Polychromophilus spp. (Haemosporida) in Malagasy bats: host specificity and insights on invertebrate vectors.

BACKGROUND: Bats are home to diverse haemosporidian parasites namely Plasmodium and Plasmodium-related. While information is available at a worldwide level, haemosporidian infection in bats from Madagascar is still scarce and recent changes in the taxonomy of the island's bat fauna, particularly the description of several new species, require a reassessment of previously described patterns, including blood parasite ecology and vectorial transmission. METHODS: A sample representing seven of the nine known bat families and 31 of the 46 currently recognized taxa from Madagascar and collected in the western and central portions of the island were screened by PCR for the presence of Polychromophilus. In addition, Nycteribiidae flies parasitizing Miniopteridae and Vespertilionidae were screened for parasites with the aim to better understand aspects of vector transmission. Phylogenetic reconstruction using the mitochondrial cytochrome b encoding gene was used in a Bayesian analysis to examine the relationship between Polychromophilus recovered from Malagasy bats and those identified elsewhere. RESULTS: Polychromophilus infection was restricted to Miniopterus spp. (Miniopteridae), Myotis goudoti (Vespertilionidae), and Paratriaenops furculus (Rhinonycteridae), with an overall infection rate of 13.5%. Polychromophilus melanipherus was found infecting Miniopterus spp. and P. furculus, whereas Polychromophilus murinus was only recovered from M. goudoti. These two protozoan parasites species were also detected in bat flies species known to parasitize Miniopterus spp. and M. goudoti, respectively. Generalized linear model analyses were conducted to elucidate the effect of species and sex on haemoparasites infection in Miniopterus spp., which revealed that males have higher risk of infection than females and prevalence differed according to the considered Miniopterus host. Molecular screening of nycteribiid flies revealed three positive species for Polychromophilus spp., including Penicillidia sp. (cf. fulvida), Penicillidia leptothrinax, and Nycteribia stylidiopsis. These three fly species are known to parasitize Miniopterus spp. and M. goudoti and should be considered as potential vectors of Polychromophilus spp. CONCLUSION: Phylogenetic analyses demonstrated the existence of at least four distinct clades within the genus Polychromophilus, two of which were documented in the present study. The screening of nycteribiid flies overlaid on the highly diversified genus Miniopterus, provides considerable insight into parasite transmission, with bat infection being associated with their roosting behaviour and the occurrence of specific arthropod vectors.

Evidence of multiple colonizations as a driver of black fly diversification in an oceanic island.

True oceanic islands typically host reduced species diversity together with high levels of endemism, which make these environmental set-ups ideal for the exploration of species diversification drivers. In the present study, we used black fly species (Diptera: Simuliidae) from Reunion Island as a model to highlight the main drivers of insect species diversification in this young and remote volcanic island located in the Southwestern Indian Ocean. Using local and regional (Comoros and Seychelles archipelagos) samples as well as specimens from continental Africa, we tested the likelihood of two distinct scenarios, i.e. multiple colonizations vs. in-situ diversification. For this, posterior odds were used to test whether species from Reunion did form a monophyletic group and we estimated divergence times between species. Three out of the four previously described Reunion black fly species could be sampled, namely Simulium ruficorne, Simulium borbonense and Simulium triplex. The phylogenies based on nuclear and mitochondrial markers showed that S. ruficorne and S. borbonense are the most closely related species. Interestingly, we report a probable mitochondrial introgression between these two species although they diverged almost six million years ago. Finally, we showed that the three Reunion species did not form a monophyletic group, and, combined with the molecular datation, the results indicated that Reunion black fly diversity resulted from multiple colonization events. Thus, multiple colonizations, rather than in-situ diversification, are likely responsible for an important part of black fly diversity found on this young Darwinian island.

Reservoir role of lizard Psammodromus algirus in transmission cycle of Borrelia burgdorferi sensu lato (Spirochaetaceae) in Tunisia.

To investigate the reservoir role of the lizard Psammodromus algirus for the Lyme disease spirochete, 199 lizards were trapped from April to October 2003 in El Jouza, northwestern Tunisia. In this site, the infection rate of free-living Ixodes ricinus (L.) by Borrelia was evaluated by immunofluorescence as 34.6% for adult ticks and 12.5% for nymphs. Eighty percent of P. algirus (117/146) captured during this study were infested by I. ricinus, the predominant tick species collected from lizards. The intensity of tick infestation of this host by larvae and nymphs ranged from 0.14 to 7.07 and from 1.5 to 6.58, respectively. These immature stages of I. ricinus were found on lizards in spring and the beginning of summer, with a peak of intensity during June (10.16 immature ticks by lizard). Tissue cultures from lizards and xenodiagnosis with larval I. ricinus were used to assess the infection and the ability, respectively, of infected lizards to transmit Borrelia to naive ticks. Seventeen percent of xenodiagnostic ticks (40/229) acquired B. lusitaniae while feeding on P. algirus. Therefore, we demonstrated the ability of the lizards to sustain Borrelia infection and to infect attached ticks, and we proved that P. algirus is a reservoir host competent to transmit B. lusitaniae.

Evolutionary history of Indian Ocean nycteribiid bat flies mirroring the ecology of their hosts.

Bats and their parasites are increasingly investigated for their role in maintenance and transmission of potentially emerging pathogens. The islands of the western Indian Ocean hold nearly 50 bat species, mostly endemic and taxonomically well studied. However, investigation of associated viral, bacterial, and external parasites has lagged behind. In the case of their ectoparasites, more detailed information should provide insights into the evolutionary history of their hosts, as well as pathogen cycles in these wild animals. Here we investigate species of Nycteribiidae, a family of obligate hematophagous wingless flies parasitizing bats. Using morphological and molecular approaches, we describe fly species diversity sampled on Madagascar and the Comoros for two cave-roosting bat genera with contrasting ecologies: Miniopterus and Rousettus. Within the sampling area, 11 endemic species of insect-feeding Miniopterus occur, two of which are common to Madagascar and Comoros, while fruit-consuming Rousettus are represented by one species endemic to each of these zones. Morphological and molecular characterization of flies reveals that nycteribiids associated with Miniopterus bats comprise three species largely shared by most host species. Flies of M. griveaudi, one of the two bats found on Madagascar and certain islands in the Comoros, belong to the same taxon, which accords with continued over-water population exchange of this bat species and the lack of inter-island genetic structuring. Flies parasitizing Rousettus belong to two distinct species, each associated with a single host species, again in accordance with the distribution of each endemic bat species.

Molecular evolutionary analysis of pH1N1 2009 influenza virus in Reunion Island, South West Indian Ocean region: a cohort study.

BACKGROUND/OBJECTIVES: Molecular epidemiology is a powerful tool to decipher the dynamics of viral transmission, quasispecies temporal evolution and origins. Little is known about the pH1N1 molecular dynamics in general population. A prospective study (CoPanFlu-RUN) was carried out in Reunion Island to characterize pH1N1 genetic variability and molecular evolution occurring in population during the pH1N1 Influenza pandemic in 2009. METHODOLOGY: We directly amplified pH1N1 genomes from 28 different nasal swabs (26 individuals from 21 households). Fifteen strains were fully sequenced and 13 partially. This includes pairs of sequences from different members of 5 separate households; and two pairs from individuals, collected at different times. We assessed the molecular evolution of pH1N1 by genetic variability and phylogenetic analyses. PRINCIPAL FINDINGS: We found that i) Reunion pH1N1 sequences stemmed from global "clade 7" but shaped two phylogenetic sub-clades; ii) D239E mutation was identified in the hemagglutinin protein of all Reunion sequences, a mutation which has been associated elsewhere with mild-, upper-respiratory tract pH1N1 infecting strains; iii) Date estimates from molecular phylogenies predicted clade emergence some time before the first detection of pH1N1 by the epidemiological surveillance system; iv) Phylogenetic relatedness was observed between Reunion pH1N1 viruses and those from other countries in South-western Indian Ocean area; v) Quasispecies populations were observed within households and individuals of the cohort-study. CONCLUSIONS: Surveillance and/or prevention systems presently based on Influenza virus sequence variation should take into account that the majority of studies of pH1N1 Influenza generate genetic data for the HA/NA viral segments obtained from hospitalized-patients, which is potentially non-representative of the overall viral diversity within whole populations. Our observations highlight the importance of collecting unbiased data at the community level and conducting whole genome analysis to accurately understand viral dynamics.

Phylogenetic analyses of mitochondrial and nuclear data in haematophagous flies support the paraphyly of the genus Stomoxys (Diptera: Muscidae).

The genus Stomoxys Geoffroy (Diptera; Muscidae) contains species of parasitic flies that are of medical and economic importance. We conducted a phylogenetic analysis including 10 representative species of the genus including multiple exemplars, together with the closely related genera Prostomoxys Zumpt, Haematobosca Bezzi, and Haematobia Lepeletier & Serville. Phylogenetic relationships were inferred using maximum likelihood and Bayesian methods from DNA fragments from the cytochrome c oxidase subunit I (COI, 753bp) and cytochrome b (CytB, 587bp) mitochondrial genes, and the nuclear ribosomal internal transcribed spacer 2 (ITS2, 426bp). The combination of mitochondrial and nuclear data strongly supports the paraphyly of the genus Stomoxys because of the inclusion of Prostomoxys saegerae Zumpt. This unexpected result suggests that Prostomoxys should be renamed into Stomoxys. Also, the deep molecular divergence observed between the subspecies Stomoxys niger niger Macquart and S. niger bilineatus Grünbreg led us to propose that they should rather be considered as distinct species, in agreement with ecological data. Bayesian phylogenetic analyses support three distinct lineages within the genus Stomoxys with a strong biogeographical component. The first lineage consists solely of the divergent Asian species S. indicus Picard which appears as the sister-group to all remaining Stomoxys species. The second clade groups the strictly African species Stomoxys inornatus Grünbreg, Stomoxys transvittatus Villeneuve, Stomoxys omega Newstead, and Stomoxys pallidus Roubaud. Finally, the third clade includes both African occurring and more widespread species such as the livestock pest Stomoxys calcitrans Linnaeus. Divergence time estimates indicate that the genus Stomoxys originated in the late Oligocene around 30 million years ago, with the major lineages diversifying in the Early Miocene between 20 and 15 million years ago at a time when temperate forests developed in the Northern Hemisphere.