Studies of hemolymph coagulation in Manduca sexta

Hemolymph coagulation is vital to insect survival because it minimizes loss of hemolymph and limits infection by potential pathogens. Insects have a rigid exoskeleton but do not have blood vessels. Rapid, localized activation of hemolymph coagulation is a necessity. Availability of information about insect hemolymph coagulation not only facilitates comparative studies of coagulation between vertebrate and invertebrate organisms to provide better insight into animal hemostasis, but also helps to identify novel targets in insects for developing new control strategies for pest management. While blood clotting in mammals has been studied extensively, information about insect systems is lacking. Research described in this thesis was conducted using M. sexta as a model insect to gather more information on insect hemolymph coagulation. It has been shown that coagulum at wound site is composed of filamentous coagulum and hemocytes; the filaments in coagulum are not cytoplasmic extensions; hemolymph coagulation in M. sexta is wound-associated; and a factor which can be extracted with saline from integument is active to promote formation of filamentous coagulum in hemolymph. The coagulum-inducing factor was partially characterized. A coagulum-forming protein, hemofibrin, was purified from hemolymph of nontreated larvae of M. sexta. Upon addition of hemolymph or cell-free hemolymph, hemofibrin gave rise to filamentous coagulum which was, in overall appearance, similar to that formed from purified bovine fibrinogen and thrombin in vitro. Hemofibrin was partially characterized. Partial amino acid sequences were obtained by automated Edman degradation of the intact protein and peptides generated by V8 protease and fractionated by HPLC. Northern blot probed with radioactive synthetic oligodeoxynucleotide probes revealed that epidermis is a rich source of hemofibrin mRNA. The contribution of hemofibrin to physiological coagulum as a structural component was also studied. A model is proposed for hemolymph coagulation in M. sexta based on the information available