The Effects of a DNA Virus Infection on the Reproductive Potential of Female Tsetse Flies, Glossina morsitans centralis and Glossina morsitans morsitans (Diptera: Glossinidae)

Reproductive anomalies associated with the tsetse DNA virus infection in the female tsetse hosts, Glossina morsitans centralis Machado and Glossina morsitans morsitans Westwood, inoculated with the virus during the 3rd instar larval stage were studied and the data compared to those obtained from the control females injected with sterile physiological saline. Virus infected flies had significantly longer first and second pregnancy cycles (P<0.0001) and produced pupae that were of significantly less weight in milligrams (P<0.0001) compared to controls. Transmission of the virus to progeny was not absolute and only 21% of G. m. centralis and 48% of G. m. morsitans first progeny flies from infected females developed salivary gland hypertrophy as a result of transmission from mother to progeny. The virus infected females produced significantly fewere pupae compared to the controls during the experimental period (P<0.00001).

MAP kinase kinase 1 (MKK1) is essential for transmission of Trypanosoma brucei by Glossina morsitans

MAP kinase kinase 1 (MKK1) is encoded by a single copy gene in Trypanosoma brucei. It has been shown recently that MKK1 is not essential for bloodstream forms [14]. To investigate the requirement for MKK1 in other life-cycle stages we generated null mutants in procyclic forms of a fly-transmissible strain. These grew normally in culture and were able to establish midgut infections in tsetse at normal rates and intensities, but were incapable of colonising the salivary glands. Transformation of null mutants with an ectopic copy of MKK1 enabled parasites to complete the life cycle in tsetse and infect mice. This is the first example of a gene that is indispensable for transmission of T. brucei. It also raises the possibility that activating the MKK1 signalling cascade in vitro might trigger the differentiation and proliferation of life-cycle stages of T. brucei that are currently refractory to culture.

Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

Wolbachia infections are distributed throughout insect somatic and germ line tissues

Wolbachia are intracellular microorganisms that form maternally-inherited infections within numerous arthropod species. These bacteria have drawn much attention, due in part to the reproductive alterations that they induce in their hosts including cytoplasmic incompatibility (CI), feminization and parthenogenesis. Although Wolbachia's presence within insect reproductive tissues has been well described, relatively few studies have examined the extent to which Wolbachia infects other tissues. We have examined Wolbachia tissue tropism in a number of representative insect hosts by western blot, dot blot hybridization and diagnostic PCR. Results from these studies indicate that Wolbachia are much more widely distributed in host tissues than previously appreciated. Furthermore, the distribution of Wolbachia in somatic tissues varied between different Wolbachia/host associations. Some associations showed Wolbachia disseminated throughout most tissues while others appeared to be much more restricted, being predominantly limited to the reproductive tissues. We discuss the relevance of these infection patterns to the evolution of Wolbachia/host symbioses and to potential applied uses of Wolbachia.

Tryptophan metabolism in tsetse flies and the consequences of its derangement

Literature comparing salmon and wild type Glossina morsitans morsitans and that comparing tan and wild type Glossina palpalis palpalis is reviewed. New information is presented on behaviour and biochemistry of salmon and wild type G. m. morsitans. The eye color mutants result from two lesions in the tryptophan to xanthommatin pathway: lack of tryptophan oxygenase in G. m morsitans and failure to produce or retain xanthommatin in eyes (but not in testes) of G. p. palpalis. The salmon allele in G. m. morsitans is pleiotropic and profoundly affects many aspects of fly biology including longevity, reproductive capacity, vision, vectorial capacity and duration of flight, but not circadian rhythms. The tan allele in G. p. palpalis has little effect upon the biology of flies under laboratory conditions, except that tan flies appear less active than normal. Adult tsetse flies metabolize tryptophan to kynurenine which is excreted; fluctuations in activities of the enzymes producing kynurenine suggest this pathway is under metabolic control.

Experimental demonstration of possible cryptic female choice on male tsetse fly genitalia

artículo -- Universidad de Costa Rica. Escuela de Biología, 2009. Este documento es privado debido a restricciones de derechos de autor del publicador.

Estrutura Genética de Populações de Glossina palpalis gambiensis (Diptera: Glossinidae) na República da Guiné-Bissau

Glossina palpalis gambiensis é o principal vector de Tripanossomose Humana Africana (THA) na África Ocidental e a mosca tsé-tsé mais comum na Guiné-Bissau. Apesar da sua ampla distribuição, nenhum caso de THA tem sido reportado no país desde finais dos anos 70 do século XX. Populações naturais do Grupo palpalis demonstraram diferentes níveis de variação intraespecífica que podem influenciar a sua capacidade vectorial. Portanto, o conhecimento exacto acerca da identidade das espécies e estrutura populacional é essencial para prever o possível restabelecimento e propagação da transmissão de THA na Guiné-Bissau. A variação genética foi analisada em amostras de Glossina palpalis gambiensis de cinco regiões da Guiné-Bissau, com recurso a microssatélites. Três das regiões pertencem à parte continental e duas representam a parte insular do país. No total, 261 moscas tsé-tsé do sexo feminino foram genotipadas para 11 loci microssatélites. Baixos níveis de diferenciação genética foram observados entre as populações de G. p. gambiensis da Guiné-Bissau (FST = 0,006, P = 0,002). Este resultado está de acordo com a análise de agrupamentos, que revelou a presença de um único cluster agrupando todas as amostras, independentemente da origem geográfica. De um modo geral, estes resultados sugerem uma baixa subestruturação populacional em G. p. gambiensis nesta região. Análises de equilíbrio mutação-deriva sugerem ainda a ocorrência de expansão populacional recente. As evidências genéticas sugerem consideráveis níveis de fluxo genético entre as populações continentais e entre as populações insulares e continentais da Guiné-Bissau. No caso do restabelecimento de focos de transmissão de THA, a possibilidade de disseminação do protozoário Trypanosoma, através da dispersão activa das moscas tsé-tsé, deve ser tida em conta no planeamento de estratégias de controlo vectorial na Guiné-Bissau. PALAVRAS-CHAVE: Glossina, Tripanossomose Humana Africana, microssatélites, diferenciação genética.

"Estrutura Genética de Populações de Glossina Palpalis gambiensis (Diptera: Glossinidae) na República da Guiné-Bissau"

Glossina palpalis gambiensis é o principal vector de Tripanossomose Humana Africana (THA) na África Ocidental e a mosca tsé-tsé mais comum na Guiné-Bissau. Apesar da sua ampla distribuição, nenhum caso de THA tem sido reportado no país desde finais dos anos 70 do século XX. Populações naturais do Grupo palpalis demonstraram diferentes níveis de variação intraespecífica que podem influenciar a sua capacidade vectorial. Portanto, o conhecimento exacto acerca da identidade das espécies e estrutura populacional é essencial para prever o possível restabelecimento e propagação da transmissão de THA na Guiné-Bissau. A variação genética foi analisada em amostras de Glossina palpalis gambiensis de cinco regiões da Guiné-Bissau, com recurso a microssatélites. Três das regiões pertencem à parte continental e duas representam a parte insular do país. No total, 261 moscas tsé-tsé do sexo feminino foram genotipadas para 11 loci microssatélites. Baixos níveis de diferenciação genética foram observados entre as populações de G. p. gambiensis da Guiné-Bissau (FST = 0,006, P = 0,002). Este resultado está de acordo com a análise de agrupamentos, que revelou a presença de um único cluster agrupando todas as amostras, independentemente da origem geográfica. De um modo geral, estes resultados sugerem uma baixa subestruturação populacional em G. p. gambiensis nesta região. Análises de equilíbrio mutação-deriva sugerem ainda a ocorrência de expansão populacional recente. As evidências genéticas sugerem consideráveis níveis de fluxo genético entre as populações continentais e entre as populações insulares e continentais da Guiné-Bissau. No caso do restabelecimento de focos de transmissão de THA, a possibilidade de disseminação do protozoário Trypanosoma, através da dispersão activa das moscas tsé-tsé, deve ser tida em conta no planeamento de estratégias de controlo vectorial na Guiné-Bissau.

Prospects for control of African trypanosomiasis by tsetse vector manipulation

The extensive antigenic variation phenomena African trypanosomes display in their mammalian host have hampered efforts to develop effective vaccines against trypanosomiasis. Human disease management aims largely to treat infected hosts by chemotherapy, whereas control of animal diseases relies on reducing tsetse populations as well as on drug therapy. The control strategies for animal diseases are carried out and financed by livestock owners, who have an obvious economic incentive. Sustaining largely insecticide-based control at a local level and relying on drugs for treatment of infected hosts for a disease for which there is no evidence of acquired immunity could prove extremely costly in the long run. It is more likely that a combination of several methods in an integrated, phased and area-wide approach would be more effective in controlling these diseases and subsequently improving agricultural output. New approaches that are environmentally acceptable, efficacious and affordable are clearly desirable for control of various medically and agriculturally important insects including tsetse. Here, Serap Aksoy and colleagues discuss molecular genetic approaches to modulate tsetse vector competence.

Desenvolvimento de métodos moleculares para a deteção de Trypanosoma spp em glossinas (Diptera: Glossinidae) da República da Guiné Bissau

As moscas do género Glossina são vetores de patologias provocadas por várias espécies de parasita do género Trypanosoma, como a Tripanossomose Humana Africana e as Tripanossomoses Animais. A correta identificação destes tripanossomas é um ponto fulcral quando se procura determinar o risco de doença que determinadas populações de glossinas podem provocar. No presente trabalho foram utilizadas e adaptadas diversas metodologias de diagnóstico molecular, nomeadamente o PCR em tempo real e o PCR-RFLP, que permitiram, através da utilização de primers genéricos, a determinação da carga parasitária e a correta identificação dos tripanossomas presentes no vetor. Os primers denominados Tryp18SF e Tryp18SR foram desenvolvidos especificamente para serem utilizados na identificação das espécies de tripanossoma utilizando PCR em tempo real com metodologia SYBR® Green I e PCR-RFLP. Os primers denominados Tryp18S2F, Tryp18S2R e sonda Tryp18S2P foram desenvolvidos para a quantificação dos parasitas presentes nas amostras utilizando PCR em tempo real com metodologia Taqman®. Foi estudada uma amostra de 762 glossinas provenientes do território da República da Guiné-Bissau, zona atualmente considerada como estando livre de Tripanossomose Humana Africana, mas onde não são realizadas atividades de recenseamento de casos desde o final da ocupação portuguesa. Das 762 glossinas utilizadas, 241 estavam infetadas com tripanossomas, o que representa uma percentagem de infeção geral de 31,6 %. Destas glossinas, 28.63 % encontravam-se infetadas com Trypanosoma grayi, 14.11 % com Trypanosoma congolense, 7.05 % com Trypanosoma vivax e 0.83 % com Trypanosoma brucei brucei, tendo as infeções mistas representado 1.66 %. Não foi possível a identificação conclusiva ao nível da espécie em 48.13 % das amostras positivas, tendo estas ficado consideradas como Trypanosoma spp. Não foram identificados vetores infetados com tripanossomas responsáveis pelas patologias humanas. A utilização de primers genéricos para a identificação permitiu obviar o número de reações necessárias para identificar corretamente o parasita responsável pela infeção, e este trabalho é de resto a primeira descrição da utilização de primers genéricos que permitam a identificação de Trypanosoma grayi. Esta é também a primeira descrição da utilização de PCR em tempo real com metodologia Taqman® para quantificação de tripanossomas, e a sua utilização com primers genéricos aumenta a aplicabilidade em estudos epidemiológicos de grande escala. Palavras-chave: PCR em tempo real; PCR-RFLP; República da Guiné-Bissau; Glossina; Trypanosoma; Tripanossomose.

Review on the Molecular Tools for the Understanding of the Epidemiology of Animal Trypanosomosis in West Africa

The epidemiology of animal trypanosomosis around Bobo-Dioulasso (Burkina Faso, West Africa) benefited a lot in the last years from the progress of molecular tools. The two most used molecular techniques were the polymerase chain reaction for the diagnosis of the disease in cattle and the characterization of the trypanosomes in the host and the vector on one hand, and the microsatellite DNA polymorphism in tsetse flies to study the intraspecific genetic variability of the vector on the other hand. The results obtained in the Sideradougou area during a recent two year survey with these techniques, associated with many other georeferenced informations concerning vector and cattle distribution, natural environment, landuse, ground occupation, livestock management, were combined in a Geographical Information System. This new approach of a complex pathogenic system led to a better evaluation of the risk of trypanosome transmission.

Can Remotely Sensed Meteorological Data Significantly Contribute to Reduce Costs of Tsetse Surveys?

A 0.125 degree raster or grid-based Geographic Information System with data on tsetse, trypanosomosis, animal production, agriculture and land use has recently been developed in Togo. This paper addresses the problem of generating tsetse distribution and abundance maps from remotely sensed data, using a restricted amount of field data. A discriminant analysis model is tested using contemporary tsetse data and remotely sensed, low resolution data acquired from the National Oceanographic and Atmospheric Administration and Meteosat platforms. A split sample technique is adopted where a randomly selected part of the field measured data (training set) serves to predict the other part (predicted set). The obtained results are then compared with field measured data per corresponding grid-square. Depending on the size of the training set the percentage of concording predictions varies from 80 to 95 for distribution figures and from 63 to 74 for abundance. These results confirm the potential of satellite data application and multivariate analysis for the prediction, not only of the tsetse distribution, but more importantly of their abundance. This opens up new avenues because satellite predictions and field data may be combined to strengthen or substitute one another and thus reduce costs of field surveys.

New Trypanosoma (Duttonella) vivax genotypes from tsetse flies in East Africa

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FELLB identified the trypanosomes in 65 of 105 (61.9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main chide of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.