Genome sequence of the tsetse fly (Glossina morsitans):Vector of African trypanosomiasis

Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein-encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology.IS

Differential expression of fat body genes in Glossina morsitans morsitans following infection with Trypanosoma brucei brucei

To determine which fat body genes were differentially expressed following infection of Glossina morsitans morsitans with Trypanosoma brucei brucei we generated four suppression subtractive hybridisation (SSH) libraries. We obtained 52 unique gene fragments (SSH clones) of which 30 had a known orthologue at E - 05 or less. Overall the characteristics of the orthologues suggest: (i) that trypanosome infection has a considerable effect on metabolism in the tsetse fly; (ii) that self-cured flies are mounting an oxidative stress response; and (iii) that self-cured flies are displaying increased energy usage. The three most consistently differentially expressed genes were further analysed by gene knockdown (RNAi). Knockdown of Glossina transferrin transcripts, which are upregulated in self-cured flies compared with flies infected with trypanosomes, results in a significant increase in the number of trypanosome infections establishing in the fly midgut, suggesting transferrin plays a role in the protection of tsetse flies from trypanosome infection. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved

Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA

Reverse genetic studies based on RNA interference (RNAi) have revolutionized analysis of gene function in most insects. However the necessity of injecting double stranded RNA (dsRNA) inevitably compromises many investigations particularly those on immunity. Additionally, injection of tsetse flies often causes significant mortality. We demonstrate, at transcript and protein level, that delivering dsRNA in the bloodmeal to Glossina morsitans morsitans is as effective as injection in knockdown of the immunoresponsive midgut-expressed gene TsetseEP. However, feeding dsRNA fails to knockdown the fat body expressed transferrin gene, 2A192, previously shown to be silenced by dsRNA injection. Mortality rates of the dsRNA fed flies were significantly reduced compared to injected flies 14 days after treatment (Fed: 10.1% +/- 1.8%; injected: 37.9% +/- 3.6% (Mean +/- SEM)). This is the first demonstration in Diptera of gene knockdown by feeding and the first example of knockdown in a blood-sucking insect by including dsRNA in the bloodmeal

Expression of chemosensory proteins in the tsetse fly Glossina morsitans morsitans is related to female host-seeking behaviour

Chemosensory proteins (CSPs) are a class of soluble proteins present in high concentrations in the sensilla of insect antennae. It has been proposed that they play an important role in insect olfaction by mediating interactions between odorants and odorant receptors. Here we report, for the first time, the presence of five CSP genes in the tsetse fly Glossina morsitans morsitans, a major vector transmitting nagana in livestock. Real-time quantitative reverse transcription PCR showed that three of the CSPs are expressed in antennae. One of them, GmmCSP2, is transcribed at a very high level and could be involved in olfaction. We also determined expression in the antennae of both males and females at different life stages and with different blood feeding regimes. The transcription of GmmCSP2 was lower in male antennae than in females, with a sharp increase in 10-week-old flies, 48 h after a bloodmeal. Thus there is a clear relationship between CSP gene transcription and host searching behaviour. Genome annotation and phylogenetic analyses comparing G. morsitans morsitans CSPs with those of other Diptera showed rapid evolution after speciation of mosquitoes

Mean (± S.E.) prevalence of midgut infections in male <i>G. m. morsitans</i> (<i>Gmm</i>) or <i>G. p. palpalis</i> (<i>Gpp</i>) after RNAi knockdown using ds<i>tsetse EP</i>.

<p>Controls were either ds<i>Ampicillin</i>, ds<i>eGFP</i> or nuclease free water*. Flies were infected with either <i>T. b. brucei</i> TSW196 or <i>T. congolense</i> 1/148 (italics) blood stream forms in the indicated bloodmeal.</p

Immunoblot film lies below the nigrosine-stained PVDF.

<p>There is a decline in tsetse EP protein levels in midguts over a 7 day starvation period following the 5^th bloodmeal. 24 h  =  Flies starved for 7 days, fed a blood meal and then sacrificed 24 hours later. L  =  molecular mass ladder. Midgut proteins (1/2 midgut equivalent from pool of 5) were blotted with mAb 247.</p

Antioxidant gene expression in the blood-feeding fly Glossina morsitans morsitans

We report the characterization of 11 antioxidant genes from the tsetse fly Glossina m. morsitans. Through similarity searches which detected homology we suggest that these genes consist of two superoxide dismutases (one with a putative signal peptide), three thioredoxin peroxidases (one with a putative signal peptide), three peroxiredoxins, one further signal peptide-containing peroxidase with its closest similarity to a glutathione peroxidase, one catalase and one thioredoxin reductase. We describe the changes occurring in the expression levels of these genes during fly development, in different adult tissues, in the adult midgut through the digestive cycle and following trypanosome infection. Overall, nine of the 11 genes studied showed responses to changes in physiological circumstance, with the peroxiredoxin group showing the smallest variations throughout

Characterisations of odorant-binding proteins in the tsetse fly Glossina morsitans morsitans

Odorant-binding proteins (OBPs) play an important role in insect olfaction by mediating interactions between odorants and odorant receptors. We report for the first time 20 OBP genes in the tsetse fly Glossina morsitans morsitans. qRT-PCR revealed that 8 of these genes were highly transcribed in the antennae. The transcription of these genes in the antennae was significantly lower in males than in females and there was a clear correlation between OBP gene transcription and feeding status. Starvation over 72 h post-blood meal (PBM) did not significantly affect the transcription. However, the transcription in the antennae of 10-week-old flies was much higher than in 3-day-old flies at 48 h PBM and decreased sharply after 72 h starvation, suggesting that the OBP gene expression is affected by the insect’s nutritional status.Sequence comparisons with OBPs of other Dipterans identified several homologs to sex pheromone-binding proteins and OBPs of Drosophila melanogaster