Co-occurrences of parasite clones and altered host phenotype in a snail-trematode system

The frequent co-occurrence of two or more genotypes of the same parasite species in the same individual hosts has often been predicted to select for higher levels of virulence. Thus, if parasites can adjust their level of host exploitation in response to competition for resources, mixed-clone infections should have more profound impacts on the host. Trematode parasites are known to induce a wide range of modifications in the morphology (size, shell shape or ornamentation) of their snail intermediate host. Still, whether mixed-clone trematode infections have additive effects on the phenotypic alterations of the host remains to be tested. Here, we used the snail Potamopyrgus antipodarum-infected by the trematode Coitocaecum parvum to test for both the general effect of the parasite on host phenotype and possible increased host exploitation in multi-clone infections. Significant differences in size, shell shape and spinosity were found between infected and uninfected snails, and we determined that one quarter of naturally infected snails supported mixed-clone infections of C. parvum. From the parasite perspective, this meant that almost half of the clones identified in this study shared their snail host with at least one other clone. Intra-host competition may be intense, with each clone in a mixed-clone infection experiencing major reductions in volume and number of sporocysts (and consequently multiplication rate and cercarial production) compared with single-clone infections. However, there was no significant difference in the intensity of host phenotype modifications between single and multiple-clone infections. These results demonstrate that competition between parasite genotypes may be strong, and suggest that the frequency of mixed-clone infections in this system may have selected for an increased level of host exploitation in the parasite population, such that a single-clone is associated with a high degree of host phenotypic alteration.     

Hebeloma species associated with Cistus

The genus Hebeloma has a number of species highly specific to Cistus and others that occur with several host genera. This paper discusses the species of Hebeloma that appear to be ectomycorrhizal with Cistus, judging from their occurrence when Cistus is the only available host. The previously unknown species H. plesiocistum spec. nov. is described. We also provide a key to the known Hebeloma associates of Cistus. Molecular analyses based on ITS sequence data further illustrate the distinctness of the newly described species and difficulties in the species delimitation with view to H. erumpens. Specific associations with Cistus may have evolved more than once within the genus Hebeloma.     

Higher rate of tissue regeneration in polyploid asexual versus diploid sexual freshwater snails

Characterizing phenotypic differences between sexual and asexual organisms is a critical step towards understanding why sexual reproduction is so common. Because asexuals are often polyploid, understanding how ploidy influences phenotype is directly relevant to the study of sex and will provide key insights into the evolution of ploidy-level variation. The well-established association between genome size and cell cycle duration, evidence for a link between genome size and tissue regeneration rate and the growing body of research showing that ploidy influences growth rate and gene expression led us to hypothesize that healing and tissue regeneration might be affected by ploidy-level variation. We evaluated this hypothesis by measuring the rate of regeneration of antenna tissue of Potamopyrgus antipodarum, a New Zealand snail characterized by frequent coexistence between diploid sexuals and polyploid asexuals. Antennae of triploid and presumptive tetraploid asexuals regenerated more rapidly than the antennae of diploid sexuals, but regeneration rate did not differ between triploids and tetraploids. These results suggest either that ploidy elevation has nonlinear positive effects on tissue regeneration and/or that factors associated directly with reproductive mode affect regeneration rate more than ploidy level. The results of this study also indicate that the lower ploidy of sexual P. antipodarum is unlikely to confer advantages associated with more rapid regeneration.     

Use of magnetically purified Plasmodium falciparum parasites improves the accuracy of erythrocyte invasion assays

Merozoite invasion of erythrocytes is a crucial step for the asexual cycle of Plasmodium falciparum. Multiple invasion pathways, which involve different ligand-receptor interactions, have been identified in P. falciparum by examining the entry of purified parasite into erythrocytes with different surface receptors, either mutant or under different enzyme treatments. The most critical step for a successful invasion assay is the isolation of erythrocytes infected with viable schizonts. Here, we applied a magnetic column to purify the schizonts for the erythrocyte invasion assay. Comparing to Percoll-sorbitol purification method, this modified approach showed great improvement on reproducibility and reliability of invasion assay, particularly for short-term, culture-adapted parasite isolates. The magnetic purification method is an excellent alternative for parasite isolates that do not tolerate or with unknown sensitivity to Percoll-sorbitol exposure.     

Asymmetrical coexistence of Nosema ceranae and Nosema apis in honey bees

Globalization has provided opportunities for parasites/pathogens to cross geographic boundaries and expand to new hosts. Recent studies showed that Nosema ceranae, originally considered a microsporidian parasite of Eastern honey bees, Apis cerana, is a disease agent of nosemosis in European honey bees, Apis mellifera, along with the resident species, Nosema apis. Further studies indicated that disease caused by N. ceranae in European honey bees is far more prevalent than that caused by N. apis. In order to gain more insight into the epidemiology of Nosema parasitism in honey bees, we conducted studies to investigate infection of Nosema in its original host, Eastern honey bees, using conventional PCR and duplex real time quantitative PCR methods. Our results showed that A. cerana was infected not only with N. ceranae as previously reported [Fries, I., Feng, F., Silva, A.D., Slemenda, S.B., Pieniazek, N.J., 1996. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol. 32, 356-365], but also with N. apis. Both microsporidia produced single and mixed infections. Overall and at each location alone, the prevalence of N. ceranae was higher than that of N. apis. In all cases of mixed infections, the number of N. ceranae gene copies (corresponding to the parasite load) significantly out numbered those of N. apis. Phylogenetic analysis based on a variable region of small subunit ribosomal RNA (SSUrRNA) showed four distinct clades of N. apis and five clades of N. ceranae and that geographical distance does not appear to influence the genetic diversity of Nosema populations. The results from this study demonstrated that duplex real-time qPCR assay developed in this study is a valuable tool for quantitative measurement of Nosema and can be used to monitor the progression of microsprodian infections of honey bees in a timely and cost efficient manner.     

Acanthamoeba polyphaga is a possible host for Vibrio cholerae in aquatic environments

Acanthamoeba is a genus of free-living amoebae found to be able to host many bacterial species living in the environment. Acanthamoebae and Vibrio cholerae are found in the aquatic environments of cholera endemic areas. Previously it has been shown that V. cholerae O1 and O139 can survive and grow in Acanthamoeba castellanii. The aim of this study was to examine the ability of Acanthamoeba polyphaga to host V. cholerae O1 and O139. The interaction between A. polyphaga and V. cholerae strains was studied by means of viable amoeba cell counts and viable count of the bacteria in the absence and presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Electron microscopy was used to determine the localization of V. cholerae inside A. polyphaga. The results showed that A. polyphaga enhanced growth and survival of V. cholerae, which grew and survived inside the amoeba cells for 2 weeks. The electron microscopy showed that A. polyphaga hosted intracellular V. cholerae localized in the vacuoles of amoeba cell. Neither the presence of V. cholerae together with A. polyphaga nor the intracellular localization of the bacteria inhibited growth and survival of A. polyphaga. The outcome of the interaction between these microorganisms may support strongly the role of A. polyphaga as host for V. cholerae O1 and O139.     

Diversimorbus metrosiderotis gen. et sp. nov. and three new species of Holocryphia (Cryphonectriaceae) associated with cankers on native Metrosideros angustifolia trees in South Africa

The Cryphonectriaceae includes important tree pathogens, especially on the Myrtales. During a routine disease survey in the Western Cape Province of South Africa, a fungus resembling the Eucalyptus pathogen Holocryphia eucalypti was observed on native Metrosideros angustifolia (Myrtales). The aims of this study were to identify the fungus and to expand surveys for fungi in the Cryphonectriaceae on M. angustifolia. Fungi were identified based on DNA sequence comparisons and morphological features, and their pathogenicity was tested on M. angustifolia under field conditions. Based on morphology and multigene phylogenetic analyses of DNA sequence data from six gene regions, we describe a new genus including a single species and three new species of Holocryphia (Cryphonectriaceae) from M. angustifolia. These fungi are provided with the names Diversimorbus metrosiderotis gen. et sp. nov., Holocryphia capensis sp. nov., Holocryphia gleniana sp. nov., and Holocryphia mzansi sp. nov. We also revise H. eucalypti, the type of the genus, to include only isolates from Eucalyptus in South Africa. Research results indicated that H. mzansi may undergo host shifts between different tree genera in the Myrtaceae. Inoculation tests showed that isolates of all the newly described species can cause lesions on the branches of M. angustifolia, indicating that they are all pathogens of this tree.     

Rhizobium pisi sv. trifolii K3.22 harboring nod genes of the Rhizobium leguminosarum sv. trifolii cluster

The taxonomic status of the Rhizobium sp. K3.22 clover nodule isolate was studied by multilocus sequence analysis (MLSA) of 16S rRNA and six housekeeping chromosomal genes, as well as by a subsequent phylogenic analysis. The results revealed full congruence with the Rhizobium pisi DSM 30132^T core genes, thus supporting the same taxonomic position for both strains. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence similarity to Rhizobium leguminosarum sv. trifolii, whereas the R. pisi DSM 30132^Tnod genes were most similar to R. leguminosarum sv. viciae. The strains differed in the host range nodulation specificity, since strain K3.22 effectively nodulated red and white clover but not vetch, in contrast to R. pisi DSM 30132^T, which effectively nodulated vetch but was not able to nodulate clover. Both strains had the ability to form nodules on pea and bean but they differed in bean cultivar specificity. The R. pisi K3.22 and DSM 30132^T strains might provide evidence for the transfer of R. leguminosarum sv. trifolii and sv. viciae symbiotic plasmids occurring in natural soil populations.     

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.     

Density and maturation of rodlet cells in brain tissue of fathead minnows (Pimephales promelas) exposed to trematode cercariae

Evidence for the presumed linkage between the enigmatic rodlet cells of fish and exposure to helminths is anecdotal and indirect. We evaluated the proliferation and development of rodlet cells in the optic lobes of fathead minnows exposed to cercariae of Ornithodiplostomum ptychocheilus. Mean rodlet cell densities (ca. 10/mm²) in the optic lobes were similar between unexposed controls and minnows with 1- and 2-week old infections. Rodlet cell densities increased at 4 weeks p.i., reaching maxima (ca. 200/mm²) at 6 weeks p.i., followed by a decline at 9 weeks. This temporal pattern of proliferation and maturation paralleled the development of metacercariae within the optic lobes. Unencysted metacercariae develop rapidly within tissues of the optic lobes for approximately 4 weeks after penetration by cercariae, then shift to the adjacent meninges to encyst. The former stage is associated with tissue damage, the latter with massive inflammation of the meninges. Thus, peak densities and maturation of rodlet cells correspond to the period when inflammation of the meninges caused by the large metacercarial cysts is at a maximum. Our results support recent contentions that rodlet cells comprise part of the host inflammatory defence response.     

Impacts of an endoparasitic copepod, Ismaila belciki, on the reproduction,growth and survivorship of its nudibranch host, Janolus fuscus

Copepods from the genus Ismaila are large endoparasites that inhabit the main body cavity and/or cerata of opisthobranch molluscs. These parasites exhibit many life history characteristics typically found in parasitic castrators, yet the actual impact of infection on reproduction, growth or survivorship of the hosts are unknown. On the Oregon (USA) coast, Ismaila belciki can infect over 80% of their hermaphroditic hosts, Janolus fuscus. In laboratory mating experiments, we compared the reproductive output (egg mass weight, number of egg capsules, number of viable embryos) and the gonadal somatic index of infected versus uninfected J. fuscus. Infected J. fuscus could produce viable sperm and copulate. Mating with an infected individual did not limit a sea slug’s reproductive output. However, infected J. fuscus had significantly lower reproductive output (by 34–54%), producing smaller egg masses with fewer capsules and viable embryos. Infected hosts had significantly lower gonadal somatic index than their uninfected counterparts, although there was no significant difference in gonadal somatic index between hosts with single and double infections. By collecting the egg sacs produced by the copepod parasite during experiments, we estimated that 25–34% of the host’s reproductive output is usurped by the parasite and re-directed to the parasite’s own reproduction. In the laboratory, infection did not alter growth in J. fuscus. However, infection significantly decreased survivorship in mature (but not immature) nudibranch hosts. These results suggest that I. belciki is not a true castrator, but it does reduce the reproductive output of its host and may therefore limit the natural population size of J. fuscus.     

Regulation of wing formation and adult development in an aphid host, Aphis fabae, by the parasitoid Aphidius colemani

Nymphs of presumptive winged gynoparae of Aphis fabae (Hemiptera: Aphididae), were exposed to female parasitoids, Aphidius colemani (Hymenoptera: Aphidiidae) and stung once with the ovipositor. Wing development was inhibited and, when aphids were parasitised during the early stages, they did not reach the adult stage but mummies with rudimentary or no wingbuds are observed in the host's fourth-stadium. These and previous studies have suggested that wing development may be inhibited by factor(s) from the maternal parasitoid injected into the host at the time of oviposition. In an attempt to identify such factor(s), saline extracts of whole female parasitoids, abdomens, ovaries and venom glands were prepared. When a saline extract of venom glands was injected into late-second-stadium aphids, many develop to fourth-stadium nymphs with rudimentary wingbuds, indicating an effect on wing formation but also showed developmental arrest and often died when attempting to moult to the adult stage. It appears that host death may be related to physiological/biochemical interactions of parasitoid and host rather than just late stage parasitoid larvae ingesting the host's vital organs. Injections with extracts into later host stadia gave similar results with regard to development to the adult, although aphids injected in the late-fourth-stadium develop normally to the adult stage with no effect on wing formation. The results indicate that the earlier the injection before the final moult the greater the effect of the injected extract on preventing adult development.Extracts prepared from head + thorax do not affect aphid development and the results indicate that there is an active factor(s) - likely a protein - in the female parasitoid's venom that disrupts wing development and/or inhibits development to the adult stage. Surprisingly, injections of extracts from male parasitoids have similar effects but the location and function of such a factor(s) in males are unknown.     

Disruption of lipid rafts by lidocaine inhibits erythrocyte invasion by Plasmodium falciparum

Membrane lipid rafts have been implicated in erythrocyte invasion process by Plasmodium falciparum. In this study, we examined the effect of lidocaine, a local anesthetic, which disrupts lipid rafts reversibly without affecting membrane cholesterol content on parasite invasion. In the presence of increasing concentrations of lidocaine in the culture medium, parasite invasion was progressively decreased with complete inhibition at 2 mM. Decreased invasion was also seen in erythrocytes pre-treated with lidocaine and cultured in the absence of lidocaine. This inhibitory effect on parasite invasion was reversed following removal of lidocaine from erythrocyte membranes. Our findings show that disruption of lipid rafts in the context of normal cholesterol content markedly inhibits parasite invasion and confirm an important role for lipid rafts in invasion of erythrocytes by P. falciparum.     

A review of global diversity in avian haemosporidians (Plasmodium and Haemoproteus: Haemosporida): new insights from molecular data

Biogeographic patterns of parasite diversity are useful for determining how host–parasite interactions can influence speciation. However, variation in methodologies and sampling effort can skew diversity estimates. Avian haemosporidians are vector-transmitted blood parasites represented by over 1300 unique genetic lineages spread across over 40 countries. We used a global database of lineage distributions for two avian haemosporidian genera, Plasmodium and Haemoproteus, to test for congruence of diversity among haemosporidians and their avian hosts across 13 geographic regions. We demonstrated that avian haemosporidians exhibit similar diversity patterns to their avian hosts; however, specific patterns differ between genera. Haemoproteus spp. diversity estimates were significantly higher than those of Plasmodium spp. in all areas where the genera co-occurred, apart from the Plasmodium spp.-rich region of South America. The geographic distributions of parasite genera also differed, with Haemoproteus spp. absent from the majority of oceanic regions while Plasmodium spp. were cosmopolitan. These findings suggest fundamental differences in the way avian haemosporidians diverge and colonise new communities. Nevertheless, a review of the literature suggests that accurate estimates of avian haemosporidian diversity patterns are limited by (i) a concentration of sampling towards passerines from Europe and North America, (ii) a frequent failure to include microscopic techniques together with molecular screening and (iii) a paucity of studies investigating distributions across vector hosts.     

Avian cestodes affect the behaviour of their intermediate host Artemia parthenogenetica: an experimental study

The brine shrimp Artemia parthenogenetica (Crustacea, Branchiopoda) is intermediate host for several cestode species whose final hosts are waterbirds. Previous field studies have shown that brine shrimps infected with cestodes have a bright red colour and are spatially segregated in the water column. However, the ethological mechanisms explaining such field observations are unknown. Changes in appearance and behaviour induced by trophically transmitted parasites have been shown to increase the risk of predation by the final host. In this experimental study, we compared the behaviour of uninfected Artemia and those infected by avian cestodes. We found that parasitised individuals behave differently from unparasitised ones in several ways. In contrast to uninfected individuals, infected brine shrimps were photophilous and showed increased surface-swimming behaviour. These observations suggest that the modified behaviour (in addition to the bright red colour of the majority of the infected individuals) results in infected brine shrimps becoming more vulnerable to avian final hosts, which facilitates parasite transmission. We discuss our results in terms of the adaptive nature of behavioural changes and their potential implications for the hypersaline ecosystem.     

A phylogenetic analysis of the phylum Fibrobacteres

Members of the phylum Fibrobacteres are highly efficient cellulolytic bacteria, best known for their role in rumen function and as potential sources of novel enzymes for bioenergy applications. Despite being key members of ruminants and other digestive microbial communities, our knowledge of this phylum remains incomplete, as much of our understanding is focused on two recognized species, Fibrobacter succinogenes and F. intestinalis. As a result, we lack insights regarding the environmental niche, host range, and phylogenetic organization of this phylum. Here, we analyzed over 1000 16S rRNA Fibrobacteres sequences available from public databases to establish a phylogenetic framework for this phylum. We identify both species- and genus-level clades that are suggestive of previously unknown taxonomic relationships between Fibrobacteres in addition to their putative lifestyles as host-associated or free-living. Our results shed light on this poorly understood phylum and will be useful for elucidating the function, distribution, and diversity of these bacteria in their niches.     

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy,morphometry and confocal laser scanning microscopy

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.     

Comparative study on the delignification of Azadirachta indica (L) Del., wood by Chrysosporium asperatum and Trichoderma harzianum

Structural alterations induced in response to degradation by two white rot Basidiomycetes on the secondary xylem of Azadirachta indica (L) Del., was compared. In vitro decay test was employed to investigate the pattern of delignification of Azadirachta wood by Trichoderma harzianum and Chrysosporium asperatum. Wood samples inoculated with both the strains were analyzed for different periods viz. 30, 60, 90 and 120 days after fungal inoculation. Initially there was no appreciable percent weight loss of the wood blocks but later on (after 60 days) it increased rapidly and was found similar for both the strains (43-46% of wood mass). Samples inoculated with both the strains showed dual pattern of degradation i.e. selective delignification in the initial stage followed by simultaneous rot during advance stage of decay. Separation of the cells due to dissolution of middle lamella was the characteristic feature of both strains but in the advanced stage of decay, formation of erosion troughs were conspicuous in all the cell types. Other features such as cell wall thinning, rounded pit erosion, formation of erosion channels and bore holes were also observed frequently. Initially, fungal invasion started through the vessel lumen, followed by all the cell types of the xylem. From the vessels, mycelia entered into the adjacent rays and parenchyma cells through the pits. In advanced stage, degradation was so pronounced that rays were partially or even completely destroyed while many cells including vessels were either deformed or destroyed due to loss of rigidity of their walls. Structural alterations induced in response to C. asperatum and T. harzianum attack is described in details.     

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%.