Significance The airway epithelia modulate the inflammatory responses to various pathogens. Pulmonary disease caused by Toxoplasma gondii infection affects human neonates, children, and immunocompromised individuals. However, it is not clear how T. gondii infection impacts airway epithelia. We report the use of a short-circuit current ( I sc ) technique to determine the Cl − secretion induced by ATP in tracheal epithelia infected by T. gondii. We surprisingly found that the ATP-evoked Cl − secretion in T. gondii -infected mouse tracheal epithelia was significantly suppressed. We also found that the mRNA expression level of the P2Y2 receptor increased significantly in T. gondii -infected mouse trachea, via real-time quantitative PCR. Our study provides previously unidentified insights into the mechanism underlying host impairment caused by T. gondii infection.
We develop a new discrete-time model, called the boundary-layer model, to describe the dynamics of single species that have a capacity for fast growth at very low population densities. The model explicitly separates the dynamics of the population at very low densities (within the “boundary layer”) and at high densities. The boundary-layer model provides a better fit than other models such as the logistic or the θ model to data from experimental populations of Drosophila willistoni and D. pseudoobscura.
We have analyzed the comportment in in vitro culture of 2 different genotypes of Trypanosoma cruzi, the agent of Chagas disease, pertaining to 2 major genetic subdivisions (near-clades) of this parasite. One of the stocks was a fast-growing one, highly virulent in mice, while the other one was slow- growing, mildly virulent in mice. The working hypothesis was that mixtures of genotypes interact, a pattern that has been observed by us in empirical experimental studies. Genotype mixtures were followed every 7 days and characterized by the DIGE technology of proteomic analysis. Proteic spots of interest were characterized by the SAMESPOT software. Patterns were compared to those of pure genotypes that were also evaluated every 7 days. One hundred and three spots exhibited changes in time by comparison with T = 0. The major part of these spots (58%) exhibited an under-expression pattern by comparison with the pure genotypes. 32% of the spots wereover-expressed; 10% of spots were not different from those of pure genotypes. Interestingly, interaction started a few minutes after the mixtures were performed. We have retained 43 different proteins that clearly exhibited either under- or over-expression. Proteins showing interaction were characterized by mass spectrometry (MALDI-TOF). Close to 50% of them were either tubulins or heat shock proteins. This study confirms that mixed genotypes of T. cruzi interact at the molecular level. This is of great interest because mixtures of genotypes are very frequent in Chagas natural cycles, both in insect vectors and in mammalian hosts, and may play an important role in the transmission and severity of Chagas disease. The methodology proposed here is potentially applicable to any micropathogen, including fungi, bacteria and viruses. It should be of great interest in the case of bacteria, for which the epidemiological and clinical consequences of mixed infections could be underestimated.
Lateral gene transfer (LGT) is a major evolutionary mechanism in prokaryotes. Knowledge about LGT— particularly, multicellular— eukaryotes has only recently started to accumulate. A widespread assumption sees the gene as the unit of LGT, largely because little is yet known about how LGT chances are affected by structural/functional features at the subgenic level. Here we trace the evolutionary trajectory of VEin Patterning 1, a novel gene family known to be essential for plant development and defense. At the subgenic level VEP1 encodes a dinucleotide-binding Rossmann-fold domain, in common with members of the short-chain dehydrogenase/reductase (SDR) protein family. We found: i) VEP1 likely originated in an aerobic, mesophilic and chemoorganotrophic α-proteobacterium, and was laterally propagated through nets of ecological interactions, including multiple LGTs between phylogenetically distant green plant/fungi-associated bacteria, and five independent LGTs to eukaryotes. Of these latest five transfers, three are ancient LGTs, implicating an ancestral fungus, the last common ancestor of land plants and an ancestral trebouxiophyte green alga, and two are recent LGTs to modern embryophytes. ii) VEP1's rampant LGT behavior was enabled by the robustness and broad utility of the dinucleotide-binding Rossmann-fold, which provided a platform for the evolution of two unprecedented departures from the canonical SDR catalytic triad. iii) The fate of VEP1 in eukaryotes has been different in different lineages, being ubiquitous and highly conserved in land plants, whereas fungi underwent multiple losses. And iv) VEP1-harboring bacteria include non-phytopathogenic and phytopathogenic symbionts which are non-randomly distributed with respect to the type of harbored VEP1 gene. Our findings suggest that VEP1 may have been instrumental for the evolutionary transition of green plants to land, and point to a LGT-mediated 'Trojan Horse' mechanism for the evolution of bacterial pathogenesis against plants. VEP1 may serve as tool for revealing microbial interactions in plant/fungi-associated environments.
We have studied by gel electrophoresis the variability of 14 structural genes in four sibling species, Drosophila willistoni, D. paulistorum, D. equinoxialis, and D. tropicalis. Samples of about 30 populations from different parts of the distribution areas of each species were examined. Genetic variants are found at every locus; 67% of the loci are polymorphic, having two or more alleles, the rarer of which has a frequency of 5% or higher. The gene frequencies are fairly uniform over the distribution area of each species, but considerably different in different species. It is estimated that individuals which belong to the different species differ on the average in somewhat more than one half of their gene loci. The morphological similarity of the four sibling species contrasts with the extensive diversity in their genetic materials.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract About 60% of emerging infectious diseases in humans are of zoonotic origin. Their increasing number requires the development of new methods for early detection and monitoring of infectious agents in wildlife. Here, we investigated whether blood meals from hematophagous flies could be used to identify the infectious agents circulating in wild vertebrates. To this aim, 1230 blood-engorged flies were caught in the forests of Gabon. Identified blood meals (30%) were from 20 vertebrate species including mammals, birds and reptiles. Among them, 9% were infected by different extant malaria parasites among which some belonged to known parasite species, others to new parasite species or to parasite lineages for which only the vector was known. This study demonstrates that using hematophagous flies as 'flying syringes' constitutes an interesting approach to investigate blood-borne pathogen diversity in wild vertebrates and could be used as an early detection tool of zoonotic pathogens. https://doi.org/10.7554/eLife.22069.001 eLife digest About 60% of new infectious diseases in humans come from animals. Their increasing number and rapid spread are linked to increasing levels of contact between humans and wildlife, as recently highlighted by the epidemics of Zika in Brazil or Ebola in West Africa. To anticipate and prevent similar outbreaks in the future, it would be ideal to develop new methods for the early detection and monitoring of infectious diseases in wild animals. Currently, three methods are mainly used to screen wild animals for infectious disease, but these all have limitations. Analyses of bushmeat and game meat only investigate those animals that are eaten by humans. Testing the organs and tissues of trapped animals can be difficult and harmful for both the humans and animals involved. Collecting and examining samples of feces, urine or saliva cannot detect all diseases and can be difficult to do for some species. Bitome-Essono et al. now demonstrate a new method for assessing the diseases carried by wild animals: using blood-sucking flies as 'flying syringes' to collect their blood. During several weeks of sampling in Gabon, Central Africa, Bitome-Essono et al. trapped thousands of these flies, about a third of which were engorged with blood. Analyses of these blood samples revealed that they had come from 20 different species, including birds, mammals and reptiles. Different malaria parasites could also be detected in the blood. Although the study performed by Bitome-Essono et al. only focused on malaria parasites, in the future the technique could be extended to analyze a number of disease-causing microbes – including viruses, bacteria, protozoa and macroparasites – that are found in the blood of wild animals. https://doi.org/10.7554/eLife.22069.002 Introduction Emerging and re-emerging human infectious diseases have increased in recent years. Around one-fourth of the 1415 pathogens known to infect humans appeared between 1940 and 2004 and their appearance has gradually increased since 1980 (Taylor et al., 2001; Woolhouse and Gaunt, 2007; Jones et al., 2008; Daszak et al., 2004). Today, seven new pathogens appear every year and this number should reach 15–20 by 2020 (Woolhouse et al., 2008), mostly due to the growth of human activities that increase contact with novel sources of pathogens and favor their spread worldwide (Murray et al., 2015). Emerging threats mainly concern viruses, such as HIV (Sharp and Hahn, 2011), SARS-CoV and MERS-CoV (de Wit et al., 2016), avian flu (Alexander, 2007) and more recently Ebola (Baize et al., 2014), chikungunya (Burt et al., 2012) and Zika (Wikan and Smith, 2016). However, disease emergence and re-emergence also concern bacteria (e.g. Helicobacter pylori, Salmonella sp., etc.) and parasites (e.g. Plasmodium knowlesi in South-East Asia). Sixty per cent of diseases emerging in humans are zoonoses and wildlife plays a key role by providing a zoonotic pool from which previously unknown pathogens may emerge (Taylor et al., 2001; Woolhouse and Gaunt, 2007; Jones et al., 2008; Daszak et al., 2004). The case of P. knowlesi in South-East Asia is a good example. This parasite emerged in the human population after a transfer from Asian macaques. It is now considered as the fifth human malaria agent after Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale (Singh and Daneshvar, 2013). Such emerging diseases constitute a massive public health issue that requires active monitoring for signs of outbreaks and rapid diagnosis of the involved pathogen. Therefore, it is crucial to anticipate and prevent potential epidemic and pandemic outbreaks by developing new methods for the early detection and monitoring of infectious agents in wild animal sources (Kuiken et al., 2005; Wolfe et al., 2005). However, in many cases, monitoring is limited or impossible due to our poor knowledge about the ecology of these pathogens (i.e. where, when and how these agents circulate in the wildlife). The case of the Ebola virus is quite exemplary. Indeed, the exact nature of its reservoir(s) remains uncertain, although thousands of animals have been screened during the last 40 years (e.g. [Marí Saéz et al., 2015]). Nowadays, pathogen circulation in wild animals is screened using mainly two methods: bushmeat analysis or direct trapping of animals for organ and tissue collection. These methods are pertinent in many cases, but present some weaknesses. Bushmeat represents only a fraction of the fauna (the one consumed by humans), whereas animal trapping can be difficult or dangerous. Moreover, such manipulation may be harmful for threatened and protected species. As a consequence, several methods were developed in the last years to study pathogen diversity from wild fauna without the need of direct contacts with animals, for example, by using fecal, urine or saliva samples (e.g. [Santiago et al., 2002; Prugnolle et al., 2010; Pesapane et al., 2013; Taberlet et al., 2012]). However, the value of these non-invasive methods remains limited because not all pathogens can be detected and not all reservoirs can be explored by these methods (for instance, it is difficult to collect feces or saliva of reptiles without trapping them). Therefore, new non-invasive methods are crucially needed to provide new opportunities for screening a larger range of hosts and pathogens. The use of hematophagous flies as 'flying syringes' may constitute a new approach to track and survey blood-borne pathogens in the wild (Calvignac-Spencer et al., 2013). Nucleic acids (DNA or RNA) of vertebrate hosts or of pathogens in arthropod blood meals are preserved and detectable for several days (Calvignac-Spencer et al., 2013; Kent, 2009; Muturi et al., 2011; Grubaugh et al., 2015; Lee et al., 2015). For example, HIV was detected 8 days and 10 to 14 days after blood ingestion by bugs and by ticks, respectively (Webb et al., 1989; Humphery-Smith et al., 1993). Recently, the H5N1 flu virus was found viable in mosquitoes (Barbazan et al., 2008), although its transmission by these insects is unproven (Sawabe et al., 2006). Grubaugh and colleagues (Grubaugh et al., 2015) applied such an idea (that they called 'xenosurveillance') using Anohpeles mosquitoes to estimate the diversity of viruses infecting human populations in remote areas. Nevertheless, blood-engorged mosquitoes are very difficult to collect in forest and often show strong host preferences (in particular for mammals). Arthropods with more generalist blood feeding patterns would be more useful to survey pathogens from a large range of vertebrates (including mammals, birds and reptiles) in these highly complex ecosystems. Hematophagous flies (tsetse flies, stomoxids and tabanids) could be good candidates for this purpose since they are usually large Diptera (length comprised between 3 and 25 mm) and hematophagous in both sexes, with the exception of male tabanids (Mullens, 2002). They are easy to trap and some studies performed on tsetse flies and stomoxids showed that 20 to 40% of trapped flies are engorged with blood (Mavoungou et al., 2008; Simo et al., 2012). These flies feed on a large spectrum of vertebrate hosts, including birds, reptiles and mammals (Muturi et al., 2011; Clausen et al., 1998; Muzari et al., 2010). The omnipresence of hematophagous flies in certain habitats and their opportunistic blood-feeding behaviour (Muturi et al., 2011; Muzari et al., 2010; Späth, 2000) make of them compelling candidates to obtain blood meals from different vertebrate hosts for pathogen detection. In the present study, we investigated the possibility of using hematophagous flies as 'flying syringes' to explore the diversity of extant malaria parasites (Haemosporida) infecting wild vertebrates living in the forests of Gabon (Central Africa). Results Host identification from blood meals A total of 4099 hematophagous flies were caught in four national parks of Gabon during dry and rainy seasons over a cumulated sampling period of 16 weeks (Figure 1a). Among them, six tsetse fly species, six stomoxid species and six tabanid species were identified (Table 1). Figure 1 Download asset Open asset Monitoring vertebrate haemosporidian diversity using haematophagous flies. (a) Localization of the sampling sites (red dots) in Gabon (Central Africa). (b) Number of blood meals originating from the different vertebrate species. (c) Position within the Cytb phylogeny of the haemosporidian Cytb sequences PCR-amplified from the blood meals of engorged flies with identified hosts (red isolates) and unidentified hosts (green isolates). Black isolates: references (Table 4). Bootstrap values at important nodes are shown. https://doi.org/10.7554/eLife.22069.003 Table 1 Number and proportion of specimens captured per fly species. The number of engorged specimens and blood meals identified in each fly species are also indicated. https://doi.org/10.7554/eLife.22069.004 Fly speciesNumber of collected specimensProportion (%)Number of engorged specimensNumber of identified blood mealsGlossinidae225254.941218423Glossina caliginea1443.518733G. fusca congolensis2105.1210442G. fuscipes fuscipes2907.0721493G. pallicera newsteadi1573.839737G. palpalis palpalis137233.47662218G. tabaniformis791.93540Muscidae136233.2394Stomoxys calcitrans2455.9852S. inornatus3348.1400S. niger niger2536.1742S. niger bilineatus2245.4600S. omega omega1974.8100S. transvittatus1092.6600Tabanidae48511.8331Ancala sp41100Atylotus sp1042.5300Chrysops sp1563.8131Haematopota sp130.3100Tabanus par521.2700Tabanus taeniola1202.9300Total40991001230428 Among the 4099 caught flies, 1230 (30%) were engorged with blood. These were mostly tsetse flies (n = 1218; 99%), particularly Glossina palpalis palpalis (n = 662; 54%) and G. fuscipes fuscipes (n = 214; 18%) specimens. The blood meal origin was successfully identified in 33% and 43% of these flies, respectively (Table 1). Overall, the blood meal origin was successfully identified in 428 fly samples (35%) using a PCR system amplifying long fragments of Cytb (450 bp) or COI genes (330 bp or 660 bp). Specifically, blood meals were from 20 vertebrate species, including 12 families and 8 orders (Figure 1b and Tables 2 and 3). Table 2 Number and origin of blood meals according to the fly species (Fsp), park and climatic season. https://doi.org/10.7554/eLife.22069.005 Number of identified blood meals by fly species (Fsp)Moukalaba-DoudouLopéRainy seasonDry seasonRainy seasonDry seasonTaxonomic group/Order/FamilyHost speciesN° IdentifiedFsp1Fsp2Fsp3Fsp4Fsp5Fsp1Fsp2Fsp3Fsp4Fsp5Fsp8Fsp1Fsp2Fsp3Fsp4Fsp5Fsp1Fsp2Fsp3Fsp4Fsp5Fsp6Fsp7Mammals Artiodactyla295143811711421233413 BovidaeCephalophus silvicultor65kobus ellipsiprymnus431Syncerus caffer12635711329211057128161Tragelaphus spekii9516415137694121 HippopotamidaeHippopotamus amphibius211 SuidaePotamochoerus porcus312 Carnivora1 HerpestidaeHerpestinae sp11 Primates67 HominidaeGorilla gorilla321Homo sapiens6411132221312141 Proboscidae10 ElephantidaeLoxodonta cyclotis10712Reptiles Crocodilia233 CrocodylidaeCrocodylus niloticus3Mecistops cataphractus19116Osteolaemus tetraspis11 Squamata12 PythonidaePython sebae82 VaranidaeVaranus sp4211 Testudines16 TestunidaeKinixys erosa11 PelomedusidaePelusios castaneus3111Pelusios chapini11Pelusios marani1138Birds Ciconiformes4 CiconiidaeCiconia sp4128 orders/12 families20 species428311144122164891671871611112262512 Fsp1 = Glossina caliginea; Fsp2 = G. fusca congolensis; Fsp3 = G. fuscipes fuscipes; Fsp4 = G. pallicera newsteadi; Fsp5 = G. palpalis palpalis; Fsp6 = Stomoxys calcitrans; Fsp7 = S. niger niger; Fsp8 = Chrysops sp. Table 3 Number and origin of blood meals according to the fly species (Fsp), park and climatic season. https://doi.org/10.7554/eLife.22069.006 Number of identified blood meals by fly species (Fsp)La LékédiIvindoRainy seasonDry seasonDry seasonTaxonomic group/Order/FamilyHost speciesN° IdentifiedFsp1Fsp3Fsp4Fsp5Fsp6Fsp1Fsp2Fsp3Fsp4Fsp5Fsp1Fsp2Fsp3Fsp5Mammals Artiodactyla295213 BovidaeCephalophus silvicultor65kobus ellipsiprymnus4Syncerus caffer126211610881311Tragelaphus spekii95131285591 HippopotamidaeHippopotamus amphibius2 SuidaePotamochoerus porcus3 Carnivora1 HerpestidaeHerpestinae sp1 Primates67 HominidaeGorilla gorilla3Homo sapiens6441511 Proboscidae10 ElephantidaeLoxodonta cyclotis10Reptiles Crocodilia23 CrocodylidaeCrocodylus niloticus3Mecistops cataphractus192423Osteolaemus tetraspis1 Squamata12 PythonidaePython sebae8222 VaranidaeVaranus sp4 Testudines16 TestunidaeKinixys erosa1 PelomedusidaePelusios castaneus3Pelusios chapini1Pelusios marani11Birds Ciconiformes4 CiconiidaeCiconia sp418 orders/12 families20 species4282411318202015321122 Fsp1 = Glossina caliginea; Fsp2 = G. fusca congolensis; Fsp3 = G. fuscipes fuscipes; Fsp4 = G. pallicera newsteadi; Fsp5 = G. palpalis palpalis; Fsp6 = Stomoxys calcitrans; Fsp7 = S. niger niger; Fsp8 = Chrysops sp. A trial study using a PCR system amplifying a shorter fragment (150 bp of the gene 16S) to deal with potential DNA degradation in the blood meal showed a high gain of sensitivity in the determination of the origin of the blood meal. Thus, out of 89 previously unidentified blood meals, the host was identified for 76% (n = 68) of them. The list of newly identified hosts is given in Figure 2. This shows a high gain of sensitivity with the new PCR system. Figure 2 Download asset Open asset Number of blood meals identified using the shorter PCR system of Boessenkool et al. (2012) out of the previously unidentified 89 blood meals. https://doi.org/10.7554/eLife.22069.007 Pathogen identification from blood meals Extant malaria parasites were detected in 37 (8.7%) of the 428 identified blood meals (Figure 1c, red isolates). Phylogenetic analyses revealed that 29.7% of these parasites belonged to Plasmodium falciparum (n = 11, Figure 1c; group 1), 8.1% to Plasmodium adleri (n = 3, Figure 1c; group 2), and 8.1% to a recently described lineage of parasites infecting wild ungulates (n = 4, Figure 1c; group 3) (Boundenga et al., 2016). For all blood meals, the identified host represented the known natural host (or one of the hosts) of such parasites. Sequences of unknown parasite lineages or of parasites for which the hosts were not known were also obtained. For instance, one sequence (Figure 1c; group 4) detected in a blood meal originating from an ungulate was related to parasites previously isolated only from Anopheles mosquitoes (Boundenga et al., 2016). One sequence detected in a blood meal originating from a bird was related to bat Haemosporida (Nycteria), (Figure 1c; group 5). Finally, 18 sequences (Figure 1c; group 6) that were amplified from blood meals originating from ungulates formed an independent and never described lineage related to groups 3 and 4. In addition, 100 additional samples for which identification of the blood meal failed were randomly chosen for malarial parasite screening. This analysis showed that 7% were infected with P. falciparum (n = 4, group 1), P. praefalciparum (n = 1, group 7), malaria parasites of antelopes from group 6 (n = 1) and parasites of tortoises (group 8, n = 1) (Figure 1c, green isolates). For the parasite, the use of a shorter PCR system led to less conclusive results than those obtained for the host identification. Out of the 91 blood meals that were negative to Plasmodium with a PCR system amplifying a long Cytb fragment, only one was found positive with the new system. The positive individual corresponded to a Tragelaphus spekii and was infected with a parasite belonging to group 3 (Figure 1c). Discussion In this study, we tested whether hematophagous flies could be used as 'flying syringes' to identify blood-borne pathogens circulating in the wild vertebrate fauna of Gabon. Our results show that the blood meals of the captured engorged flies can be successfully used to analyze the diversity of extant malaria parasites. Despite a limited sampling effort (a total of 4 weeks of sampling for each park), we could screen the diversity of haemosporidian parasites from a large range of vertebrate hosts, including mammals, birds and reptiles. Parasites were detected in more than 8% of the analyzed samples. These malaria parasites belonged to already known, but also to never previously described lineages. In addition, the method allowed identifying the natural hosts of parasites for which only the vectors were known. Concerning the method efficiency, 30% of blood meals were obtained from 4099 hematophagous flies. This result is consistent with previous studies (Mavoungou et al., 2008; Simo et al., 2012) showing that most hematophagous flies caught using traps are often seeking hosts for a blood meal. Other methods using a dip net seem to have a better capture efficiency with more than 40% of engorged flies caught on their resting places (Gouteux et al., 1984). However, this method requires spending a lot of time in the field because of difficulties in finding their resting sites and catching the flies. Tsetse flies provided 99% of the collected blood meals (54% by Glossina palpalis palpalis) and they are an interesting candidate as 'flying syringes'. Indeed, differently from stomoxids and tabanids, both sexes are exclusively hematophagous in tsetse flies. In addition, G. p. palpalis is considered to be an opportunistic species concerning its feeding behaviour, thus explaining the large diversity of blood meals (Clausen et al., 1998; Simo et al., 2008; Weitz, 1963). Conversely, stomoxids and tabanids show sex-specific differences in feeding behaviour and this may partly explain the smaller number of blood meals collected in these two families. In stomoxids, both sexes are hematophagous, but males sometimes feed on nectar (Wall and Shearer, 1997). Moreover, the digestion of stomoxids starts more rapidly than in the other hematophagous flies (Moffatt et al., 1995). Male and female tabanids feed on nectar just after their emergence as adults. Only after having been fertilized, females start sucking blood (Mullens, 2002). Therefore, engorged stomoxid and tabanid flies are more difficult to capture. Additionally, the lack of engorged stomoxids and tabanids could be explained by the fact that we sampled flies only at floor level. Indeed, some stomoxid species readily feed on arboreal monkeys that are mostly found higher in the tree layer (Mavoungou et al., 2008). The low rate (35%) of blood meal identifications could be explained by the degradation of host DNA during digestion in the fly midgut or by a too small blood quantity in the midgut. The stage of digestion might influence DNA degradation and the host identification efficiency. Nevertheless, the diversity of hosts we successfully identified, mainly in tsetse fly blood meals, was large, including big terrestrial (elephants) and semi-aquatic mammals (hippopotamus) and also reptiles and birds. As previously noted, the diversity of blood meals can be due to the fly high mobility, their opportunistic feeding behaviour and their frequent feeding. In our study, most blood meals were from terrestrial animals (i.e. that live primarily on the ground) and very few from arboreal species. As mentioned above, this result is potentially biased by the trophic preferences of tsetse flies and by the capture method that excluded canopy levels. Previous studies have shown that hematophagous flies sampled in canopies mainly feed on arboreal species (Mavoungou et al., 2008). Therefore, changes in trap position could broaden the range of host species analysed. We can also notice the absence of small mammals (e.g., rodents or bats) within the diversity of host vertebrates we identified. This may be explained by the trophic preferences of the flies we sampled which could have a preferential taste for large vertebrates as previously documented for tsetse flies (e.g. [Muturi et al., 2011; Späth, 2000]). Concerning pathogen detection, we detected extant haemosporidian parasites in 8.65% of the 428 blood meals for which the host origin was successfully identified. Moreover, we also detected parasites in blood meals of unknown origin, thus increasing the number of detected parasites. Together, these results show that blood meals collected from hematophagous flies are suitable for tracking blood-borne pathogens from wild animals. Haemosporidian pathogens ingested by hematophagous flies during their blood meal can remain detectable in the fly digestive tract even after partial digestion of the blood meal. We observed congruence between the identified hosts and the detected pathogens. As expected, P. falciparum was detected in human blood and P. adleri in gorilla blood. Haemosporidian lineages are often host-specific or restricted to certain classes of vertebrate hosts. Therefore, the unknown host could be inferred from the detected haemosporidian species (Figure 1c). For example, the blood meal from unknown host N°110 could have originated from a Kinixys turtle (Kinixys sp.). Similarly, the blood meals from the unknown hosts N°649, 520, 665, 512 and 819 could have originated from humans (Homo sapiens). The present study demonstrates the possibility to use hematophagous flies as 'flying syringes' to analyze the diversity of pathogens circulating in wildlife. We think that there is now room for improvement of the tool; for instance, by improving the methods used to identify the blood meals and the pathogens. Since DNA is likely to be degraded in many blood meals (Calvignac-Spencer et al., 2013; Schnell et al., 2012), the use of PCR systems targeting fragments of shorter size could potentially improve the performance of detection. A trial study based on 89 previously unidentified blood meals using a PCR system amplifying a shorter fragment (<150 bp) (Boessenkool et al., 2012) than the one used in the present study allowed the identification of 76% (n = 68) of the hosts (Figure 2). This represents an important gain of sensitivity. However, these primers are still not ideal for our purpose as they were designed for optimal amplification of mammal DNA and often fail to properly amplify the DNA of other classes of vertebrates. A similar PCR system targeting the entire range of vertebrates still remains to be developed. For Plasmodium, our trial for amplifying a shorter fragment of Cytb (<200 bp) using a combination of previously published primers did not increase the sensitivity. Indeed, out of 91 samples for which the blood meal was successfully identified but in which no haemosporidian infection was detected with our long Cytb PCR system, only one was shown to be positive with the short PCR system. However, it is possible that other PCR systems, more optimized, could indeed improve the sensitivity of Plasmodium detection. Another direction of improvement could be the use of high-throughput sequencing technologies on pools of blood-engorged flies or amplicons to ease the identification of both hosts and parasites (especially in the case of mixed blood meals or mixed infections). Finally, another way to improve the tool could be to use high-throughput multiplexed pathogen detection methods for the simultaneous testing of many samples in rapid succession. With such improvements, this approach of 'xenorsurveillance' could usefully complete recently developed methods based on the analysis of other invertebrates (carrion flies (Hoffmann et al., 2016), mosquitoes [Grubaugh et al., 2015]) and become an innovative way for the concomitant surveillance of many enzootic blood-borne pathogens, such as viruses (chikungunya, Zika), bacteria, protozoa and macro-parasites. The use of hematophagous flies as 'flying syringes' could indeed improve public health management by allowing the surveillance and early detection of zoonotic pathogens and thus prevent they spread to humans before they cause massive infections. This tool could also help to better understand the circulation in wildlife of other enzootic viruses, such as chikungunya or Zika, especially at the interface between natural/sylvan environments and, consequently, improving our knowledge of their natural history. From a broader perspective, this method could also be useful for people interested in wildlife biodiversity and conservation. Indeed, it could help monitoring the wildlife diversity within a specific region as demonstrated with other invertebrate systems (Calvignac-Spencer et al., 2013; Lee et al., 2015; Schnell et al., 2012; Schubert et al., 2015). More importantly, it could also allow detecting the emergence of new diseases in wild animals that may threaten their long-term survival. Conclusion Despite the significant scientific advances in the medical field, humans are still unable to predict where, when and how epidemics arise. Around 60% of emerging diseases in humans are of zoonotic origin. The progressive reduction of wild habitats will increase the contacts between humans and species that are potential reservoirs of diseases. We propose here a new non-invasive tool that can help identifying pathogens that circulate in wildlife before they spread in humans. Materials and methods Study sites Request a detailed protocol The fly sampling was carried out in four wildlife reserves in Gabon (Figure 1a): Moukalaba-Doudou National Park (MDNP; S: 2° 26' 08"/E: 10° 25' 18''), La Lopé National Park (LNP; S: 0° 31' 31"/E: 11° 32' 34"), La Lékédi Park (LP; S: 1° 45' 32"/E: 13° 03' 16") and Ivindo National Park (INP; N: 0° 30' 82"/E: 12° 48' 20"). Both MDNP and LNP are dominated by mature forests and mosaic forest-savannah. The INP is largely dominated by mature forest with some open biotopes that characterize the secondary forest. The LP is a private park dominated by large savannahs and some secondary forest and primary forest patches. Sampling strategy Request a detailed protocol Hematophagous flies were sampled during the rainy and dry seasons between 2012 and 2014. In INP and MDNP, sampling was done during two years following a gradient of human activity from primary forest to villages. In the other parks, flies were sampled during a single year. Flies were collected by using Vavoua and Nzi traps (Laveissiere and Grebaut, 1990; Acapovi et al., 2001; Mihok, 2002; Gilles et al., 2007). The Vavoua trap, initially developed for the capture of tsetse flies was also successfully used for the capture of stomoxids at La Réunion Island (Laveissiere and Grebaut, 1990; Gilles et al., 2007). The Nzi trap was more adapted to the capture of Glossina pallidipes and tabanids in Africa (Acapovi et al., 2001; Mihok, 2002). In each park, we placed 24 traps (12 Vavoua and 12 Nzi) during 2 weeks per climatic season. Each trap was activated from 7:00 AM to 5:00 PM. Identification and dissection of hematophagous flies Request a detailed protocol Freshly collected hematophagous flies were identified using a stereo-microscope and taxonomic procedure. The fly species (tsetse, stomoxids and tabanids) was determined following the determination keys of Pollock (1982), Brunhes et al., 1998, Zumpt, 1973, Garros et al. (2004) and Oldroyd (1973), on the basis of their morphological characteristics, such as size, color, wing venation structure and proboscis. After species identification, engorged flies were dissected individually in a drop of Dulbecco's phosphate buffered saline solution (1x DPBS) to isolate blood meals from midgut. Each hematophagous fly was dissected on a slide using one forceps and one scalpel that were changed each time to avoid contaminations. Each blood meal was transferred in a 1.5-ml microtube containing 50 µl of RNAlater stabilization solution (Qiagen: Store at RT Tissue Collection) to stabilize and protect nucleic acids of vertebrate hosts and pathogens contained in the blood meals. Samples were kept at ambient temperature during field session and then frozen at −80°C until DNA extraction. DNA extraction Request a detailed protocol Samples were centrifuged at 15,000 rpm at 4°C for 10 min to remove the RNAlater solution. Pellets were used to extract DNA using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. Extracted DNA was eluted in 100 µl of buffer AE and stored at −20°C. Blood meal identification Re
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