Structural Modification and Expression of Attacin A from Glossina morsitans morsitans in E. coli and its Antibacterial Activities

Attacin A from Glossina morsitans morsitans belongs to the type of Gly-rich antimicrobial peptides, with mature peptide of 188 amino acids and molecular weight of 19.4 kDa. In this work, a truncated form of Attacin A was studied to evaluate its N-terminal and G1 domain for antibacterial activities with the help of genetic engineering technology. Genes coding for mature full length Attacin A together with its truncated form Attacin AP1 containing 1–99 amino acids of Attacin A were cloned, expressed and purified. The expression level for Attacin A and Attacin AP1 were about 10 and 20% of total cell protein, respectively. The purity of bioactive recombinant proteins, which showed virtually homogenous as determined by SDS–PAGE, was over 90%. Antibacterial testing shows that Attacin A could inhibit the growth of Gram-negative bacteria such as E. coli, Enterobacter cloacae and Klebsiella pneumoniae at relatively high concentration comparing with Ampicillin, but had little activity against Gram-positive Staphylococcus aureus. Attacin AP1 maintained the inhibitory activities against E. coli, E. cloacae and K. pneumoniae. This result makes it possible for better understanding of the structure–activity relationship of antimicrobial peptides.     

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.     

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.     

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.     

Horizontal transmission of mycotic infection in adult tsetse,Glossina morsitans morsitans

AdultGlossina morsitans morsitans exposed to wet conidia ofBeauveria bassiana andMetarhizium anisopliae suffered high mortalities ranging from 90 to 100% by 2 weeks post-exposure. Infected ♂ ♂ maintained in the same cages with non-infected ♀♀ throughout the experimental period transmitted the fungal infection to the ♀♀ resulting in mortalities of 65% withB. bassiana and 55% withM. anisopliae. Likewise, infected ♀♀ maintained together with non-infected ♂♂ transmitted the infection to the ♂♂ resulting in mortalities of 75% withB. bassiana and 45% withM. anisopliae. Female tsetse flies infected withB. bassiana andM. anisopliae and maintained in the same cages with non-infected ♀♀ also transmitted infection to the non-infected tsetse resulting in mortalities of 62% and 48% withB. bassiana andM. anisopliae respectively. Infected tsetse exposed to non-infected tsetse of the opposite sex for only 30 min were also able to transmit the fungal infection. Pupae produced by female tsetse infected withB. bassiana andM. anisopliae exhibited higher pupal mortality than those produced by non-infected ♀♀. However, pupae exposed directly to dry spores ofB. bassiana andM. anisopliae had no increase in pupal mortality but adults emerging from theB. bassiana-exposed pupae had markedly reduced longevity.      

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.     

Fine structure of the female reproductive system in a viviparous insect, Glossina morsitans morsitans (Diptera, Glossinidae)

The female reproductive system of the tsetse fly Glossina morsitans morsitans is analysed by scanning electron microscopy (SEM). The study focuses in particular on the choriothete, a peculiar uterine structure involved in the viviparous mode of reproduction of Glossina morsitans morsitans.Under light microscopy, the choriothete appears formed by numerous tongue-like folds projecting towards the uterine lumen and lined by a thin cuticle. SEM analysis highlights for the first time a distinctive new feature that is not visible by traditional histological methods. That is a cuticular covering of the choriothete, which shows numerous thorns in the form of crest-like structures arranged in nearly parallel lines. The role of the choriothete in pregnancy and in larval nourishment is discussed.     

A novel vehicle-mounted sticky trap; an effective sampling tool for savannah tsetse flies Glossina morsitans morsitans Westwood and Glossina morsitans centralis Machado

BackgroundBlack screen fly round (BFR) is a mobile sampling method for Glossina morsitans. This technique relies on the ability of operator(s) to capture flies landing on the screen with hand nets. In this study, we aimed to evaluate a vehicle-mounted sticky panel trap (VST) that is independent of the operator’s ability to capture flies against BFR, for effective and rapid sampling of G. m. morsitans Westwood and G. m. centralis Machado. We also determined the influence of the VST colour (all-blue, all-black or 1:1 blue-black), orientation and presence of odour attractants on tsetse catch.Methodology/Principal findingsUsing randomised block design experiments conducted in Zambia, we compared and modelled the number of tsetse flies caught in the treatment arms using negative binomial regression. There were no significant differences in the catch indices of the three colour designs and for in-line or transversely oriented panels for both subspecies (P > 0.05). When baited with butanone and 1-octen-3-ol, VST caught 1.38 (1.11–1.72; P < 0.01) times more G. m. centralis flies than the un-baited trap. Attractants did not significantly increase the VST catch index for G. m. morsitans (P > 0.05). Overall, the VST caught 2.42 (1.91–3.10; P < 0.001) and 2.60 (1.50–3.21; P < 0.001) times more G. m. centralis and G. m. morsitans respectively, than the BFR. The VST and BFR took 10 and 35 min respectively to cover a 1 km transect.Conclusion/SignificanceThe VST is several times more effective for sampling G. m. morsitans and G. m. centralis than the BFR and we recommend its use as an alternative sampling tool.     

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.     

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.

     

Spirostomum spp. (Ciliophora, Protista), a suitable system for endocytobiosis research

Summary. Among ciliate genera, only Paramecium and Euplotes species have been studied extensively as host organisms of bacterial endocytobionts. In this article, we show that members of the genus Spirostomum may also serve as a suitable system for endocytobiosis research. Two strains of Spirostomum minus (Heterotrichea, Ciliophora) collected in Germany and Italy, respectively, were found to harbor different types of bacterial infections. Bacteria of various sizes and shapes were observed in the cytoplasm or in the nuclei of the ciliates. The bacteria in the cytoplasm were either surrounded by a peribacterial membrane or lay naked. One of the bacterial species was found in the vicinity of the contractile fibrillar system (myonemes) of the ciliates. In rare cases, another type of bacteria was observed associated with mitochondria. The macronuclei of both the Italian and the German strains were crowded with endocytobionts. The endonuclear bacteria in the two S. minus strains differed with respect to their cytoplasmic structures but they were of similar size and both were rod shaped. According to the results of in situ hybridization, the endonuclear bacteria of the Italian strain belong to the subgroup of alphaproteobacteria, whereas the bacteria associated with the fibrillar system appeared to be gram-positive bacteria with high G+C content. While both the German and the Italian strains were found to permanently maintain their endocytobionts, they were at least partly colonized by different bacteria. This is taken as an indication that geographically separated populations of ciliates may be stably infected by different endocytobionts, possibly due to different ecological conditions. For S. minus and S. ambiguum a total of 7 different bacterial endocytobionts have now been recorded. We recommend the members of the genus Spirostomum as a suitable system for endocytobiosis research. Correspondence and reprints (present address): Laboratorio di Protistologia, Dipartimento di Etologia, Ecologia ed Evoluzione, Università di Pisa, Via A. Volta 4, 56126 Pisa, Italy.     

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.     

Effect of temperature on the reproduction of Glossina pallidipes, with reference to G. m. morsitans

The reproductive biology of G. pallidipes Austen was studied at 28°, 25° and 22° C. Experiments showed that incubation of puparia at 28° C resulted in sterility of both males and females. Incubation at 22° C resulted in a reduced fecundity of the females due to egg retention; the fertility of the males was not affected.Comparative studies with G. m. morsitans Westw. showed that G. m. morsitans puparia are less affected by a temperature of 28° C than are G. pallidipes puparia.

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Effet de la température sur la reproduction de Glossina pallidipes, avec référence à G. m. morsitans

Résumé Les productivités de G. pallidipes Austen élevés au laboratoire pendant tout leur cycle à 22, 25 et 28° C, ont été comparées.A 28° C, la vie intrapupale est réduite à environ 23 jours, contre 30 jours environ à 25° C; la survie des adultes est plus brève qu'à 25° C et les mouches ne s'accouplent pas. Les ovaires présentent une rétention d'oeufs et seulement 1/3 des mâles contient des spermatozoïdes mobiles. A 22° C, le cycle est considérablement prolongé, la vie intrapupale durant environ 40 jours. Les femelles s'accouplaient environ 14 jours après l'émergence. Les ovaires présentaient une rétention d'oeufs, bien que moins souvent qu'à 28° C. Les mâles contenaient des spermatozoïdes mobiles.Des expériences avec changements de température à différents moments du cycle ont montré que la stérilité des mâles et des femelles est provoquée par l'incubation de pupes de G. pallidipes à 28° C. La mensuration des ovocytes montre à 28° C un effet nocif sur leur maturation. Des observations sur les testicules dans les pupes révèlent, par comparaison avec 25° C, que l'enroulement des testicules et des spermatozoïdes est retardé à 28° C, tandis que la pigmentation des testicules est retardée à 22° C. Les pupes de G. m. morsitans sont moins affectées à 28° C que celles de G. pallidipes.

     

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     

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.     

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.     

Towards improving tsetse fly paratransgenesis: stable colonization of Glossina morsitans morsitans with genetically modified Sodalis

Background

Tsetse flies (Glossina sp.) refractory to trypanosome infection are currently being explored as potential tools to contribute in the control of human and animal African trypanosomiasis. One approach to disrupt trypanosome transmission by the tsetse fly vector involves the use of paratransgenesis, a technique that aims to reduce vector competence of disease vectors via genetic modification of their microbiota. An important prerequisite for developing paratransgenic tsetse flies is the stable repopulation of tsetse flies and their progeny with its genetically modified Sodalis symbiont without interfering with host fitness.

Results

In this study, we assessed by qPCR analysis the ability of a chromosomally GFP-tagged Sodalis (recSodalis) strain to efficiently colonize various tsetse tissues and its transmission to the next generation of offspring using different introduction approaches. When introduced in the adult stage of the fly via thoracic microinjection, recSodalis is maintained at high densities for at least 21 days. However, no vertical transmission to the offspring was observed. Oral administration of recSodalis did not lead to the colonization of either adult flies or their offspring. Finally, introduction of recSodalis via microinjection of third-instar larvae resulted in stably colonized adult tsetse flies. Moreover, the subsequent generations of offspring were also efficiently colonized with recSodalis. We show that proper colonization of the female reproductive tissues by recSodalis is an important determinant for vertical transmission.

Conclusions

Intralarval microinjection of recSodalis proves to be essential to achieve optimal colonization of flies with genetically modified Sodalis and its subsequent dissemination into the following generations of progeny. This study provides the proof-of-concept that Sodalis can be used to drive expression of exogenous transgenes in Glossina morsitans morsitans colonies representing a valuable contribution to the development of a paratransgenic tsetse fly based control strategy.

     

A study on isoenzyme polymorphism in the tsetse fly Glossina morsitans

Polymorphism was studied for a number of enzyme systems in the tsetse fly Glossina morsitans. Enzyme polymorphism was observed for -glycerophospate dehydrogenase, aldehyde oxidase and esterases. For esterases, the operation of null alleles was assumed, as otherwise no explanation could be given for the observed frequencies of the variants.Two laboratory colonies and two field populations were compared with respect to their variation at the leucine aminopeptidase (Lap) loci, for which polymorphism was shown to occur in previous work. Conspicuous differences were found between material originating from Tanzania and from Rhodesia. In addition, allelic relationships were established for the Lap ₃-locus.

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Résumé Faisant suite à des études antérieures, le polymorphisme d'un certain nombre de systèmes enzymatiques a été étudié chez Glossina morsitans. Un polymorphisme a été observé pour l' -glycerophosphate déhydrogénase, pour l'aldéhyde-oxydase et pour les estérases. Pour les estérases, on a supposé l'intervention d'allèles nuls, les fréquences observées chez les variants ne pouvant être expliquées d'autre façon.Deux colonies élevées au laboratoire et deux populations naturelles ont été comparées quant à leur variation au niveau des loci (Lap) pour la leucine aminopeptidase, pour lesquels un travail antérieur avait mis en évidence un polymorphisme. Des différences nettes ont été trouvées entre le matériel provenant de Tanzanie et celui de Rhodésie. En outre des parentés alléliques ont été établies en ce qui concerne le locus Lap ₃.

     

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.