Effects Of Several Immunostimulants On The Yield, Activities Of Phenoloxidase And The Number, Ultrastructure Of Hemocytes In Clam Ruditapes Philippinarum

Immunostimulants are well-known for their stimulating potentials to reduce losses in aquaculture. Direct stimulation of nonspecific immunity seems to be the most promising methods for microbial control in marine invertebrates. Studies have been performed on stimulation of the nonspecific immune defence of shrimps, describing a wide range of attractive methods for prophylaxis in aquaculture, but no study has been performed in clams by now.To study the stimulating mechanisms of immunostimulates in clam autogenous immunocompetence, the immunostimulating effects ofβ-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on the phenoloxidase (PO) from clam (Ruditapes philippinarum) were investigated in this study. Using L-dihydroxyphenylalanine (L-DOPA) as a specific substrate, PO from clam (Ruditapes philippinarum) was purified by Q Sepharose Fast Flow ion-exchange chromatography and Sephacryl S-100 gel-filtration, and characterized biochemically and enzymatically in this study. The PO molecule had a high oxidative activity, and the proPO molecule had almost no oxidative activity.Under the stimulation ofβ-glucan, LPS, inactivated Vibrio Harvey and inactivated Vibrio anguillaru, the amount of PO synthesized increased about103.45 %, 65.52%, 17.24 % and 27.59 %, respectively and PO activity increased about105.9%, 64.7%, 17.65% and 29.41%, respectively, but the unit activity of PO remains almost unchanged. The immunostimulation ofβ-glucan is most efficient on the amount of PO synthesized and PO activity.Under the stimulation ofβ-glucan, LPS, inactivated Vibrio Harvey and inactivated Vibrio anguillaru, the number of all hemocytes increased about 42.7%, 39.56%, 42.0% and 83.0 %, respectively and the number of semigranular cells increased about 49.49 %, 41.08 %, 44.78 % and 69.02 %, respectively. The immunostimulation of the inactivated Vibrio anguillaru is most efficient on the the number of all hemocytes and the number of semigranular cells. The highest percentege of semigranular cells in all kinds of hemocytes after stimulation implies that it plays a key role in the innate immune system of Ruditapes philippinarum.The unltrastructure of hemocytes also changed after stimulation.The amout of rough endoplasmic reticulum (RER), ribosomes and mitochondria in three kinds of hemocytes all increased obviously after the stimulation ofβ-glucan and LPS while only the unltrastructure of semigranular obviously changed after the stimulation of inactivated Vibrio Harvey and inactivated Vibrio anguillaru.Therefore, it can be concluded that, the enhancement of clam autogenous immunocompetence is finally realized via stimulating the proliferation of hemocytes, changing their unltrastructure to increase PO production and then oxidizing activities after stimulated by immunostimulates, which is put forward in the world at the first time.