Effect of the axenic nematode Steinernema carpocapsae on the immune responses of two lepidopteran larvae, Galleria mellonella (F. Pyralidae) and Malacosoma disstria (F. Lasiocampidae)

Live adult and juvenile entomopathogenic as opposed to freeze-killed Steinernema carpocapsue DD136 (P. Nematoda) did not have adhering haemocytes of lepidopteran insect larvae of Galleria mellonella or Malacosoma dissiria in vitro or in vivo. Haemocyte accumulation on dead nematodes was associated with cuticular activation of host phenoloxidase. Live nematodes and their exudates did not activate the enzyme. Live-nematode exudate but not dead-nematode exudate inhibited granular cell and some plasmatocyte adhesion to slides, increased granular cell but not plasmatocyte dissociation from preformed haemocyte monolayers and in vivo elevated total haemocyte counts while impairing bacterial removal from the haemolymph. Immunosuppressant activity by third stage juvenile nematodes may represent the release of non-cuticular inhibitors.;Initial characterization of the immunosuppressant properties revealed the suppressant(s) were weakly hydrophobic, were not affected by repeated freeze-thawing but most were destroyed at 65°C. A residual heat-stable suppressant remained in the exudate, attributed to lipopolysaccharides. Exudate with a cocktail of protease inhibitors partially increased haemocyte adhesion with a small decline in total protein occurring indicating that non-enzymic proteins are also involved. A decrease in exudate total protein but not immunosuppression in exudate with trypsin and chymotrypsin-conjugated agarose beads indicated the presence of non-suppressant trypsin and chymotrypsin sensitive proteins. Correlation analysis between enzyme activities and level of adhering haemocytes implied several enzyme types including trypsin and chymotrypsin may participate in immunosuppression.;Herein, is reported for the first time that the serine proteases, trypsin and chymotrypsin released by live axenic Steinernema carpocapsae inhibits haemocyte adhesion to slides. Increasing concentrations of enzyme-specific inhibitors and polyclonal antibodies to trypsin and chymotrypsin in the exudate inhibited the enzymes and increased haemocyte adhesion, compared to the controls. Incubating exudate with chymotrypsin specific inhibitor and trypsin substrate showed no reaction indicating that trypsin-like and chymotrypsin-like enzymes react only with their respective substrates. Antibodies targeting specific serine proteases in exudate elevated haemocyte adhesion but in PBS alone, neither enzyme antibody affected haemocyte adhesion, suggesting the haemocytes do not react with these foreign proteins. There was no observed cross-reactions of antibodies with the opposite enzyme targets. In conjunction with chemical inhibitors, the antibodies confirmed the presence of suppressive trypsin-like and chymotrypsin-like enzymes versus non-suppressive chemically inhibited enzymes. Both chemical inhibitors and antibodies in the exudate increased bacterial removal from the haemolymph in vivo. The release time of both enzymes from the nematode in artificial serum was 2 h with a peak release at 4 h for trypsin and 6 h for chymotrypsin.;Index descriptors: Steinernema earpocapsae, Galleria mellonella, Malacosoma disstria, insect, inhibition haemocytes, adhesion, phenoloxidase, nematode encapsulation, protease inhibitor, trypsin, chymotrypsin, Apizym, chemical inhibitor, polyclonal antibody, trypsin, chymotrypsin, serine protease.