Protein components of the blood meal of tsetse flies during a digestion cycle
Due to the economic importance of the type of trypanosomiasis that infects man, wild and domestic animals, there is growing interest in studies of tsetse flies that are known to be vectors of this disease. Trypanosomes of the brucei sub-group have a complicated life cycle in Glossina, part of which is spent in the alimentary canal. Tsetse flies are infected by ingestion of a blood meal containing trypanosomes. Biochemical conditions that might influence the development of these organisms in the tsetse fly gut have not been fully established. It was the purpose of this study to make basic investigations of the components of the blood meal during a complete digestion cycle in a normal fly, in order to provide preliminary information for future studies. Methods of disc acrylamide gel electrophoresis and thin layer chromatography were used for the separation of components. The protein components of blood of a goat obtained from Limuru in Kenya, on which the tsetse flies were fed were typed by means of starch gel electrophoresis, and by visual comparison of the bands to pictures of known types. Twenty seven protein bands were revealed on acrylamide gels of serum. samples of the Limuru breed, and some of these bands were identified according to the nomenclature of Davis (1964). The albumins, pre-albumins and several post-albumins were located on the gels. A number of transferrins were well separated, and haptoglobins, lipoproteins and carbohydrate-bound protein bands were stained. Electropherograms of pure haemo-globinrevealed six bands. The blood of a Saanan breed was analyzed in a similar manner, and from comparison of the mobilities of the bands, polymorphism of the protein components of the two breeds was demonstrated. A schedule of the blood meal throughput in the alimentary canal of the tsetse fly under the normal laboratory conditions was as follows: a large meal was engorged causing the distention of the crop and anterior section of the midgut. The crop was then rapidly emptied, the blood meal being completely transferred to the anterior section of the midgut within 30 min. At the same time, fluid was removed from the meal at a high rate, appearing at the anus as colourless drops of urine. The blood in the anterior section became increasingly concentrated and was passed on to the middle section by peristaltic movements. mood was present in the middle section within 10 min. and became increasingly darker and thicker, but the climax of digestion was not reached until several hours later. By-products of digestion were passed on to the posterior section of the midgut which did not distend and appeared greenish black under the microscope. 3 hr. after feeding, small portions of contents of the posterior section were occasionally emptied into the hindgut, and thereby voided at the anus as pasty faeces. Samples Of contents of the blood meal from various sections of the alimentary canal were analyzed to obtain an indication of the processes occurring in the meal during the entire digestion cycle: Visual inspection of gels of crop contents revealed no changes in the protetn band pattern of the original host blood. However, an extra band was observed, which was thought to represent a component of the tsetse fly saliva. Densttometric measurement of protein concentration of the crop contents at various time intervals after feeding demonstrated that there was no significant change, and that fluid had not been removed from the contents. Protein bands of the blood meal sample from the anterior section clearly illustrated the concentration process. 1 hr. after feeding the total concentration of the proteins had doubled, showing that fluid had been removed from the blood meal. This fluid has been referred to as "urine" in this study. The rate of urine secretion was discussed, and some of the components determined. Radioisotope investigations confirmed that the blood meal was the origin of the components of urine, and that these substances were eliminated by way of the haemolymph. During the concentration of proteins in the anterior section, lysing of erythrocytes occurred, the haemoglobin band becoming so intense and broad that it overlapped several other bands. Further inspection of the band pattern revealed that a few changes, other than of concentration of the proteins, did occur in this section. A number of bands were noted to be absent on gels run 16 hr. after engorgement. Explanation for this could only be that there was some other unknown activity in the anterior section. The rate of emptying of this section was low, and depended on the rate of digestion of the blood meal further down the alimentary canal. The middle section of the midgut is the region of active digestion of the proteins. The rate of digestion was found to be low and fairly constant for the first 24 hr. Then there was a steady in- . crease which continued until digestion was completed. It was also determined that digestion proceeded, at different rates for the various protein zones, haemoglobin being broken down fastest, and albumin slowest. Analysis of the contents of the middle section 72 hr. after feeding showed that except for small amounts of albumin, lipoprotein and glycoprotein all other proteins were absent. This implied that their digestion had been completed thin this time. Electrophoretically separable by-products of digestion were located on the gels as faint bands 3 hr. after feeding, and many more bands of these products were revealed 24 hr. after engorgement. A number of the bands were coloured, did not react with Amido Black, and were referred to as "pigments". Parallel runs of samples containing pure bilirubin indicated that one of the coloured pigment bands in samples of the middle section was due to bilirubin. Thin layer chromatograms also suggested the presence of bilirubin and biliverdin in these samples. Other iron-containing by-products were located on the gels and these, together with the pigments, were thought to be breakdown products of haemoglobin. After 24 hr.; blood meal material in the posterior section contained very little undigested proteins showing that digestion was completed in the middle section. Material passed on to the posterior section during this time contained only very small amounts of albumin, lipoprotein and glycoprotein, all other components having been digested. The predominantly black- green substances of this section gave several pigment bands. Occasionally, waste products of digestion were passed on to the hindgut by waves of peristaltic contractions. Fluid in the ileum . was a mixture of waste ·products from the midgut and the Malpighian tubules. Sometimes this fluid was observed to flow backwards and forwards at the proximal ends of the Malpighian tubules, indicating that the pyloric valve probably did not prevent the back-flow of fluid into the tubules. The white granular solids of faecal matter collected between 0 and 2 hr. after feeding revealed no bands, showing that no material from the midgut had been discharged within this time. Between 3 and.5 hr., the pale yellow and grey faeces were probably a mixture of remains of meconium seen at the base of the posterior section on dissection of the fly soon after feeding, and of excretory products of the Malpighian tubules. From 5 to 96 hr., the brown, dark brown and black pasty faecal matter contained only small amounts of undigested albumin, lipoprotein and glycoprotein. Thin layer chromatograms suggested the presence of bilirubin, blliverdin and a number of unidentified coloured pigments. It was concluded that the digestive mechanism of proteins in the tsetse flies was highly efficient, with only 3.4% of the original amount of protein left undigested. Analysis of the haemolymph proteins after a blood meal showed that there was a small increase in the concentration of the proteins corresponding to the increased intensities of a few fast-moving bands. The level of protein concentration remained fairly constant between 24 and 48 hr. after feeding, indicating that if any proteins were formed or released into the haemolymph, an equal amount was removed. From 48 to 72 hr. there was a drop in the protein level which was thought to be probably due to the decrease in the nutrients available from the midgut at the completion of digestion. 96 hr. after feeding, there was a considerable drop in concentration, and it was suggested that this was the result of starvation of the insect. 1.e whole process of digestion in the tsetse flies is indicative of the high utilization of the ingested blood meal. The varying environmental conditions of the alimentary canal could have vital Influences on the development and survival of the trypanosomes.
School of Biological Sciences, University of Nairobi
Master of Science