Host-parasite relationships during a biologic invasion: 75 years postinvasion, cane toads and sympatric Australian frogs retain separate lungworm faunas.

Invasive species may carry with them parasites from their native range, differing from parasite taxa found in the invaded range. Host switching by parasites (either from the invader to native fauna or from native fauna to the invader) may have important consequences for the viability of either type of host (e.g., their survivorship, fecundity, dispersal ability, or geographic distribution). Rhabdias pseudosphaerocephala (Nematoda) is a common parasite of cane toads (Rhinella marina) in the toad's native range (South and Central America) and also in its introduced Australian range. This lungworm can depress host viability and is capable of infecting Australian frogs in laboratory trials. Despite syntopy between toads and frogs for up to 75 yr, our analyses, based on DNA sequence data of lungworms from 80 frogs and 56 toads, collected from 2008 to 2011, did not reveal any cases of host switching in nature: toads and native frogs retain entirely different lungworm faunas. All lungworms in cane toads were the South and Central American species Rhabdias pseudosphaerocephala, whereas Australian frogs contained at least four taxa (mostly undescribed and currently lumped under the name Rhabdias cf. hylae). General patterns of prevalence and intensity, based on the dissection of 1,315 frogs collected between 1989 and 2011 across the toads' Australian range, show that these Australian endemic Rhabdias spp. are widely distributed geographically and across host taxa but are more common in some frog species (especially, large-bodied species) than they are in others.

Implication of TNF family members in the immune response to the infection with the intracellular protozoan parasite "Leishmania major"

Suite à une infection avec le protozoaire Leishmania major (L. major), les souris sensibles de souche BALB/c développent des lésions progressives associées à une maturation des cellules CD4+ TH2 sécrétant de l'IL-4. A l'inverse, les souris résistantes de souche C57BL/6 guérissent à terme, sous l'influence de l'expansion des cellules CD4+ TH1 produisant de l'IFNy qui a un effet synergique avec le TNF ("tumor necrosis factor") sur l'activation des macrophages et leur fonction leishmanicide. Lors de notre étude nous avons montré que des souris C57BL/6 doublement déficientes en TNF et FasL ("Fas ligand") infectées par L. major ne guérissaient ni leur lésions ni ne contrôlaient la réplication de parasites malgré une réponse de type TH1. Bien que l'activité de synthétase inductible de l'oxyde nitrique ("iNOs") soit comparable chez les souris doublement ou simplement déficientes, seules celles déficientes en FasL ont démontré une incapacité à contrôler la réplication parasitaire. De surcroît il est apparu que le FasL a un effet synergique avec l'IFNy. L'adjonction de FasL à une culture cellulaire de macrophages stimulés par l'IFNy conduit à une activation de ces cellules. Celle-ci est démontrée par l'augmentation de la production de TNF et de NO par les macrophages ainsi que par l'élimination des parasites intracellulaires par ces mêmes cellules. Alors que le FasL et l'IFNy semblent essentiels au contrôle de la réplication des pathogènes intracellulaires, la contribution de TNF s'oriente davantage vers le contrôle de l'inflammation. L'activation macrophagique via Fas précède la mort cellulaire qui survient quelques jours plus tard. Cette mort cellulaire programmée était indépendante de la cascade enzymatique des caspases, au vu de l'absence d'effet de l'inhibiteur non-spécifique ZVAD-fmk des caspases. Ces résultats suggèrent que l'interaction Fas-FasL agit comme une costimulation nécessaire à une activation efficace des macrophages, la mort cellulaire survenant consécutivement à l'activation des macrophages.¦-¦Upon infection with the protozoan parasite Leishmania major (L. major), susceptible BALB/c mice develop non healing lesions associated with the maturation of CD4+ TH2 cells secreting IL-4. In contrast, resistant C57BL/6 mice are able to heal their lesions, because of CD4+ TH1 cell expansion and production of high levels of IFNy, which synergizes with tumour necrosis factor (TNF) in activating macrophages to their microbicidal state. In our study we showed that C57BL/6 mice lacking both TNF and Fas ligand (FasL) infected with L. major neither resolved their lesions nor controlled L. major replication despite a strong TH1 response. Although comparable inducible nitric oxide synthase (iNOs) was measured in single or double deficient mice, only mice deficient in FasL failed to control the parasite replication. Moreover FasL synergized with IFNy for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Addition of FasL to IFNy stimulated macrophages led to their activation, as reflected by the secretion of tumour necrosis factor and nitrite oxide, as well as the induction of their microbicidal activity, resulting in the killing of intracellular L. major. While FasL along with IFNy and iNOs appeared to be essential for the complete control of intracellular pathogen replication, the contribution of TNF appeared more important in controlling the inflammation on the site of infection. Macrophage activation via Fas pathway preceded cell death, which occurred a few days after Fas mediated activation. This program cell death was independent of caspase enzymatic activities as revealed by the lack of effect of ZVAD-fmk, a pan-caspase inhibitor. These results suggested that the Fas-FasL pathway, as part of the classical activation pathway of the macrophages, is essential in the stimulation of macrophage leading to a microbicidal state and to AICD, and may thus contribute to the pathogenesis of L. major infection.

Testing the predictive adaptive response in a host-parasite system

1. Harsh environmental conditions experienced during development can reduce the performance of the same individuals in adulthood. However, the 'predictive adaptive response' hypothesis postulates that if individuals adapt their phenotype during development to the environments where they are likely to live in the future, individuals exposed to harsh conditions in early life perform better when encountering the same harsh conditions in adulthood compared to those never exposed to these conditions before. 2. Using the common vole (Microtus arvalis) as study organism, we tested how exposure to flea parasitism during the juvenile stage affects the physiology (haematocrit, resistance to oxidative stress, resting metabolism, spleen mass, and testosterone), morphology (body mass, testis mass) and motor performance (open field activity and swimming speed) of the same individuals when infested with fleas in adulthood. According to the 'predictive adaptive response' hypothesis, we predicted that voles parasitized at the adult stage would perform better if they had already been parasitized with fleas at the juvenile stage. 3. We found that voles exposed to fleas in adulthood had a higher metabolic rate if already exposed to fleas when juvenile, compared to voles free of fleas when juvenile and voles free of fleas in adulthood. Independently of juvenile parasitism, adult parasitism impaired adult haematocrit and motor performances. Independently of adult parasitism, juvenile parasitism slowed down crawling speed in adult female voles. 4. Our results suggest that juvenile parasitism has long-term effects that do not protect from the detrimental effects of adult parasitism. On the contrary, experiencing parasitism in early-life incurs additional costs upon adult parasitism measured in terms of higher energy expenditure, rather than inducing an adaptive shift in the developmental trajectory. 5. Hence, our study provides experimental evidence for long term costs of parasitism. We found no support for a predictive adaptive response in this host-parasite system.

Residual antimalarials in malaria patients from Tanzania--implications on drug efficacy assessment and spread of parasite resistance.

Repeated antimalarial treatment for febrile episodes and self-treatment are common in malaria-endemic areas. The intake of antimalarials prior to participating in an in vivo study may alter treatment outcome and affect the interpretation of both efficacy and safety outcomes. We report the findings from baseline plasma sampling of malaria patients prior to inclusion into an in vivo study in Tanzania and discuss the implications of residual concentrations of antimalarials in this setting. In an in vivo study conducted in a rural area of Tanzania in 2008, baseline plasma samples from patients reporting no antimalarial intake within the last 28 days were screened for the presence of 14 antimalarials (parent drugs or metabolites) using liquid chromatography-tandem mass spectrometry. Among the 148 patients enrolled, 110 (74.3%) had at least one antimalarial in their plasma: 80 (54.1%) had lumefantrine above the lower limit of calibration (LLC = 4 ng/mL), 7 (4.7%) desbutyl-lumefantrine (4 ng/mL), 77 (52.0%) sulfadoxine (0.5 ng/mL), 15 (10.1%) pyrimethamine (0.5 ng/mL), 16 (10.8%) quinine (2.5 ng/mL) and none chloroquine (2.5 ng/mL). The proportion of patients with detectable antimalarial drug levels prior to enrollment into the study is worrying. Indeed artemether-lumefantrine was supposed to be available only at government health facilities. Although sulfadoxine-pyrimethamine is only recommended for intermittent preventive treatment in pregnancy (IPTp), it was still widely used in public and private health facilities and sold in drug shops. Self-reporting of previous drug intake is unreliable and thus screening for the presence of antimalarial drug levels should be considered in future in vivo studies to allow for accurate assessment of treatment outcome. Furthermore, persisting sub-therapeutic drug levels of antimalarials in a population could promote the spread of drug resistance. The knowledge on drug pressure in a given population is important to monitor standard treatment policy implementation.

The economics of anti-parasite defences in animal societies

RESUME : De nombreuses espèces animales vivent en groupe. Du simple grégarisme aux colonies hautement intégrées de fourmis, la vie sociale a atteint des degrés divers de complexité. Les nombreuses interactions entre membres d'une société favorisent la transmission de parasites. Cela représente un coût potentiel de la vie sociale. Cette thèse s'intéresse aux défenses permettant de réduire le coût du parasitisme dans les colonies de fourmis ainsi qu'à la manière dont le parasitisme a pu façonner certains aspects de ces sociétés. Les colonies de fourmis des bois (Forimica paralugubris) contiennent de grandes quantités de résine de conifères. Cette résine réduit la densité microbienne dans le nid et augmente la survie des ouvrières lors d'infections parasitaires. Dans cette thèse, nous montrons, d'une part, que les ouvrières collectent activement la résine et que ce comportement est plutôt préventif que curatif et, d'autre part, que la résine permet aux ouvrières une utilisation moindre de leurs défenses immunitaires. Ces résultats permettent de conclure que ce comportement réduit l'exposition au parasitisme et qu'il a une fonction adaptative. L'émergence d'un tel comportement de médication chez une espèce d'insectes sociaux illustre le fait que la socialité, bien yue provoquant une exposition accrue au parasitisme, permet également l'émergence de mécanismes sociaux de défense. II a été suggéré que la présence de plusieurs reines au sein d'un même nid (polygynie) améliore la résistance aux parasites en augmentant la diversité génétique au sein de la colonie. En accord avec cette hypothèse, nous montrons qu'une augmentation de la diversité génétique au sein de groupes expérimentaux de Formica selysi améliore leur survie lors d'une infection parasitaire. Cependant, nous suggérons également que sur le terrain, d'autres facteurs corrélés à la polygynie ont des effets antagoniques sur la résistance. Nous montrons par exemple que les ouvrières polygynes semblent avoir une capacité moindre à monter une réponse immunitaire. Certains aspects de la reproduction des fourmis ont pu également être façonnés par le parasitisme. L'accouplement n'a lieu que lors d'une courte période au début de la vie adulte, généralement à l'extérieur de la colonie. Les reines stockent ensuite le sperme et l'utilisent parcimonieusement au cours de leur vie alors que les males meurent rapidement. Nous montrons que les défenses immunitaires des reines de fourmis des bois (F. paralugubris) sont fortement affectées par l'accouplement. Ces modulations immunitaires sont probablement liées à une augmentation de l'exposition au parasitisme lors de l'accouplement ainsi qu'à des blessures copulatoires. I1 semble donc que l'accouplement soit accompagné de coûts immunitaires pour les reines. Dans son ensemble, cette thèse illustre la diversité des mécanismes de défenses contre les parasites dans les sociétés de fourmis. La vie sociale, en offrant un nouveau niveau d'interaction, permet en effet l'émergence d'adaptations originales. Cela explique probablement le grand succès écologique des espèces sociales. SUMMARY : Sociality is widespread among animals and has reached variable degrees of complexity, from loose social Groups to highly integrated ant colonies. The many interactions between members of a social group promote the spread of parasites, but social life also permits the evolution of original defence mechanisms. This thesis sheds light on how ant colonies defend themselves against parasites, and on how parasitism shapes certain aspects of these societies. Wood ants nests (Formica paralugubris) contain large amounts of conifer resin which reduces the microbial density in ant nests and enhances the survival of ants challenged by some pathogens. We show that resin is actively collected by workers and that resin collection is rather a prophylactic than a curative behaviour. Moreover, we suggest that resin reduces the use of the immune defences of workers. Altogether, these results indicate that the use of resin is a collective adaptation to prevent the spread of parasites. The emergence of medication in a social insect species illustrates that sociality does not only increase the exposure to parasites but also allows the emergence of social mechanisms to counter this threat. The number of reproducing queens per colony is a variable trait in ants. It has been suggested that polygyny (the occurrence of multiple queens within a colony), by increasing the colonial genetic diversity, improves disease resistance. In line with this hypothesis, we show that in a socially polymorphic ant (Formica selysi), an experimental increase of colony genetic diversity enhances disease resistance. However, we also suggest that factors covarying with queen number variation in the field have antagonistic effects on parasite resistance. We show for instance that polygyne workers seem to have lower immune defences. Parasites may also shape some aspects of ant queen reproductive biology. Ant queens mate at the beginning of their adult life, usually outside of the colony, and store sperm for several years to fertilize eggs. Males die shortly after mating and queens never remate later in life, which drastically reduces sexual conflicts. Moreover, mating and nest founding occur away from the collective defence mechanisms of the natal colony and might be associated with an increased risk of parasitism. We show that mating affects the immune defences of wood ant queens (F. paralugubris) in multiple ways that are consistent with mating wounds and increased risk of parasitism. We suggest that mating is associated with immunity costs in ants, despite the reduced level of sexual conflicts. Altogether, my thesis illustrates the diversity of anti-parasite mechanisms in ant societies. This sheds light on how sociality, by offering a new level of interactions, allows the evolution of original adaptations, which may explain the wide ecological success of social species.

Correlative light and electron microscopy in parasite research.

The interaction of a parasite and a host cell is a complex process, which involves several steps: (1) attachment to the plasma membrane, (2) entry inside the host cell, and (3) hijacking of the metabolism of the host. In biochemical experiments, only an event averaged over the whole cell population can be analyzed. The power of microscopy, however, is to investigate individual events in individual cells. Therefore, parasitologists frequently perform experiments with fluorescence microscopy using different dyes to label structures of the parasite or the host cell. Though the resolution of light microscopy has greatly improved, it is not sufficient to reveal interactions at the ultrastructural level. Furthermore, only specifically labeled structures can be seen and related to each other. Here, we want to demonstrate the additional value of electron microscopy in this area of research. Investigation of the different steps of parasite-host cell interaction by electron microscopy, however, is often hampered by the fact that there are only a few cells infected, and therefore it is difficult to find enough cells to study. A solution is to profit from low magnification, hence large overview, and specific location of the players by fluorescence labels in a light microscope with the high power resolution and structural information provided by an electron microscope, in short by correlative light and electron microscopy.

Characterisation of two soluble metalloexopeptidases in the protozoan parasite Leishmania major.

Two soluble exopeptidases were identified in promastigotes of Leishmania major, using an iodinated model tetrapeptide (LIAY) as substrate. Similar activities were also detected in L. major amastigotes and in different species of Leishmania promastigotes. A carboxy- and an aminopeptidase activity were resolved and isolated by anion exchange and gel permeation chromatographies. A single polypeptide of 62 kDa co-purified with the aminopeptidase activity. Optimum pH was neutral for the carboxypeptidase and neutral to alkaline for the aminopeptidase. Both activities were able to hydrolyse a dipeptide substrate (YL), and were inhibited by 20 microM bestatin and 200 microM 1,10-phenanthroline, but not by leupeptin, iodoacetamide and a range of other inhibitors. These results strongly suggest that both enzymes are metalloexopeptidases and thus represent a novel class of soluble peptidases in Leishmania.

Ultrastructural data on the life cycle of the parasite, Perkinsus atlanticus (Apicomplexa), on the clam, Ruditapes phillipinarum, in the Mediterranean

An Apicomplexan Perkinsus species has been found parasitizing the clam Ruditapes philippinarum (= Tapes semidecussatus) collected on the Mediterranean coast in the region of the Ebro Delta (Tarragona, Spain). Light and transmission electron microscopy were used to study different stages of this parasite during zoosporulation induced by incubation in thioglycollate medium and seawater. During incubation the trophozoites began zoosporulation, which originated prezoosporangia and zoosporangia at different developmental stages. Successive cytokinesis and nucleokinesis gave rise to prezoospores, which became elongate and differentiated in biflagellated zoospores. The latter presented large mitochondria and an apical complex formed by a conoid, polar ring, micronemes, rhophtries and subpellicular microtubules. The zoosporangium wall showed some typical lamosomes and a discharge tube developed in early phases of incubation. Ultrastructural data were compared with the only four species of the genus Perkinsus previously described. The morphological data, the host and the geographic proximity suggest that the species located on the Mediterranean coast was Perkinsus atlanticus.

Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the contribution of tumor necrosis factor is minimal.

Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.

The evolutionary host switches of Polychromophilus: a multi-gene phylogeny of the bat malaria genus suggests a second invasion of mammals by a haemosporidian parasite.

BACKGROUND: The majority of Haemosporida species infect birds or reptiles, but many important genera, including Plasmodium, infect mammals. Dipteran vectors shared by avian, reptilian and mammalian Haemosporida, suggest multiple invasions of Mammalia during haemosporidian evolution; yet, phylogenetic analyses have detected only a single invasion event. Until now, several important mammal-infecting genera have been absent in these analyses. This study focuses on the evolutionary origin of Polychromophilus, a unique malaria genus that only infects bats (Microchiroptera) and is transmitted by bat flies (Nycteribiidae). METHODS: Two species of Polychromophilus were obtained from wild bats caught in Switzerland. These were molecularly characterized using four genes (asl, clpc, coI, cytb) from the three different genomes (nucleus, apicoplast, mitochondrion). These data were then combined with data of 60 taxa of Haemosporida available in GenBank. Bayesian inference, maximum likelihood and a range of rooting methods were used to test specific hypotheses concerning the phylogenetic relationships between Polychromophilus and the other haemosporidian genera. RESULTS: The Polychromophilus melanipherus and Polychromophilus murinus samples show genetically distinct patterns and group according to species. The Bayesian tree topology suggests that the monophyletic clade of Polychromophilus falls within the avian/saurian clade of Plasmodium and directed hypothesis testing confirms the Plasmodium origin. CONCLUSION: Polychromophilus' ancestor was most likely a bird- or reptile-infecting Plasmodium before it switched to bats. The invasion of mammals as hosts has, therefore, not been a unique event in the evolutionary history of Haemosporida, despite the suspected costs of adapting to a new host. This was, moreover, accompanied by a switch in dipteran host.

Altitudinal variation in haemosporidian parasite distribution in great tit populations.

BACKGROUND: One of the major issues concerning disease ecology and conservation is knowledge of the factors that influence the distribution of parasites and consequently disease outbreaks. This study aimed to investigate avian haemosporidian composition and the distribution of these parasites in three altitudinally separated great tit (Parus major) populations in western Switzerland over a three-year period. The objectives were to determine the lineage diversity of parasites occuring across the study populations and to investigate whether altitudinal gradients govern the distribution of haemosporidian parasites by lineage. METHODS: In this study molecular approaches (PCR and sequencing) were used to detect avian blood parasites (Plasmodium sp., Haemoproteus sp. and Leucocytozoon sp.) in populations of adult great tits caught on their nests during three consecutive breeding seasons. RESULTS: High levels of parasite prevalence (88-96%) were found across all of the study populations with no significant altitude effect. Altitude did, however, govern the distribution of parasites belonging to different genera, with Plasmodium parasites being more prevalent at lower altitudes, Leucocytozoon parasites more at high altitude and Haemoproteus parasite prevalence increasing with altitude. A total of 27 haemosporidian parasite lineages were recorded across all study sites, with diversity showing a positive correlation to altitude. Parasites belonging to lineage SGS1 (P. relictum) and PARUS4 and PARUS19 (Leucocytozoon sp.) dominated lower altitudes. SW2 (P. polare) was the second most prevalent lineage of parasite detected overall and these parasites were responsible for 68% of infections at intermediate altitude, but were only documented at this one study site. CONCLUSIONS: Avian haemosporidian parasites are not homogeneously distributed across host populations, but differ by altitude. This difference is most probably brought about by environmental factors influencing vector prevalence and distribution. The high occurrence of co-infection by different genera of parasites might have pronounced effects on host fitness and should consequently be investigated more rigorously.

The importance of host spatial distribution for parasite specialization and speciation: a comparative study of bird fleas (Siphonaptera : Ceratophyllidae)

1. The environment of parasites is determined largely by their hosts. Variation in host quality, abundance and spatial distribution affects the balance between selection within hosts and gene flow between hosts, and this should determine the evolution of a parasite's host-range and its propensity to locally adapt and speciate. 2. We investigated the relationship between host spatial distribution and (1) parasite host range, (2) parasite mobility and (3) parasite geographical range, in a comparative study of a major group of avian ectoparasites, the birds fleas belonging to the Ceratophyllidae (Siphonaptera). 3. Flea species parasitizing colonial birds had narrower host ranges than those infesting territorial nesters or birds with an intermediate level of nest aggregation. 4. The potential mobility and geographical ranges of fleas decreased with increasing level of aggregation of their hosts and increased with the fleas' host ranges. 5. Birds with aggregated nest distribution harboured more flea species mainly due to a larger number of specialists than solitarily nesting hosts. 6. These results emphasize the importance of host spatial distribution for the evolution of specialization, and for local adaptation and speciation in Ceratophyllid bird fleas.