Storage and sexual separation of Glossina morsitans morsitans Westwood puparia.

Maintenance of unknown-age tsetse puparia at 4 degrees C for six days substantially reduced emergence; normal emergence occurred when puparia were held at temperatures of 12 degrees, 14 degrees, or 16 degrees C for two, three or four weeks, respectively. In-storage eclosion of adults was either suppressed completely at the temperatures used or was slight. Weight, puparial duration, and density of puparia were examined in an attempt to detect sexual differences in puparia. There appeared to be no usable correlation between pupal weight and sex, but the difference between the puparial duration of each sex offers a mechanism for retaining 81% of the females produced and releasing 84% of the males. The flotation of unknown-or known-age puparia in methanol-water solutions of varying specific gravities demonstrated some diffences in the density of male and female puparia. However, these differences were not great or consistent enough to provide an efficient system of separating puparia by age or sex.     

Selection for drug resistance in Trypanosoma congolense during cyclic transmissions through Glossina morsitans morsitans and drug treated rabbits

A drug-sensitive Trypanosoma congolense (IL 1180 strain), with a known CD50 and CD90 (doses required to cure 50 and 90% of the infected animals) was cyclically passaged through tsetse flies. The infected flies were then fed on rabbits which received weekly prophylactic treatment of Samorin. It was observed that the infections arising from flies maintained for over 60 days on drug-treated rabbits required higher curative doses to achieve a 50 and 90% cure. The results of this work suggest that a selection for drug resistance occurs when trypanosome stage in Glossina is continuously exposed to drug-treated animals.     

Nutrition of Glossina morsitans: metabolism of U-14C threonine during pregnancy.

Following injection of U-14C threonine into the haemolymph of adult female Glossina morsitans during late pregnancy, radioactivity was detected in the postparturient female and in its offspring, in threonine, lipids, and a range of non-essential amino acids. The level of radioactivity recovered from the larva was higher than that remaining in the injected adult, and the radioactivity recovered was considerably higher in the amino acid than in the lipid fraction. Administration of labelled threonine into maternal haemolymph on each of the first 8 days of the 9-10 day long pregnancy cycle was followed 24 h later by measurement of radioactivity in the developing o?cyte and in the intra-uterine progeny. The patterns of nutrient uptake are discussed in relation to vitellogenesis in the o?cyte and to growth of the larva. Analysis of the expired carbon dioxide and excreta was carried out 24 h after maternal injection of labelled threonine on the first or eighth day of pregnancy. Carbon dioxide and excreta from females in early pregnancy showed significantly higher radioactivity than those from females in late pregnancy. In both cases, radioactivity in the amino acid fraction from the excreta was extremely small and about 95% of the total activity was in uric acid. These results are discussed in terms of the utilization of threonine in relation to the metabolic demands for various nutriments by the pregnant female.     

Effect of amino acids on the plasma concentration and urinary excretion of uric acid and uridine.

To determine the effect of amino acids on the plasma level and urinary excretion of uric acid and uridine, 200 mL 12% amino acid solution, and 2 weeks later, 100 mL physiological saline solution containing glucagon (1.2 microg/kg weight), was infused into five healthy men. Both increased the urinary excretion of uric acid and the concentration of glucagon, insulin, and glucose in plasma and pyruvic acid in blood, whereas they decreased the concentration of uridine and inorganic phosphate in plasma. However, neither the amino acid infusion nor glucagon infusion affected the concentration of purine bases (hypoxanthine, xanthine, and uric acid), cyclic adenosine monophosphate (cAMP) in plasma, or lactic acid in blood or the urinary excretion of oxypurines (hypoxanthine and xanthine), uridine, or sodium. These results suggest that glucagon may have an important role in the amino acid-induced increase in urinary excretion of uric acid and decrease in plasma uridine.     

Investigations on the transmissibility of Trypanosoma congolense by the tsetse fly Glossina morsitans morsitans during its development in a mammalian host

Experiments were conducted to investigate the effect of the developmental stage of a monomorphic T. congolense IL1180 strain, in a vertebrate host, on its transmissibility by the tsetse fly Glossina morsitans morsitans Westwood (Diptera: Glossinidae). Batches of 160 male teneral tsetse flies were given a single bloodmeal on mice infected with this T. congolense strain 4, 5, 6, 7 or 10 days post-infection. The proportion of infected flies in each of those batches showed that the stage of development of the trypanosome does affect the proportion of flies that develop a mature or immature infection with immature and mature infection rates of flies infected on days 5 or 10 significantly higher. The proportion of infected flies was not affected by the parasitaemia at the moment of infection. Results show that tsetse flies can become infected at any phase of the development of the T. congolense IL 1180 strain but the ease with which trypanosomes develop in the fly depends on the phase in the parasite's development in the host. Those observations suggest that in analogy with the pleomorphic T. brucei s.l. adaptation of the monomorphic T. congolense to development in the fly may also determine the parasite's transmissibility. Moreover, the findings stress the importance of standardising experiments in which the vectorial capacity of tsetse flies is determined and compared.     

Infection rates in Glossina morsitans morsitans fed on waterbuck and Boran cattle infected with Trypanosoma congolense.

Teneral Glossina morsitans morsitans were fed on waterbuck (Kobus defassa) and Boran cattle (Bos indicus) infected experimentally with Trypanosoma congolense clone IL2895. Infection rates in tsetse varied from 9 to 31% when fed on cattle, and from 2 to 59% when fed on waterbuck. In waterbuck, infections were often detected through the development of parasites in tsetse at times when parasitaemia could not be detected through microscopic examination of blood. Male and female, and 1- and 2-day-old flies were equally susceptible to infection on both hosts. Infection in tsetse was associated with a 14% absolute reduction in survival during the month following the infective feed.     

Nuclear coat and viruslike particles in the midgut epithelium of Glossina morsitans sspp.

The ultrastructural aspects of the nuclear coat formation in midgut epithelial cells of pupae and adult flies of G. morsitans sspp. are described. Out of four different species of Glossina examined, this peculiar structure was only found in G. morsitans sspp. Three different types of viruslike particles were found in midgut epithelial cells. One type which is of the same kind of particle found in the salivary glands, lies inside of cytoplasmic vesicles. Two other types of particles were detected in the nuclei.     

Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host’s brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.     

The effect of Trypanosoma brucei infection of the localization of salivary gland cholinesterase in Glossina morsitans morsitans

When salivary glands of the tsetse fly, Glossina morsitans morsitans, are stained for cholinesterase (ChE) activity, a net-like pattern of reaction product is observed surrounding each epithelial cell of the gland's secretory region. Glands infected with Trypanosoma brucei brucei show a progressive reduction in this ChE activity as the parasites develop. When the infection is mature, ChE is rarely detected in the epithelial layer but appears in the lumen of gland. The luminal ChE responds to substrates and inhibitors in the same manner as the epithelium-associated enzyme and appears to have leaked from the epithelium due to cellular damage in epithelium of the infected gland. The possible effect of glandular damage on feeding behaviour and state of health is discussed.     

Some observations on factors associated with the development of Trypanosoma brucei brucei infections in Glossina morsitans morsitans

The susceptibility of Glossina morsitans morsitans to Trypanosoma brucei brucei infection was shown to be age-dependent during the first 12 h: the youngest age group (1-8 h after emergence) being more susceptible than the older ones. The susceptibility was enhanced by cooling the young flies to a temperature of 0-5 degrees C for 30 min. Male flies were found to be more susceptible than females. The number of trypanosomes ingested did not influence the subsequent salivary-gland infection rates observed in G.m. morsitans; however, there was a relationship between the number ingested and subsequent T.b. brucei midgut infections in the flies.     

Defence reactions of Glossina morsitans morsitans against different species of bacteria and Trypanosoma brucei brucei

Tsetse flies, Glossina morsitans morsitans, fed on rats infected with Trypanosoma brucei brucei showed wide fluctuations in total and differential haemocyte counts. Similar fluctuations occurred in controls fed on non-infected rats and also between the two groups without showing any difference which could be attributed to the infection. Trypanosome infection of the tsetse haemocoel occurred in 16.25% of the flies, starting from the second day after feeding on the infected rats, but salivary glands and proboscis became infected only after the eleventh day. About 2% of bloodstream forms of T. b. brucei injected into tsetse haemocoels completed their developmental cycle successfully. Injection of tsetse homogenates into teneral G. m. morsitans prior to exposure to trypanosome-infected feed increased T. b. brucei infections in the flies significantly. Injection of live Escherichia coli, Enterobacter cloacae and Acinetobacter calcoaceticus into tsetse induced a remarkable increase in two pre-existing haemolymph proteins with molecular weights of about 70 and 17 kilodaltons, while live Bacillus subtilis and Micrococcus luteus induced a very weak response or sometimes none at all. T. b. brucei also failed to induce any increase in these proteins. Inoculation of G. m. morsitans with live E. coli und T. b. brucei prior to feeding on trypanosome-infected rats had no effect on the salivary gland and proboscis infection rates by T. b. brucei. Injection of live T. b. brucei into the haemocoels of tsetse caused no change in total haemocyte counts, but the trypanosomes disappeared from the haemolymph so rapidly that by 48 h post-injection, only about 1% were left.