude that protein coagulation is an important response, along with increased PO activity, that could manifest in Sudanonautes africanus after exposure to 'free' LPS or select LPS-bearing organisms in their environment.