| dc.description.abstract | 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. | en |
