Subcellular degeneration of mycetomal endocytobionts in tsetse,Glossina morsitans morsitans,inoculated twice withEscherichia coli

Specimens of mycetome, a portion of anterior midgut harboring intracellular bacterioids (endocytobionts), obtained from both untreated control female tsetse,Glossina morsitans morsitans, and those inoculated twice with strain D31 ofEscherichia coli, were processed for routine electron microscopy, and the endocytobionts were examined for structural alterations. In the controls, mycetocytes contained intact bacterioids with numerous, electron-dense ribosomal particles in the cytoplasm. FemaleG. m. morsitans subjected to two hemocoelic inoculations with the liveE. coli showed severe degeneration of the subcellular components of the endocytobionts characterized by advanced lysis and rarefaction. The observed endocytobiotic degeneration is attributed to effects of induced humoral antibacterial factors.     

Two Tsetse Fly Species, Glossina palpalis gambiensis and Glossina morsitans morsitans, Carry Genetically Distinct Populations of the Secondary Symbiont Sodalis glossinidius

Genetic diversity among Sodalis glossinidius populations was investigated using amplified fragment length polymorphism markers. Strains collected from Glossina palpalis gambiensis and Glossina morsitans morsitans flies group into separate clusters, being differentially structured. This differential structuring may reflect different host-related selection pressures and may be related to the different vector competences of Glossina spp.     

Sodalis glossinidius (Enterobacteriaceae) and Vectorial Competence of Glossina palpalis gambiensis and Glossina morsitans morsitans for Trypanosoma congolense Savannah Type

Sodalis glossinidius is an endosymbiont of Glossina palpalis gambiensis and Glossina morsitans morsitans, the vectors of Trypanosoma congolense. The presence of the symbiont was investigated by PCR in Trypanosoma congolense savannah type-infected and noninfected midguts of both fly species, and into the probosces of flies displaying either mature or immature infection, to investigate possible correlation with the vectorial competence of tsetse flies. Sodalis glossinidius was detected in all midguts, infected or not, from both Glossina species. It was also detected in probosces from Glossina palpalis gambiensis flies displaying mature or immature infection, but never in probosces from Glossina morsitans morsitans. These results suggest that, a) there might be no direct correlation between the presence of Sodalis glossinidius and the vectorial competence of Glossina, and b) the symbiont is probably not involved in Trypanosoma congolense savannah type maturation. It could however participate in the establishment process of the parasite.     

Insect immunity: Induction of cecropin and attacin-like antibacterial factors in the haemolymph of Glossina morsitans morsitans

Injections of live Escherichia coli into adult tsetse flies, Glossina morsitans morsitans induced an antibacterial activity in the haemolymph after a lag period of 6–18 hr. Peak activity occurred after 24–72 hr with a dose of 10⁴ bacteria/fly. Acidic electrophoresis of immune haemolymph from G. m. morsitans followed by an antibacterial assay on the gel revealed the presence of cecropin- and attacin-like factors. The induction of antibacterial activity in tsetse was completely blocked by injection of cycloheximide, a known inhibitor of protein synthesis in eukaryotic organisms. Purified InA from Bacillus thuringiensis, a proteolytic enzyme with specificity for cecropins and attacins in haemolymph, inactivated the antibacterial activity in tesetse immune haemolymph. When tested against 10 different bacterial species, the spectrum was the same for the antibacterial activity in immune haemolymph from tsetse and Cecropia.     

Precocene-induced sterility in F1 generation of Glossina morsitans morsitans

Precocene treatment does not disrupt the events of reproduction in Glossina morsitans morsitans or induce any apparent changes in treated tsetse. However, some females of the F₁ generation are either sterile or show retardations in follicle development. Sterility is not reversed spontaneously or with juvenile hormone analogues. The critical period for precocene action is related to each ovulation. The corpora allata of precocene-treated tsetse are normal, but those of F₁ sterile females are degenerate. The occurrence of retardation has enabled the characterisation of stages in follicle development in G. m. morsitans.     

Studies on the transmission of a West African stock of Trypanosoma vivax to rabbits,rats, mice and goats by Glossina morsitans morsitans and G. m. centralis

The cyclical transmission of a West African stock of Trypanosoma vivax by Glossina morsitans morsitans and G. m. centralis was studied. It was possible to transmit this parasite amongst rabbits, rats, mice and goats. Whereas goats and mice succumbed to the disease very rapidly, rats and rabbits showed scant and transient parasitaemia and frequently suppressed the infection. A trypanosome challenge using single infected tsetse showed that goats became infected much more readily than mice. The infected vector can transmit the infection throughout its life but not at every feed.     

Tibial campaniform sensilla of Glossina morsitans

On the basis of behavioural tests and light microscopy, a pair of dome shaped sensilla occuring on the outer and inner surfaces of Glossina morsitans morsitans tibiae were considered to be sex pheromone receptors. Recent observations using electrophysiological tests and ultrastructural methods have revealed that those receptors have no olfactory function; they are ampaniform sensilla sensitive to mechanical stimulation.     

Ultrastructural Studies of Certain Aspects of the Development of Trypanosoma congolense in Glossina morsitans morsitans*

SYNOPSIS The course of Trypanosoma congolense infections in Glossina morsitans morsitans was followed by electron-microscopic examination of ultrathin sections of the guts and proboscises of infected flies. Guts dissected from flies 7 days after infection with culture procyclic forms of T. congolense had heavy trypanosome infections in the midgut involving both the endo- and ectoperitrophic spaces. Trypanosomes were also seen in the process of penetrating the fully formed peritrophic membrane in the central region of the midgut. By post infection day 21, trypanosomes had reached the proboscis of the fly and were found as clumps of epimastigote forms attached to the labrum by hemidesmosomes between their flagella and the chitinous lining of the food canal. Desmosome connections were observed between the flagella of adjacent epimastigotes. Flies examined at postinfection days 28 and 42 had, in addition to the attached forms in the labrum, free forms in the hypopharynx.     

Glossina morsitans morsitansandGlossina palpalis palpalis:Dosage Compensation Raises Questions about the Milligan Model for Control of Trypanosome Development

Gooding, R. H., and McIntyre, G. S. 1998.Glossina morsitans morsitansandGlossina palpalis palpalis: Dosage compensation raises questions about the Milligan model for control of trypanosome development.Experimental Parasitology90, 244–249. Evidence that dosage compensation occurs in tsetse flies was obtained by comparing the activities of X chromosome-linked enzymes, arginine phosphokinase and glucose-6-phosphate dehydrogenase inGlossina m. morsitansand hexokinase and phosphoglucomutase inGlossina p. palpalis, with the activity of an autosome-linked enzyme, malate dehydrogenase, in each species. The shortcomings of the X chromosome model for the control ofTrypanozoonmaturation in tsetse are discussed in light of these findings and previously published reports on the lack of fitness effects of matureTrypanozooninfections in tsetse and on published results on antitrypanosomal factors in male and female tsetse flies.     

Odorant and Gustatory Receptors in the Tsetse Fly Glossina morsitans morsitans

Tsetse flies use olfactory and gustatory responses, through odorant and gustatory receptors (ORs and GRs), to interact with their environment. Glossina morsitans morsitans genome ORs and GRs were annotated using homologs of these genes in Drosophila melanogaster and an ab initio approach based on OR and GR specific motifs in G. m. morsitans gene models coupled to gene ontology (GO). Phylogenetic relationships among the ORs or GRs and the homologs were determined using Maximum Likelihood estimates. Relative expression levels among the G. m. morsitans ORs or GRs were established using RNA-seq data derived from adult female fly. Overall, 46 and 14 putative G. m. morsitans ORs and GRs respectively were recovered. These were reduced by 12 and 59 ORs and GRs respectively compared to D. melanogaster. Six of the ORs were homologous to a single D. melanogaster OR (DmOr67d) associated with mating deterrence in females. Sweet taste GRs, present in all the other Diptera, were not recovered in G. m. morsitans. The GRs associated with detection of CO₂ were conserved in G. m. morsitans relative to D. melanogaster. RNA-sequence data analysis revealed expression of GmmOR15 locus represented over 90% of expression profiles for the ORs. The G. m. morsitans ORs or GRs were phylogenetically closer to those in D. melanogaster than to other insects assessed. We found the chemoreceptor repertoire in G. m. morsitans smaller than other Diptera, and we postulate that this may be related to the restricted diet of blood-meal for both sexes of tsetse flies. However, the clade of some specific receptors has been expanded, indicative of their potential importance in chemoreception in the tsetse.     

Polytene chromosome maps in four species of tsetse flies Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans (Diptera: Glossinidae): a comparative analysis

Photographic polytene chromosome maps from pupal trichogen cells of four tsetse species, Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans were constructed and compared. The homology of chromosomal elements between the species was achieved by comparing banding patterns. The telomeric and subtelomeric chromosome regions were found to be identical in all species. The pericentromeric regions were found to be similar in the X chromosome and the left arm of L1 chromosome (L1L) but different in L2 chromosome and the right arm of L1 chromosome (L1R). The L2 chromosome differs by a pericentric inversion that is fixed in the three species, G. pallidipes, G. morsitans morsitans and G. m. submorsitans. Moreover, the two morsitans subspecies appeared to be homosequential and differ only by two paracentric inversions on XL and L2L arm. Although a degree of similarity was observed across the homologous chromosomes in the four species, the relative position of specific chromosome regions was different due to chromosome inversions established during their phylogeny. However, there are regions that show no apparent homology between the species, an observation that may be attributed to the considerable intra—chromosomal rearrangements that have occurred following the species divergence. The results of this comparative analysis support the current phylogenetic relationships of the genus Glossina.     

Biosynthesis of contact sex pheromone in the female tsetse fly, Glossina morsitans morsitans Westwood

Injection of [2,3 ¹⁴C] sodium succinate into recently emerged, unfed females of Glossina morsitans morsitans resulted in incorporation of radiolabel mainly into surface cuticular alkanes. In vivo experiments with intact flies showed that the distribution of labelled alkanes depended on fly mobility, the legs of unrestrained flies possessing proportionately larger amounts of radioactive hydrocarbon material than those of flies whose legs were tied together with a silk suture. The heads of both restrained and unrestrained flies contained proportionately more material per unit surface area than did any other body part. However, ablation experiments and in vitro incubation showed that the most active incorporation of label into alkanes occurred in the abdomen and that all dorsal abdominal segments were equally active. The ventral abdomen also incorporated label into cuticular alkanes in vitro, but other body parts were apparently less able to do so. The sex pheromone of G. m. morsitans is a trimethyl-substituted alkane, the labelling of which appeared to be in proportion to the relative abundance of its methyl groups among those of the other alkanes of the cuticle following injection of either intact or legless flies. Hence it is proposed that sex pheromone is synthesized along with other cuticular alkanes mainly by cells closely associated with the abdominal cuticle of females and that it is spread over the external surface both by diffusion and by grooming which leads to accumulations of hydrocarbon material on the legs.     

Experiments on the elimination of symbionts from the tsetse fly,Glossina morsitans morsitans (Diptera: Glossinidae), by antibiotics and lysozyme

One sulfonamide and ten antibiotics were tested for symbiont elimination in Glossina morsitans morsitans. Only tetracycline, penicillin, and kanamycin could be used in concentrations which destroyed the symbionts and impeded host reproduction, but did not affect host longevity. The later the treatment with penicillin and kanamycin began, the smaller was the damage to symbionts from these two antibiotics. Small doses damaged the symbionts to an extent that flies could produce offspring, but these were free of symbionts. Lysozyme was used as an alternative to antibiotics. After both injection and oral administration of lysozyme the symbionts were damaged, and host reproduction ceased, although host longevity remained unaffected. It can be concluded from the results of these experiments that tsetse flies do not require their symbionts for survival but probably do need them for reproduction.     

Spectral responses of the tsetse fly, Glossina morsitans morsitans

Spectral responses of male Glossina morsitans morsitans Westw, were investigated using behavioural and electrophysiological techniques. Phototactic responses to monochromatic lights of different wavelengths but equivalent intensities (measured either in energy or quantal units) were tested in an apparatus which permitted the simultaneous presentation of pairs of lights to groups of flies. Near ultraviolet light was the most attractive, followed by blue and red, with green and far red light giving no significant responses above control levels. The spectral sensitivity of the compound eye was also determined from the electroretinogram (ERG) recorded from electrodes implanted in the eye which was stimulated with flashes of monochromatic light. Three areas of maximum sensitivity were found by this method in the UV, blue/green and red. The relationship between the behavioural and electrophysiological sensitivity characteristics is explained with reference to visual systems of other Diptera which have previously been described.     

Performance of the tsetse, Glossina morsitans morsitans reared under various feeding regimens in Zambia

Groups of Glossina morsitans morsitans Westwood fed at emergence and thereafter daily, every second or third day respectively, up to the end of their first pregnancy cycle, survived well (73–79%) and produced virtually the same number of puparia/ (0.83–0.85) in the same puparial weight class (23–24 mg). However, adult survival (29%), number of puparia/ (0.30) and puparial weight (19 mg) were much lower in the group consistently fed every 4th day after the initial meal at emergence. It is proposed that tsetse colonies could be fed on Mondays, Wednesdays and Fridays without jeopardising adult survival, puparial production/ and the size (weight) of puparia produced.

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Performances en Zambie de la mouche Tsé-Tsé (Glossina morsitans morsitans) élevée en utilisant différents régimes alimentaires

Résumé Afin d'estimer si une alimentation quotidienne de G. morsitans morsitans présente un avantage sur une alimentation moins fréquente, nous avons enrigestré la survie des adultes, la production et le poids des pupes chez des individus nourris à l'émergence et, ensuite, tous les jours ou tous les 2, 3 ou 4 jours. Les femelles alimentées quotidiennement, ou tous les 2 ou 3 jours, ont produit le même nombre de pupes (0,83–0,85), avec des poids de même ordre (23–24 mg), et avec une aussi bonne survie (73 à 79%). Alimentées tous les 4 jours, elles ont produit 0,30 pupe/femelle, pesant 19 mg/pupe et avec une plus faible survie (29%). Ces résultats montrent qu'il est inutile de nourrir les mouches chaque jour au lieu de tous les 2 ou 3 jours. Cependant une alimentation à des intervalles supérieurs à 3 jours, a eu des conséquences clairement défavorables. Ainsi, peuvent être considérablement réduits et le travail et le coût des élevages, sans porter préjudice à la production, en alimentant les mouches 3 fois par semaine, par ex. les lundis, mercredis et vendredis.

     

An Investigation into the Protein Composition of the Teneral Glossina morsitans morsitans Peritrophic Matrix

Background

Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism.

Methods

PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS.

Results

Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM.

Conclusion

To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse.     

Cloning and Expression of a Bacillus thuringiensis (L1-2) Gene Encoding a Crystal Protein Active Against Glossina morsitans morsitans and Chilo partellus

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 M_r 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. Received: 29 June 1996 / Accepted: 1 August 1996