Purification and Properties of the Low Molecular Weight Protein from Haemolymph of the Tsetse Fly, Glossina Morsitans Morsitans

Abstract

Tsetse fly is an insect of great economic importance to man because it transmits sleeping sickness to both man and his livestock. The insect feeds on a bloodmeal thereby transmitting the disease from infected to healthy individuals. The tsetse fly, Glossina spp. belongs to the order Diptera and family Glossinidae and 22 species have been identified. The fly is mainly found in humid areas especially along the river valleys and bushes in Sub-Saharan Africa . Insects are known to posses haemolymph as the circulatory fluid equivalent to blood and lymph in vertebrates. In both cases, the circulating fluids have been shown to contain lipoproteins as the major component. Various haemolymph proteins serving a wide range of functiong have been described in many insect species. Whereas some of these proteins have been well studied in some insect species, the same has not been done in the tsetse fly despite its great economic importance. This study therefore, investigates the properties of a low molecular weight protein isolated from haemolymph of adult male G.m morsitans. The protein was purified from haemolymph by a combination of density gradient ultracentrifugation and gel permeation chromatography. The protein has a molecular weight of 23,000 dalton as determined by electrophoresis on polyacrylamide gels. The hydrated density of the low molecular weight protein, determined by density gradient equilibration was = 1.29 g/ml. Staining of the protein with Sudan Black confirmed that it is lipidated. Carbohydrate analysis showed the low molecular weight protein to be non-glycosylated since it neither bound onto concanavalin A-Sepharose affinity chromatography column nor stained with periodic acid Schiff's stain on SDS-gels. Amino acid analysis of the low molecular weight protein showed very high content of acidic amino acids, glutamate (16%) and aspartate (13%) . The protein also contained high proportions of serine (11%) and glycine (10%). The content of basic amino acid, lysine (10%) was also high. De novo synthesis using radiolabelled [35s] methionine and [14c]- leucine shows the low molecular weight protein is synthesized by the fat body and then released into the haemolymph. An investigation was carried out to determine whether there was immunological cross-reactivity between other haemolymph proteins from eleven insect species representing six orders. From the results obtained by both double radial immunodiffusions and immunoblots, the low molecular weight protein was not detected in any species other than the order Diptera, family Glossinidae to which the tsetse fly belongs. Thus, no cross-reactivity was observed with other insect haemolymph proteins, suggesting this protein could be unique to tsetse fly.