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.     

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.     

Wolbachia symbiont infections induce strong cytoplasmic incompatibility in the tsetse fly Glossina morsitans

Tsetse flies are vectors of the protozoan parasite African trypanosomes, which cause sleeping sickness disease in humans and nagana in livestock. Although there are no effective vaccines and efficacious drugs against this parasite, vector reduction methods have been successful in curbing the disease, especially for nagana. Potential vector control methods that do not involve use of chemicals is a genetic modification approach where flies engineered to be parasite resistant are allowed to replace their susceptible natural counterparts, and Sterile Insect technique (SIT) where males sterilized by chemical means are released to suppress female fecundity. The success of genetic modification approaches requires identification of strong drive systems to spread the desirable traits and the efficacy of SIT can be enhanced by identification of natural mating incompatibility. One such drive mechanism results from the cytoplasmic incompatibility (CI) phenomenon induced by the symbiont Wolbachia. CI can also be used to induce natural mating incompatibility between release males and natural populations. Although Wolbachia infections have been reported in tsetse, it has been a challenge to understand their functional biology as attempts to cure tsetse of Wolbachia infections by antibiotic treatment damages the obligate mutualistic symbiont (Wigglesworthia), without which the flies are sterile. Here, we developed aposymbiotic (symbiont-free) and fertile tsetse lines by dietary provisioning of tetracycline supplemented blood meals with yeast extract, which rescues Wigglesworthia-induced sterility. Our results reveal that Wolbachia infections confer strong CI during embryogenesis in Wolbachia-free (Gmm(Apo)) females when mated with Wolbachia-infected (Gmm(Wt)) males. These results are the first demonstration of the biological significance of Wolbachia infections in tsetse. Furthermore, when incorporated into a mathematical model, our results confirm that Wolbachia can be used successfully as a gene driver. This lays the foundation for new disease control methods including a population replacement approach with parasite resistant flies. Alternatively, the availability of males that are reproductively incompatible with natural populations can enhance the efficacy of the ongoing sterile insect technique (SIT) applications by eliminating the need for chemical irradiation.     

Development of Real Time PCR to Study Experimental Mixed Infections of T. congolense Savannah and T. b. brucei in Glossina morsitans morsitans

Tsetse flies are able to acquire mixed infections naturally or experimentally either simultaneously or sequentially. Traditionally, natural infection rates in tsetse flies are estimated by microscopic examination of different parts of the fly after dissection, together with the isolation of the parasite in vivo. However, until the advent of molecular techniques it was difficult to speciate trypanosomes infections and to quantify trypanosome numbers within tsetse flies. Although more expensive, qPCR allows the quantification of DNA and is less time consuming due to real time visualization and validation of the results. The current study evaluated the application of qPCR to quantify the infection load of tsetse flies with T. b. brucei and T. congolense savannah and to study the possibility of competition between the two species. The results revealed that the two qPCR reactions are of acceptable efficiency (99.1% and 95.6%, respectively), sensitivity and specificity and can be used for quantification of infection load with trypanosomes in experimentally infected Glossina morsitans morsitans. The mixed infection of laboratory Glossina species and quantification of the infection suggests the possibility that a form of competition exists between the isolates of T. b. brucei and T. congolense savannah that we used when they co-exist in the fly midgut.     

Asymmetric Wolbachia Segregation during Early Brugia malayi Embryogenesis Determines Its Distribution in Adult Host Tissues

Wolbachia are required for filarial nematode survival andfertility and contribute to the immune responses associated with human filarialdiseases. Here we developed whole-mount immunofluorescence techniques tocharacterize Wolbachia somatic and germline transmissionpatterns and tissue distribution in Brugia malayi, a nematoderesponsible for lymphatic filariasis. In the initial embryonic divisions,Wolbachia segregate asymmetrically such that they occupyonly a small subset of cells in the developing embryo, facilitating theirconcentration in the adult hypodermal chords and female germline.Wolbachia are not found in male reproductive tissues andthe absence of Wolbachia from embryonic germline precursors inhalf of the embryos indicates Wolbachia loss from the malegermline may occur in early embryogenesis. Wolbachia rely onfusion of hypodermal cells to populate adult chords. Finally, we detectWolbachia in the secretory canal lumen suggesting livingworms may release bacteria and/or their products into their host.     

Wolbachia in the Flesh: Symbiont Intensities in Germ-Line and Somatic Tissues Challenge the Conventional View of Wolbachia Transmission Routes

Symbionts can substantially affect the evolution and ecology of their hosts. The investigation of the tissue-specific distribution of symbionts (tissue tropism) can provide important insight into host-symbiont interactions. Among other things, it can help to discern the importance of specific transmission routes and potential phenotypic effects. The intracellular bacterial symbiont Wolbachia has been described as the greatest ever panzootic, due to the wide array of arthropods that it infects. Being primarily vertically transmitted, it is expected that the transmission of Wolbachia would be enhanced by focusing infection in the reproductive tissues. In social insect hosts, this tropism would logically extend to reproductive rather than sterile castes, since the latter constitute a dead-end for vertically transmission. Here, we show that Wolbachia are not focused on reproductive tissues of eusocial insects, and that non-reproductive tissues of queens and workers of the ant Acromyrmex echinatior, harbour substantial infections. In particular, the comparatively high intensities of Wolbachia in the haemolymph, fat body, and faeces, suggest potential for horizontal transmission via parasitoids and the faecal-oral route, or a role for Wolbachia modulating the immune response of this host. It may be that somatic tissues and castes are not the evolutionary dead-end for Wolbachia that is commonly thought.     

Thermal effect of blood feeding in the telmophagous fly Glossina morsitans morsitans

During feeding on warm-blooded hosts, haematophagous insects are exposed to thermal stress due to the ingestion of a meal which temperature may highly exceed their own body temperature. In order to avoid overheating and its subsequent deleterious effects, these insects respond by setting up molecular protective mechanisms such as heat shock proteins synthesis or by using thermoregulative strategies. Moreover, the duration of contact with the host depends on the way of feeding displayed by the different species (either telmophagous or solenophagous) and thus also impacts their exposure to heat. Solenophagous insects feed directly on blood vessels and are relatively slow feeders while telmophagous insects by lacerating capillaries, facilitate their access to blood and thus feed more quickly. The aim of this work was to investigate to what extent strictly telmophagous insects such as tsetse flies are exposed to thermal stress during feeding and consequently to evaluate the impact of the feeding strategy on the exposition to overheating in haematophagous insects in general. Real time thermographic analysis during feeding revealed that the flies’ body significantly heat up quite homogeneously. At the end of feeding, however, a marked regional heterothermy occurs as a consequence of the alary muscles warm up that precedes take-off. Feeding strategies, either solenophagy or telmophagy, thus appear to have a great impact on both exposition to predation risks and to thermal stress.     

Octopamine distribution in the tsetse fly Glossina morsitans

Tissues of Glossina morsitans were assayed for octopamine using an enzymatic technique. Octopamine was detected at the highest concentration in the brain (7.06-7.99 ng mg-1 tissue protein) and thoracic ganglion (10.9-13.89 ng mg-1 tissue protein). Octopamine was present in haemolymph at a concentration of 1.0-1.27 X 10(-7) M. This was not found to vary when insects were flown or mechanically stressed. Nervous tissue, flight muscle and haemolymph showed a significant ability to metabolize octopamine. The greatest enzyme activity was present in the haemolymph.     

昆虫共生细菌Wolbachia的研究进展

Wolbachia是广泛分布在节肢动物生殖组织的一类细胞质遗传的细菌,它们能通过多种机制调节寄生的生殖,包括诱导细胞质不亲和,孤雌生殖和遗传上的雄性雌性化,目前对这些微生物的研究已取了可喜的进展,通过主要综述Wolbachia的分布,功能,系统发育,多样性及时空动态,也讨论了将来的研究方向     

Concordant Evolution of a Symbiont with Its Host Insect Species: Molecular Phylogeny of Genus Glossina and Its Bacteriome-Associated Endosymbiont, Wigglesworthia glossinidia

Many arthropods with restricted diets rely on symbiotic associations for full nutrition and fecundity. Tsetse flies (Diptera: Glossinidae) harbor three symbiotic organisms in addition to the parasitic African trypanosomes they transmit. Two of these microorganisms reside in different gut cells, while the third organism is harbored in reproductive tissues and belongs to the genus Wolbachia. The primary symbiont (genus Wigglesworthia glossinidia) lives in differentiated epithelial cells (bacteriocytes) which form an organ (bacteriome) in the anterior gut, while the secondary (S) symbionts are present in midgut cells. Here we have characterized the phylogeny of Wigglesworthia based on their 16S rDNA sequence analysis from eight species representing the three subgenera of Glossina: Austenina (=fusca group), Nemorhina (=palpalis group), and Glossina (=morsitans group). Independently, the ribosomal DNA internal transcribed spacer-2 (ITS-2) regions from these species were analyzed. The analysis of Wigglesworthia indicated that they form a distinct lineage in the γ subdivision of Proteobacteria and display concordance with their host insect species. The trees generated by parsimony confirmed the monophyletic taxonomic placement of Glossina, where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of the species Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on both the ITS-2 and the symbiont 16S rDNA sequence analysis, suggest that Glossina austeni should be placed into a separate fourth subgenus, Machadomyia, which forms a sister-group relationship with the morsitans group species. Received: 17 March 1998 / Accepted: 1 May 1998     

Wolbachia Utilize Host Actin for Efficient Maternal Transmission in Drosophila melanogaster

Wolbachia pipientis is a ubiquitous, maternally transmitted bacterium that infects the germline of insect hosts. Estimates are that Wolbachia infect nearly 40% of insect species on the planet, making it the most prevalent infection on Earth. The bacterium, infamous for the reproductive phenotypes it induces in arthropod hosts, has risen to recent prominence due to its use in vector control. Wolbachia infection prevents the colonization of vectors by RNA viruses, including Drosophila C virus and important human pathogens such as Dengue and Chikungunya. Here we present data indicating that Wolbachia utilize the host actin cytoskeleton during oogenesis for persistence within and transmission between Drosophila melanogaster generations. We show that phenotypically wild type flies heterozygous for cytoskeletal mutations in Drosophila profilin (chic²²¹/+ and chic¹³²⁰/+) or villin (qua^6-396/+) either clear a Wolbachia infection, or result in significantly reduced infection levels. This reduction of Wolbachia is supported by PCR evidence, Western blot results and cytological examination. This phenotype is unlikely to be the result of maternal loading defects, defects in oocyte polarization, or germline stem cell proliferation, as the flies are phenotypically wild type in egg size, shape, and number. Importantly, however, heterozygous mutant flies exhibit decreased total G-actin in the ovary, compared to control flies and chic²²¹ heterozygous mutants exhibit decreased expression of profilin. Additionally, RNAi knockdown of profilin during development decreases Wolbachia titers. We analyze evidence in support of alternative theories to explain this Wolbachia phenotype and conclude that our results support the hypothesis that Wolbachia utilize the actin skeleton for efficient transmission and maintenance within Drosophila.     

Cloning and expression of the yolk protein of the tsetse fly Glossina morsitans morsitans

Two major families of nutritional proteins exist in insects, namely the vitellogenins and the yolk proteins. While in other insects only vitellogenins are found, cyclorraphan flies only contain yolk proteins. Possible sites of yolk protein synthesis are the fat body and the follicle cells surrounding the oocyte. We report the cloning of the yolk protein of the tsetse fly Glossina morsitans morsitans, a species with adenotrophic viviparity. The tsetse fly yolk protein could be aligned with other dipteran yolk proteins and with some vertebrate lipases. In contrast to the situation in most fly species, only a single yolk protein gene was found in the tsetse fly. Northern blot analysis showed that only the ovarian follicle cells, and not the fat body represents the site of yolk protein synthesis.     

Post-feed buzzing in the tsetse, Glossina morsitans morsitans, is an endothermic mechanism

ABSTRACT. Post-feed buzzing in Glossina morsitans morsitans Westw. causes a rise in thoracic temperature relative to the length of the buzz. As lift is proportional to the square of wing-beat frequency, which increases with temperature up to 32°C, buzzing results in an increase in the lift which the fly can produce. Heat generated by buzzing, in combination with the heat received from the host at the time of feeding, may well allow the fly to maximize lift generated in the immediate post-feeding period. Buzzing flies excrete excess water from the meal more rapidly than non-buzzing flies. It is argued that this is due to a rise in abdominal temperature. Maximized lift in the immediate post-feeding period and the rapid elimination of water from the very large blood meals taken by these flies may be expected to have strong selective advantages for the flies.     

Ultrastructural studies of certain aspects of the development of Trypanosoma congolense in Glossina morsitans morsitans

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.     

Dynamics of the pregnancy cycle in the tsetse Glossina morsitans

A normal pregnancy in tsetse involves the successful integration of larval development with maternal activity. At 25°C, ovulation in Glossina morsitans occurs 1 hr after the previous larviposition, the egg hatches on day 3·8 (1·57 mm length, 0·09 mg dry wt.), ecdysis to second instar occurs on day 4·9 (2·3 mm, 0·30 mg), the third instar cuticle is formed on day 6·8 (4·5 mm, 5·0 mg), and parturition occurs on day 9·0 (6·0 mm, 10·0 mg). Melanization of the in utero third instar follows a regular sequence over a 2 day period. Parturition follows a circadian pattern with a peak 9 hr after lights on (12 hr daily photophase). All instars receive nutriment from the female's milk gland. During early pregnancy the rate of milk synthesis is greater than rate of uptake by the larva, thus causing expansion of the secretory reservoirs. After day 6, the volume of the secretory reservoirs decreases, but as is indicated by nuclear volume and larval growth the rate of synthesis remains high until day 8. Feeding activity of the adult female is maximal on day 1, levels off at 60 per cent up to day 6, and then declines sharply towards the end of pregnancy. Oöcyte development proceeds in phase with larval development and thus minimizes a lag period between successive pregnancies.     

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.