Ellie O. Osir

Amplification of 1-amino-cyclopropane-1-carboxylic (ACC) deaminase from plant growth promoting rhizobacteria in Striga-infested soil

Experiments were conducted in pots to determine the growth effect of different rhizobacteria on maize under Striga hermonthica infestation. Three bacteria were selected based on their plant growth promoting effects. Whole bacterial cells of the rhizobacteria were used to amplify 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene by polymerase chain reaction (PCR). Each bacterial inoculation increased agronomic characteristics of maize although not always to a statistically significant extent. The extent of growth enhancement differs between the isolates. Enterobacter sakazakii 8MR5 had the ability to stimulate plant growth, however in the PCR study, ACC deaminase was not amplified from this isolate, indicating that not all plant growth-promoting rhizobacteria contain the enzyme ACC deaminase. In contrast, an ACC deaminase specific product was amplified from Pseudomonas sp. 4MKS8 and Klebsiella oxytoca 10MKR7. This is the first report of ACC deaminase in K. oxytoca.

Studies on tsetse midgut factors that induce differentiation of blood-stream Trypanosoma brucei brucei in vitro

An in vitro system for studying the transformation of bloodstream forms ofTrypanosoma brucei brucei into procylic (midgut) forms is described. In this system, transformation of the parasites was stimulated byGlossina morsitans morsitans midgut homogenates at 27° C but not at 4° C. The transformation-stimulating capacity was irreversibly destroyed by heating the midgut homogenates at 60° C for 1 h. A correlation was established between the transformation activity of the midgut homogenates and trypsin activity. The protease inhibitors (soybean trypsin inhibitor andN-p-tosyl-l-lysine-chloromethyl-ketone) inhibited trypsin activity and completely blocked the transformation of the parasites. Furthermore, the midgut homogenates could induce transformation only in the presence of blood. These results provide evidence for the involvement of trypsin or trypsin-like enzymes within the tsetse midgut in stimulation of the transformation of bloodstream trypanosomes.

Time-course haemolymph juvenile hormone titres in solitarious and gregarious adults of Schistocerca gregaria, and their relation to pheromone emission, CA volumetric changes and oocyte growth.

The haemolymph JH III titres in solitarious and gregarious adult desert locusts, Schistocerca gregaria, were examined in relation to corpus allatum (CA) volumes, aggregation-maturation pheromone production in males and oocyte growth in females. The JH titres of gregarious females were generally higher than those of solitarious females at all ages studied. The titre patterns, however, were similar: relatively high on day 10, dropping to low levels between days 20 and 25, before rising again by day 25. In the solitarious males, the JH titre was very low on day 10 after fledging, but increased gradually and reached a maximal amount on day 30. The JH titre in gregarious males was low on day 10, elevated on day 15 coinciding with the start of the production of the pheromone, and dropped to a relatively low level on day 20 around the time of maximal pheromone production, then rising again by day 25. These results suggest that biosynthesis of the pheromone is associated with a high JH titre peak in the haemolymph. Although a clear relationship was found during the first gonadotropic cycle between JH titres, on one hand, and CA volume and oocyte growth, on the other, in both phases, no such correlation could be discerned in the second cycle.

Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly Glossina longipennis

A blood-meal-induced lectin (agglutinin) with proteolytic activity was isolated from midgut extracts ofGlossina longipennis by a two-step procedure involving anion-exchange chromatography. It is a glycoprotein [native molecular weight (Mr, 61000±3000 da) composed of two noncovalently-linked subunits designated α (Mr, ∼27000 da) and β (Mr, ∼33000 da). The trypsin activity and the glycosyl residues were present on the α- and β-subunits, respectively. The native protein was capable of agglutinating both bloodstream-form and procyclic trypanosomes as well as rabbit red blood cells. This activity was strongly inhibited byD-glucosamine and weakly inhibited byN-acetyl-D-glucosamine. Similarly, soybean trypsin inhibitor abrogated agglutination of bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination activity was sensitive to temperatures above 40° C but was unaffected by chelators of metal ions. Antibodies raised against the protein were used in immunoblotting experiments to show the presence of a similar protein in several members of theGlossina species. However, no cross-reactivity was detected with midgut extracts prepared from sandflies, mosquitoes, or stable flies. It is proposed that this molecule might play an important role in differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms.

Inhibition of Glossina morsitans midgut trypsin activity by D-glucosamine

The effect of the amino sugard-glucosamine on trypsin in crude midgut homogenates ofGlossina morsitans morsitans was studied in vitro. The results showed that the midgut trypsin was specifically and competitively inhibited byd-Glucosamine. Glucose, fructose, mannose, inositol, galactose, galactosamine,N-acetyl-d-glucosamine, and methyl-α-d-glucosamine were ineffective as inhibitors, even at concentrations exceeding 600mm.d-glucosamine also had a similar inhibitory effect on bovine pancreatic trypsin. In both cases, the inhibition was incomplete as shown by nonlinear Dixon plots. The Michaelis and inhibition constants estimated for the midgut trypsin were 41±2 μm and 68±3mm, respectively. These results suggest that the susceptibility of tsetse flies to trypanosome infection, which is associated with high midgut glucosamine levels, could be due to inhibition of trypsin or trypsin-like enzymes by this sugar.

FAT BODY TRIACYLGLYCEROL LIPASE IN SOLITARY AND GREGARIOUS PHASES OF SCHISTOCERCA GREGARIA (FORSKAL) (ORTHOPTERA: ACRIDIDAE)

The activities of the fat body triacylglycerol lipase were determined using 14C-trioleoylglycerol. Resting activities were estimated at 1.98 ± 0.24 and 1.95 ± 0.53 nmol free fatty acid (FFA)/hr/mg protein for gregarious and solitary locusts, respectively. Administration of adipokinetic hormone (AKH) I led to the activation of lipase with peak activities occurring after 30 min. In the solitary locusts, activities of 2.28 ± 0.16 and 2.30 ± 0.43 nmol FFA/hr/mg were obtained following administration of 10 and 2 pmol AKH I, respectively. In the gregarious locusts, enzyme activities of 3.17 ± 0.66 and 2.47 ± 0.39 nmol FFA/hr/mg were obtained after administration of 10 and 2 pmol AKH I, respectively. The Km values were estimated at 46.67 and 18.75 μM for gregarious and solitary locusts, respectively. Similarly, the Vmax values were estimated at 10.29 and 2.52 nmol/hr/mg for gregarious and solitary locusts, respectively. These results confirm phase-dependent differences in lipase properties with the gregarious form having a higher catalytic ability, but a lower affinity for the substrate than the solitary type.

Cloning and expression of a Bacillus thuringiensis (L1-2) gene encoding a crystal protein active against Glossina morsitans morsitans and Chilo partellus.

Abstract. A local isolate of Bacillus thuringiensis, designated L1-2, that is toxic to Chilo partellus was found to be toxic to the adult tsetse fly, Glossina morsitans morsitans. The δ-endotoxin crystals derived from the isolate gave a major protein band with a molecular weight of Mr 130,000–140,000 on denaturing polyacrylamide gel electrophoresis. The sequence of the cloned gene was found to be similar to that of the B. thuringiensis subsp. kurstaki HD-73 cryIA(c) gene, having one amino acid difference at position 148 and four additional DNA differences.

The effect of host blood in the in vitrotransformation of bloodstream trypanosomes by tsetse midgut homogenates

Abstract. Midgut homogenates prepared from Glossina morsitans morsitans, that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma bruceiinto procyclic (midgut) forms in vitro.Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.

Properties of a blood-meal-induced midgut lectin from the tsetse fly Glossina morsitans

The properties of a blood-meal-induced lectin (agglutinin) from the midgut ofGlossina morsitans capable of agglutinatingTrypanosoma brucei were studied in vitro. The midgut homogenate from flies that had been fed twice had the highest agglutination activity, followed by that from the once-fed flies and that from the unfed insects. As compared with the bloodstream-form trypanosomes, a much lower concentration of the midgut homogenate was required for agglutination of the procyclic parasites. Furthermore, the agglutination process was specifically inhibited byD-glucosamine. Soybean trypsin inhibitor abrogated agglutination of the bloodstreamform parasites, whereas the procyclics were unaffected. The agglutination process was temperature-sensitive, with little activity being evident between 4° and 15°C. Similarly, heating the midguts to 60°–100°C led to loss of activity. When the midgut homogenate was separated by anion-exchange chromatography, the agglutination activity co-eluted with trypsin activity at approximately 50% NaCl. These results suggest a very close relationship between midgut trypsin-like enzyme and the agglutinin. Since successful agglutination of bloodstream-form trypanosomes requires protease activity, it may be that the enzyme cleaves off some surface molecules on the parasite surface, thus exposing the lectin-binding sites.

Effects of juvenile hormone treatment on phase changes and pheromone production in the desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae).

The roles of juvenile hormone III (JH III) on phase changes and pheromone production were examined in laboratory-reared gregarious desert locust, Schistocerca gregaria (Forskal). The hormone was applied to 5th instar nymphs and newly emerged adult locusts. Generally, the 5th instar nymphs exhibited a higher sensitivity to hormone treatments than the adults. Hormone applications inhibited pheromone production (as measured by the amounts of phenylacetonitrile released). In addition, JH III had a significant effect on the external colouration and absorbance ratios of the haemolymph pigments. It is concluded that the effects of exogenous JH III on gregarious locusts represent a shift towards the solitarious phase.