Institute of Parasitology

Marine plankton support global biological and geochemical processes. Surveys of their biodiversity have hitherto been geographically restricted and have not accounted for the full range of plankton size. We assessed eukaryotic diversity from 334 size-fractionated photic-zone plankton communities collected across tropical and temperate oceans during the circumglobal Tara Oceans expedition. We analyzed 18S ribosomal DNA sequences across the intermediate plankton-size spectrum from the smallest unicellular eukaryotes (protists, >0.8 micrometers) to small animals of a few millimeters. Eukaryotic ribosomal diversity saturated at ~150,000 operational taxonomic units, about one-third of which could not be assigned to known eukaryotic groups. Diversity emerged at all taxonomic levels, both within the groups comprising the ~11,200 cataloged morphospecies of eukaryotic plankton and among twice as many other deep-branching lineages of unappreciated importance in plankton ecology studies. Most eukaryotic plankton biodiversity belonged to heterotrophic protistan groups, particularly those known to be parasites or symbiotic hosts.

This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re-introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under-sampled areas and from environmental genomic information.

The increase of allergic diseases in the industrialized world has often been explained by a decline in infections during childhood. The immunological explanation has been put into the context of the functional T cell subsets known as T helper 1 (TH1) and T helper 2 (TH2) that display polarized cytokine profiles. It has been argued that bacterial and viral infections during early life direct the maturing immune system toward TH1, which counterbalance proallergic responses of TH2 cells. Thus, a reduction in the overall microbial burden will result in weak TH1 imprinting and unrestrained TH2 responses that allow an increase in allergy. This notion is contradicted by observations that the prevalence of TH1-autoimmune diseases is also increasing and that TH2-skewed parasitic worm (helminth) infections are not associated with allergy. More recently, elevations of anti-inflammatory cytokines, such as interleukin-10, that occur during long-term helminth infections have been shown to be inversely correlated with allergy. The induction of a robust anti-inflammatory regulatory network by persistent immune challenge offers a unifying explanation for the observed inverse association of many infections with allergic disorders.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

BACKGROUND: The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. METHODOLOGY AND PRINCIPAL FINDINGS: Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? CONCLUSIONS AND SIGNIFICANCE: We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

The beneficial effects of statins are the result of their capacity to reduce cholesterol biosyntesis, mainly in the liver, where they are selectively distributed, as well as to the modulation of lipid metabolism, derived from their effect of inhibition upon HMG-CoA reductase. Statins have antiatherosclerotic effects, that positively correlate with the percent decrease in LDL cholesterol. In addition, they can exert antiatherosclerotic effects independently of their hypolipidemic action. Because the mevalonate metabolism generates a series of isoprenoids vital for different cellular functions, from cholesterol synthesis to the control of cell growth and differentiation, HMG-CoA reductase inhibition has beneficial pleiotropic effects. Consequently, statins reduce significantly the incidence of coronary events, both in primary and secondary prevention, being the most efficient hypolipidemic compounds that have reduced the rate of mortality in coronary patients. Independent of their hypolipidemic properties, statins interfere with events involved in bone formation and impede tumor cell growth.

A list of myxozoan genera is presented in the current taxonomical scheme. These genera are defined; their type species and most important pathogens along with their hosts are listed. Simultaneously, definitions of actinospore stages representing sexual stages of the myxosporean life cycle are given; altogether, 17 actinospore collective groups with 180 types have been described. Life cycles of the two classes of the phylum Myxozoa, Malacosporea and Myxosporea, are briefly outlined with specification of the appropriate terms. Up to now, 4 malacosporean and 2,180 myxosporean species assigned to a total of 62 genera, have been established. The surviving classification of myxosporeans, based on spore morphology, is discussed in the context of the still fragmentary data resulting from SSU rDNA sequence analyses. The main task for the future is a rigorous, detailed morphological description combined with molecular techniques in establishment of new species and in revision of the existing ones. Establishment of a classification acceptable from morphological, biological and phylogenetical viewpoints is necessary.

The wild-type p53 protein exhibits a common polymorphism at amino acid 72, resulting in either a proline residue (p53Pro) or an arginine residue (p53Arg) at this position. Despite the difference that this change makes in the primary structure of the protein resulting in a difference in migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no differences in the biochemical or biological characteristics of these wild-type p53 variants have been reported. We have recently shown that p53Arg is significantly more susceptible than p53Pro to the degradation induced by human papillomavirus (HPV) E6 protein. Moreover, this may result in an increased susceptibility to HPV-induced tumors in homozygous p53Arg individuals. In further investigating the characteristics of these p53 variants, we now show that both forms are morphologically wild type and do not differ in their ability to bind to DNA in a sequence-specific manner. However, there are a number of differences between the p53 variants in their abilities to bind components of the transcriptional machinery, to activate transcription, to induce apoptosis, and to repress the transformation of primary cells. These observations may have implications for the development of cancers which harbor wild-type p53 sequences and possibly for the ability of such tumors to respond to therapy, depending on their p53 genotype.

Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipids (GIPLs) from parasitic protozoa have been shown to exert a wide variety of effects on cells of the host innate immune system. However, the receptor(s) that are triggered by these protozoan glycolipids has not been identified. Here we present evidence that Trypanosoma cruzi-derived GPI anchors and GIPLs trigger CD25 expression on Chinese hamster ovary-K1 cells transfected with CD14 and Toll-like receptor-2 (TLR-2), but not wild-type (TLR-2-deficient) Chinese hamster ovary cells. The protozoan-derived GPI anchors and GIPLs containing alkylacylglycerol and saturated fatty acid chains or ceramide were found to be active in a concentration range of 100 nM to 1 microM. More importantly, the GPI anchors purified from T. cruzi trypomastigotes, which contain a longer glycan core and unsaturated fatty acids in the sn-2 position of the alkylacylglycerolipid component, triggered TLR-2 at subnanomolar concentrations. We performed experiments with macrophages from TLR-2 knockout and TLR-4 knockout mice, and found that TLR-2 expression appears to be essential for induction of IL-12, TNF-alpha, and NO by GPI anchors derived from T. cruzi trypomastigotes. Thus, highly purified GPI anchors from T. cruzi parasites are potent activators of TLR-2 from both mouse and human origin. The activation of TLR-2 may initiate host innate defense mechanisms and inflammatory response during protozoan infection, and may provide new strategies for immune intervention during protozoan infections.

Animals, plants, and fungi—the three traditional kingdoms of multicellular eukaryotic life—make up almost all of the visible biosphere, and they account for the majority of catalogued species on Earth [1]. The remaining eukaryotes have been assembled for convenience into the protists, a group composed of many diverse lineages, single-celled for the most part, that diverged after Archaea and Bacteria evolved but before plants, animals, or fungi appeared on Earth. Given their single-celled nature, discovering and describing new species has been difficult, and many protistan lineages contain a relatively small number of formally described species (Figure 1A), despite the critical importance of several groups as pathogens, environmental quality indicators, and markers of past environmental changes. It would seem natural to apply molecular techniques such as DNA barcoding to the taxonomy of protists to compensate for the lack of diagnostic morphological features, but this has been hampered by the extreme diversity within the group. The genetic divergence observed between and within major protistan groups greatly exceeds that found in each of the three multicellular kingdoms. No single set of molecular markers has been identified that will work in all lineages, but an international working group is now close to a solution. A universal DNA barcode for protists coupled with group-specific barcodes will enable an explosion of taxonomic research that will catalyze diverse applications.

BACKGROUND: A malaria vaccine could be an important addition to current control strategies. We report the safety and vaccine efficacy (VE) of the RTS,S/AS01 vaccine during 18 mo following vaccination at 11 African sites with varying malaria transmission. METHODS AND FINDINGS: 6,537 infants aged 6-12 wk and 8,923 children aged 5-17 mo were randomized to receive three doses of RTS,S/AS01 or comparator vaccine. VE against clinical malaria in children during the 18 mo after vaccine dose 3 (per protocol) was 46% (95% CI 42% to 50%) (range 40% to 77%; VE, p<0.01 across all sites). VE during the 20 mo after vaccine dose 1 (intention to treat [ITT]) was 45% (95% CI 41% to 49%). VE against severe malaria, malaria hospitalization, and all-cause hospitalization was 34% (95% CI 15% to 48%), 41% (95% CI 30% to 50%), and 19% (95% CI 11% to 27%), respectively (ITT). VE against clinical malaria in infants was 27% (95% CI 20% to 32%, per protocol; 27% [95% CI 21% to 33%], ITT), with no significant protection against severe malaria, malaria hospitalization, or all-cause hospitalization. Post-vaccination anti-circumsporozoite antibody geometric mean titer varied from 348 to 787 EU/ml across sites in children and from 117 to 335 EU/ml in infants (per protocol). VE waned over time in both age categories (Schoenfeld residuals p<0.001). The number of clinical and severe malaria cases averted per 1,000 children vaccinated ranged across sites from 37 to 2,365 and from -1 to 49, respectively; corresponding ranges among infants were -10 to 1,402 and -13 to 37, respectively (ITT). Meningitis was reported as a serious adverse event in 16/5,949 and 1/2,974 children and in 9/4,358 and 3/2,179 infants in the RTS,S/AS01 and control groups, respectively. CONCLUSIONS: RTS,S/AS01 prevented many cases of clinical and severe malaria over the 18 mo after vaccine dose 3, with the highest impact in areas with the greatest malaria incidence. VE was higher in children than in infants, but even at modest levels of VE, the number of malaria cases averted was substantial. RTS,S/AS01 could be an important addition to current malaria control in Africa. TRIAL REGISTRATION: www.ClinicalTrials.gov NCT00866619 Please see later in the article for the Editors' Summary.

For multicellular organisms, the rigorous control of programmed cell death is as important as that of cell proliferation. The mechanisms involved in the regulation of cell death are not yet understood, but a key component is the family of caspases which are activated in a cascade and are responsible for the apoptotic-specific changes and disassembly of the cell. Although the caspases represent a central point in apoptosis, their activation is regulated by a variety of other factors. Among these, Bcl-2 family plays a pivotal role in caspases activation, by this deciding whether a cell will live or die. Bcl-2 family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, but their functions are not yet well defined. This review summarizes the recent data regarding the Bcl-2 proteins and the ways they regulate the apoptosis.

Ticks and the pathogens they transmit constitute a growing burden for human and animal health worldwide. Vector competence is a component of vectorial capacity and depends on genetic determinants affecting the ability of a vector to transmit a pathogen. These determinants affect traits such as tick-host-pathogen and susceptibility to pathogen infection. Therefore, the elucidation of the mechanisms involved in tick-pathogen interactions that affect vector competence is essential for the identification of molecular drivers for tick-borne diseases. In this review, we provide a comprehensive overview of tick-pathogen molecular interactions for bacteria, viruses, and protozoa affecting human and animal health. Additionally, the impact of tick microbiome on these interactions was considered. Results show that different pathogens evolved similar strategies such as manipulation of the immune response to infect vectors and facilitate multiplication and transmission. Furthermore, some of these strategies may be used by pathogens to infect both tick and mammalian hosts. Identification of interactions that promote tick survival, spread, and pathogen transmission provides the opportunity to disrupt these interactions and lead to a reduction in tick burden and the prevalence of tick-borne diseases. Targeting some of the similar mechanisms used by the pathogens for infection and transmission by ticks may assist in development of preventative strategies against multiple tick-borne diseases.

Ancient migrations in Southeast Asia The past movements and peopling of Southeast Asia have been poorly represented in ancient DNA studies (see the Perspective by Bellwood). Lipson et al. generated sequences from people inhabiting Southeast Asia from about 1700 to 4100 years ago. Screening of more than a hundred individuals from five sites yielded ancient DNA from 18 individuals. Comparisons with present-day populations suggest two waves of mixing between resident populations. The first mix was between local hunter-gatherers and incoming farmers associated with the Neolithic spreading from South China. A second event resulted in an additional pulse of genetic material from China to Southeast Asia associated with a Bronze Age migration. McColl et al. sequenced 26 ancient genomes from Southeast Asia and Japan spanning from the late Neolithic to the Iron Age. They found that present-day populations are the result of mixing among four ancient populations, including multiple waves of genetic material from more northern East Asian populations. Science , this issue p. 92 , p. 88 ; see also p. 31

Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs.

We present Procrustean Approach to Cophylogeny (PACo), a novel statistical tool to test for congruence between phylogenetic trees, or between phylogenetic distance matrices of associated taxa. Unlike previous tests, PACo evaluates the dependence of one phylogeny upon the other. This makes it especially appropriate to test the classical coevolutionary model that assumes that parasites that spend part of their life in or on their hosts track the phylogeny of their hosts. The new method does not require fully resolved phylogenies and allows for multiple host-parasite associations. PACo produces a Procrustes superimposition plot enabling a graphical assessment of the fit of the parasite phylogeny onto the host phylogeny and a goodness-of-fit statistic, whose significance is established by randomization of the host-parasite association data. The contribution of each individual host-parasite association to the global fit is measured by means of jackknife estimation of their respective squared residuals and confidence intervals associated to each host-parasite link. We carried out different simulations to evaluate the performance of PACo in terms of Type I and Type II errors with respect to two similar published tests. In most instances, PACo performed at least as well as the other tests and showed higher overall statistical power. In addition, the jackknife estimation of squared residuals enabled more elaborate validations about the nature of individual links than the ParaFitLink1 test of the program ParaFit. In order to demonstrate how it can be used in real biological situations, we applied PACo to two published studies using a script written in the public-domain statistical software R.

The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga.

BACKGROUND: Robust reference values for fecal egg count reduction (FECR) rates of the most widely used anthelmintic drugs in preventive chemotherapy (PC) programs for controlling soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, and hookworm) are still lacking. However, they are urgently needed to ensure detection of reduced efficacies that are predicted to occur due to growing drug pressure. Here, using a standardized methodology, we assessed the FECR rate of a single oral dose of mebendazole (MEB; 500 mg) against STHs in six trials in school children in different locations around the world. Our results are compared with those previously obtained for similarly conducted trials of a single oral dose of albendazole (ALB; 400 mg). METHODOLOGY: The efficacy of MEB, as assessed by FECR, was determined in six trials involving 5,830 school children in Brazil, Cambodia, Cameroon, Ethiopia, United Republic of Tanzania, and Vietnam. The efficacy of MEB was compared to that of ALB as previously assessed in 8,841 school children in India and all the above-mentioned study sites, using identical methodologies. PRINCIPAL FINDINGS: The estimated FECR rate [95% confidence interval] of MEB was highest for A. lumbricoides (97.6% [95.8; 99.5]), followed by hookworm (79.6% [71.0; 88.3]). For T. trichiura, the estimated FECR rate was 63.1% [51.6; 74.6]. Compared to MEB, ALB was significantly more efficacious against hookworm (96.2% [91.1; 100], p<0.001) and only marginally, although significantly, better against A. lumbricoides infections (99.9% [99.0; 100], p = 0.012), but equally efficacious for T. trichiura infections (64.5% [44.4; 84.7], p = 0.906). CONCLUSIONS/SIGNIFICANCE: A minimum FECR rate of 95% for A. lumbricoides, 70% for hookworm, and 50% for T. trichiura is expected in MEB-dependent PC programs. Lower FECR results may indicate the development of potential drug resistance.

Naturally acquired immunity to malaria develops slowly, requiring several years of repeated exposure to be effective. The cellular and molecular factors underlying this observation are only partially understood. Recent studies suggest that chronic Plasmodium falciparum exposure may induce functional exhaustion of lymphocytes, potentially impeding optimal control of infection. However, it remains unclear whether the "atypical" memory B cells (MBCs) and "exhausted" CD4 T cells described in humans exposed to endemic malaria are driven by P. falciparum per se or by other factors commonly associated with malaria, such as coinfections and malnutrition. To address this critical question we took advantage of a "natural" experiment near Kilifi, Kenya, and compared profiles of B and T cells of children living in a rural community where P. falciparum transmission is ongoing to the profiles of age-matched children living under similar conditions in a nearby community where P. falciparum transmission ceased 5 y prior to this study. We found that continuous exposure to P. falciparum drives the expansion of atypical MBCs. Persistent P. falciparum exposure was associated with an increased frequency of CD4 T cells expressing phenotypic markers of exhaustion, both programmed cell death-1 (PD-1) alone and PD-1 in combination with lymphocyte-activation gene-3 (LAG-3). This expansion of PD-1-expressing and PD-1/LAG-3-coexpressing CD4 T cells was largely confined to CD45RA(+) CD4 T cells. The percentage of CD45RA(+)CD27(+) CD4 T cells coexpressing PD-1 and LAG-3 was inversely correlated with frequencies of activated and classical MBCs. Taken together, these results suggest that P. falciparum infection per se drives the expansion of atypical MBCs and phenotypically exhausted CD4 T cells, which has been reported in other endemic areas.

SUMMARY: Tapeworms (Cestoda) continue to be an important cause of morbidity in humans worldwide. Diphyllobothriosis, a human disease caused by tapeworms of the genus Diphyllobothrium, is the most important fish-borne zoonosis caused by a cestode parasite. Up to 20 million humans are estimated to be infected worldwide. Besides humans, definitive hosts of Diphyllobothrium include piscivorous birds and mammals, which represent a significant zoonotic reservoir. The second intermediate hosts include both freshwater and marine fish, especially anadromous species such as salmonids. The zoonosis occurs most commonly in countries where the consumption of raw or marinated fish is a frequent practice. Due to the increasing popularity of dishes utilizing uncooked fish, numerous cases of human infections have appeared recently, even in the most developed countries. As many as 14 valid species of Diphyllobothrium can cause human diphyllobothriosis, with D. latum and D. nihonkaiense being the most important pathogens. In this paper, all taxa from humans reported are reviewed, with brief information on their life history and their current distribution. Data on diagnostics, epidemiology, clinical relevance, and control of the disease are also summarized. The importance of reliable identification of human-infecting species with molecular tools (sequences of mitochondrial genes) as well as the necessity of epidemiological studies aimed at determining the sources of infections are pointed out.