Specific calpain activity evaluation in Plasmodium parasites

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.     

Host associations and evolutionary relationships of avian blood parasites from West Africa

The host specificity of blood parasites recovered from a survey of 527 birds in Cameroon and Gabon was examined at several levels within an evolutionary framework. Unique mitochondrial lineages of Haemoproteus were recovered from an average of 1.3 host species (maximum = 3) and 1.2 host families (maximum = 3) while lineages of Plasmodium were recovered from an average of 2.5 species (maximum = 27) and 1.6 families (maximum = 9). Averaged within genera, lineages of both Plasmodium and Haemoproteus were constrained in their host distribution relative to random expectations. However, while several individual lineages within both genera exhibited significant host constraint, host breadth varied widely among related lineages, particularly within the genus Plasmodium. Several lineages of Plasmodium exhibited extreme generalist host-parasitism strategies while other lineages appeared to have been constrained to certain host families over recent evolutionary history. Sequence data from two nuclear genes recovered from a limited sample of Plasmodium parasites indicated that, at the resolution of this study, inferences regarding host breadth were unlikely to be grossly affected by the use of parasite mitochondrial lineages as a proxy for biological species. The use of divergent host-parasitism strategies among closely related parasite lineages suggests that host range is a relatively labile character. Since host specificity may also influence parasite virulence, these results argue for considering the impact of haematozoa on avian hosts on a lineage-specific basis.     

Genetic characterisation of uninucleated cyst-producing Entamoeba spp. from ruminants

Six ssrRNA gene sequences were obtained by PCR amplification of DNA from uninucleated Entamoeba cysts isolated from fresh faeces of sheep, cows, a roe deer and a reindeer. Phylogenetic analysis using sequences of non-, uni-, quadri- and octonucleate cyst-producing Entamoeba spp. for comparison showed that all six isolates formed a separate clade nested within the clade of quadrinucleate cyst producers. The data indicate that Entamoeba bovis can be isolated from ruminant hosts other than cattle, and we suggest that organisms clustering with the sheep and cattle isolates analysed in the present study be named E. bovis.     

Cysteamine, the natural metabolite of pantetheinase, shows specific activity against Plasmodium

In mice, loss of pantetheinase activity causes susceptibility to infection with Plasmodium chabaudi AS. Treatment of mice with the pantetheinase metabolite cysteamine reduces blood-stage replication of P. chabaudi and significantly increases survival. Similarly, a short exposure of Plasmodium to cysteamine ex vivo is sufficient to suppress parasite infectivity in vivo. This effect of cysteamine is specific and not observed with a related thiol (dimercaptosuccinic acid) or with the pantethine precursor of cysteamine. Also, cysteamine does not protect against infection with the parasite Trypanosoma cruzi or the fungal pathogen Candida albicans, suggesting cysteamine acts directly against the parasite and does not modulate host inflammatory response. Cysteamine exposure also blocks replication of P. falciparum in vitro; moreover, these treated parasites show higher levels of intact hemoglobin. This study highlights the in vivo action of cysteamine against Plasmodium and provides further evidence for the involvement of pantetheinase in host response to this infection.     

Consequences of chronic infections with three different avian malaria lineages on reproductive performance of Lesser Kestrels (Falco naumanni)

We studied the consequences of chronic infections by three different lineages of avian malaria, two Plasmodium (RTSR1, LK6) and one Haemoproteus (LK2), on reproductive performance of Lesser Kestrels (Falco naumanni). Malaria infections in male and female parents had no effect on clutch size, hatching success or nesting success. However, when only successful nests were considered, we found that males parasitized by LK6 raised a lower number of fledglings, suggesting that the level of parental effort by males may be limited by this particular lineage of Plasmodium. This effect was not evident in females, probably due to the higher investment of males during the chick rearing period in this species. Overall, we have found that chronic stages of specific malaria lineages have certain negative consequences on host reproductive performance, highlighting the importance of considering genetic differences among malaria parasites to study their consequences on natural bird populations.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.     

Food availability and competition do not modulate the costs of Plasmodium infection in dominant male canaries

Understanding the different factors that may influence parasite virulence is of fundamental interest to ecologists and evolutionary biologists. It has recently been demonstrated that parasite virulence may occur partly through manipulation of host competitive ability. Differences in competitive ability associated with the social status (dominant or subordinate) of a host may determine the extent of this competition-mediated parasite virulence. We proposed that differences between subordinate and dominant birds in the physiological costs of infection may change depending on the level of competition in social groups. We observed flocks of domestic canaries to determine dominant or subordinate birds, and modified competition by providing restricted (high competition) or ad libitum food (low competition). Entire flocks were then infected with either the avian malaria parasite, Plasmodium relictum or a control. Contrary to our predictions we found that the level of competition had no effect on the outcome of infection for dominant or subordinate birds. We found that dominant birds appeared to suffer greater infection mediated morbidity in both dietary treatments, with a higher and more sustained reduction in haematocrit, and higher parasitaemia, than subordinates. Our results show that dominance status in birds can certainly alter parasite virulence, though the links between food availability, competition, nutrition and virulence are likely to be complex and multifaceted.     

The effect of parental quality and malaria infection on nestling performance in the Collared Flycatcher (Ficedula albicollis)

Plumage ornamentation often signals the quality of males and, therefore, female birds may choose elaborately ornamented mates to increase their fitness. Such mate choice may confer both direct and indirect benefits to the offspring. Males with elaborate ornaments may provide good genes, which can result in better nestling growth, survival or resistance against parasitic infections. However, these males may also provision their offspring with more food or food of better quality, resulting in nestlings growing at a higher rate or fledging in better condition. In this study, we examined if there was an association between male ornamentation and malaria infection in Collared Flycatchers (Ficedula albicollis). We also investigated offspring performance in relation to malaria infection in the parents and the quality of the genetic and rearing fathers (assessed by the size of two secondary sexual characters) under simulated good and bad conditions (using brood size manipulation). We found that secondary sexual characters did not signal the ability of males to avoid parasitic infections, and malaria infection in the genetic and the rearing parents had no effect on nestling growth and fledging size. Our results do show, however, that it may be beneficial for the females to mate with males with a large forehead patch because wing feathers of nestlings reared by large-patched males grew at a higher rate. Fast feather growth can result in earlier fledging which, in turn, could improve nestling survival in highly variable environments or under strong nest predation.     

Distribution,diversity and drivers of blood-borne parasite co-infections in Alaskan bird populations

Avian species are commonly infected by multiple parasites, however few studies have investigated the environmental determinants of the prevalence of co-infection over a large scale. Here we believe that we report the first, detailed ecological study of the prevalence, diversity and co-infections of four avian blood-borne parasite genera: Plasmodium spp., Haemoproteus spp., Leucocytozoon spp. and Trypanosoma spp. We collected blood samples from 47 resident and migratory bird species across a latitudinal gradient in Alaska. From the patterns observed at collection sites, random forest models were used to provide evidence of associations between bioclimatic conditions and the prevalence of parasite co-infection distribution. Molecular screening revealed a higher prevalence of haematozoa (53%) in Alaska than previously reported. Leucocytozoons had the highest diversity, prevalence and prevalence of co-infection. Leucocytozoon prevalence (35%) positively correlated with Trypanosoma prevalence (11%), negatively correlated with Haemoproteus prevalence (14%) and had no correlation with Plasmodium prevalence (7%). We found temperature, precipitation and tree cover to be the primary environmental drivers that show a relationship with the prevalence of co-infection. The results provide insight into the impacts of bioclimatic drivers on parasite ecology and intra-host interactions, and have implications for the study of infectious diseases in rapidly changing environments.     

Initiation of Plasmodium sporozoite motility by albumin is associated with induction of intracellular signalling

Malaria infection is initiated when a mosquito injects Plasmodium sporozoites into a mammalian host. Sporozoites exhibit gliding motility both in vitro and in vivo. This motility is associated with the secretion of at least two proteins, circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Both derive from micronemes, which are organelles that empty out of the apical end of the sporozoite. Sporozoite motility can be initiated in vitro by albumin added to the medium. To investigate how albumin functions in this process, we studied second messenger signalling within the sporozoite. Using pharmacological activators and inhibitors, we have concluded that gliding motility is initiated when albumin interacts with the surface of the sporozoite and that this leads to a signal transduction cascade within the sporozoite, including the elevation of intracellular cAMP, the modulation of sporozoite motility by Ca²⁺ and the release of microneme proteins.     

A molecular analysis of some Eastern Atlantic grouper from the Epinephelus and Mycteroperca genus

Mitochondrial cytochrome b (397 bp) and 16S rDNA (516 bp) sequences analysis was used to investigate the phylogenetic relationships among some Eastern Atlantic Epinephelinae species. Six species of Epinephelus (E. aeneus, E. caninus, E. costae, E. haifensis, E. marginatus and E. tauvina) and two species of Mycteroperca (M. rubra and M. fusca) were analysed. Neighbour-joining and maximum-parsimony analysis support the paraphyletic grouping of the Epinephelus and Mycteroperca analysed. The maximum pairwise nucleotide divergence value in cyt b among all taxa was 0.196 between E. aeneus and E. marginatus and the minimum value was 0.006 between E. costae and M. rubra. Meanwhile, in 16S sequence analysis, the maximum value is 0.093 between E. aeneus and E. tauvina and the minimum value is 0.011 between E. marginatus and M. rubra. Molecular clock estimates for the species suggest a divergence time of 20-24 mya, which coincides with the Miocene period. A molecular analysis was also conducted, using other Epinephelinae sequences from GenBank in order to improve our understanding of the phyletic status of the Epinephelus and Mycteroperca species analysed.     

Systematics of Alloteuthis (Cephalopoda:Loliginidae) based on molecular and morphometric data

Alloteuthis is a group of small, slender loliginid squids of minor fisheries importance. There are three nominal Alloteuthis species—A. media (Linnaeus), A. subulata (Lamarck) and A. africana Adam. Two of these species (A. media and A. subulata) have largely overlapping ranges in the Mediterranean and northeastern Atlantic, while A. africana is found along the west coast of Africa. Despite the low level of species diversity, Alloteuthis taxonomy and systematics are confused, and assignment of specimens to species can be difficult. To clarify Alloteuthis systematics, we gathered morphometric data and DNA sequence data from two mitochondrial loci and a nuclear locus from Alloteuthis specimens collected from several localities. Analyses of the morphometric data suggest that head width is the main variable allowing separation of A. africana from the other two species, and central club sucker size separates A. media from A. subulata. One easily diagnosable character often used to distinguish Alloteuthis species—relative fin length—appears to be of little taxonomic value. Only three specimens assignable to A. subulata both morphologically and genetically were found, all from the Adriatic; possible reasons for this apparent rarity are discussed. Gene tree parsimony and coalescent-based methods were used to estimate species relationships from the molecular data, and both supported a sister-species relationship between A. media and A. subulata. Analyses of molecular variation (AMOVA's) revealed significant genetic differentiation between Atlantic and Mediterranean A. media. This study highlights the importance of 1) sampling multiple individuals, locations and loci for species-level phylogenetic studies, 2) using morphometric analyses to reveal taxonomically meaningful morphological characters and 3) accounting for the stochastic nature of the coalescent process when estimating species phylogenies for closely related taxa.     

Regulation of immunity to Plasmodium: Implications from mouse models for blood stage malaria vaccine design

Malaria, a disease caused by the protozoan parasite Plasmodium, remains a serious healthcare problem in developing countries worldwide. While the host-parasite relationship in humans has been difficult to determine, the pliability of murine malaria models has enabled valuable contributions to the understanding of the pathogenesis of disease. Although no single model reflects precisely malaria infection of the human, different models collectively provide important information on the mechanisms of protective immunity and immunopathogenesis. This review summarizes progress towards understanding the broad spectrum of immune responsiveness to the blood stages of the malaria parasite during experimental infections in mice and highlights how examination of murine malarias sheds light on the factors involved in the modulation of vaccine-potentiated immunity.     

Dynamics of parasitemia of malaria parasites in a naturally and experimentally infected migratory songbird, the great reed warbler Acrocephalus arundinaceus

Little is known about the development of infection of malaria parasites of the genus Plasmodium in wild birds. We used qPCR, targeting specific mitochondrial lineages of Plasmodium ashfordi (GRW2) and Plasmodium relictum (GRW4), to monitor changes in intensities of parasitemia in captive great reed warblers Acrocephalus arundinaceus from summer to spring. The study involved both naturally infected adults and experimentally infected juveniles. The experiment demonstrated that P. ashfordi and P. relictum lineages differ substantially in several life-history traits (e.g. prepatent period and dynamics of parasitemia) and that individual hosts show substantial differences in responses to these infections. The intensity of parasitemia of lineages in mixed infections co-varied positively, suggesting a control mechanism by the host that is general across the parasite lineages. The intensity of parasitemia for individual hosts was highly repeatable suggesting variation between the host individuals in their genetic or acquired control of the infections. In future studies, care must be taken to avoid mixed infections in wild caught donors, and when possible use mosquitoes for the experiments as inoculation of infectious blood ignores important initial stages of the contact between the bird and the parasite.     

Species delimitation in the European species of Clavulina (Cantharellales, Basidiomycota) inferred from phylogenetic analyses of ITS region and morphological data

The identification of the conventionally accepted species of Clavulina (Cantharellales, Basidiomycota) in Europe (Clavulina amethystina, Clavulina cinerea, Clavulina cristata, and Clavulina rugosa) is often difficult and many specimens are not straightforwardly assignable to any of those four species, which is why some authors have questioned their identity. In order to assess the status of those species, a morphological examination was combined with the molecular analysis of the ITS region. The same six major clades were obtained in the Bayesian and parsimony phylogenetic analyses, and all six clades were well-supported at least by one of the analyses. Morphological characters, such as the overall branching pattern, the presence and intensity of grey colour, the cristation of the apices, and basidiospore size and shape were to various extents correlated with the phylogenetic signal obtained from the ITS region. The congruence between the molecular analyses and morphology, rather than geographical origin, suggests the existence of several species that can be delimited using a combined phylogenetic and morphological species recognition. The analyses revealed that C. cristata and C. rugosa are well-delimited species. In contrast, more than one taxa could be subsumed under the names C. amethystina and C. cinerea, the taxonomical complexity of which is discussed. The ITS region is proved to be adequate to separate phylogenetic species of Clavulina.