Haplosporidians are rhizarian parasites of mostly marine invertebrates. They include the causative agents of diseases of commercially important molluscs, including MSX disease in oysters. Despite their importance for food security, their diversity and distributions are poorly known. We used a combination of group-specific PCR primers to probe environmental DNA samples from planktonic and benthic environments in Europe, South Africa and Panama. This revealed several highly distinct novel clades, novel lineages within known clades and seasonal (spring vs autumn) and habitat-related (brackish vs littoral) variation in assemblage composition. High frequencies of haplosporidian lineages in the water column provide the first evidence for life cycles involving planktonic hosts, host-free stages or both. The general absence of haplosporidian lineages from all large online sequence data sets emphasises the importance of lineage-specific approaches for studying these highly divergent and diverse lineages. Combined with host-based field surveys, environmental sampling for pathogens will enhance future detection of known and novel pathogens and the assessment of disease risk.
Ascetosporea; Haplosporidia; Haplosporidium; Minchinia; Bonamia; Urosporidium
Emotional events are often remembered better than neutral events, a type of memory prioritization by affective salience that depends on the amygdala. Studies with rats have indicated that direct activation of the basolateral complex of the amygdala (BLA) can enhance memory for neutral events, and if the activation is brief and temporally targeted, can do so in way that benefits memories for specific events. The essential targets of BLA activation in the case of event-specific memory enhancement were unknown, but the hippocampus was known to receive direct projections from the BLA and to support memory for events. In the present study, rats received counterbalanced infusions of either muscimol, a GABAA receptor agonist, or saline into the hippocampus prior to performing a novel object recognition memory task during which initial encounters with some of the objects were immediately followed by brief electrical stimulation to the BLA. When memory was tested 1 day later in the saline condition, rats remembered these objects well but showed no memory for objects for which the initial encounter had not been followed by BLA stimulation. In contrast, no benefit to memory of BLA stimulation was observed in the muscimol condition. The results indicated that brief activation of the BLA can prioritize memories for events by enhancing memory for some object encounters but not others and that this benefit to memory depends on interactions between the amygdala and the hippocampus.
basolateral amygdala; hippocampus; object recognition memory; memory enhancement; electrical stimulation; fluorophore-conjugated muscimol
Vampire amoebae (vampyrellids) are predators of algae, fungi, protozoa and small metazoans known primarily from soils and in freshwater habitats. They are among the very few heterotrophic naked, filose and reticulose protists that have received some attention from a morphological and ecological point of view over the last few decades, because of the peculiar mode of feeding of known species. Yet, the true extent of their biodiversity remains largely unknown. Here we use a complementary approach of culturing and sequence database mining to address this issue, focusing our efforts on marine environments, where vampyrellids are very poorly known. We present 10 new vampyrellid isolates, 8 from marine or brackish sediments, and 2 from soil or freshwater sediment. Two of the former correspond to the genera Thalassomyxa Grell and Penardia Cash for which sequence data were previously unavailable. Small-subunit ribosomal DNA analysis confirms they are all related to previously sequenced vampyrellids. An exhaustive screening of the NCBI GenBank database and of 454 sequence data generated by the European BioMarKs consortium revealed hundreds of distinct environmental vampyrellid sequences. We show that vampyrellids are much more diverse than previously thought, especially in marine habitats. Our new isolates, which cover almost the full phylogenetic range of vampyrellid sequences revealed in this study, offer a rare opportunity to integrate data from environmental DNA surveys with phenotypic information. However, the very large genetic diversity we highlight within vampyrellids (especially in marine sediments and soils) contrasts with the paradoxically low morphological distinctiveness we observed across our isolates.
BioMarKs; environmental clones; Penardia; SSU rDNA; Thalassomyxa; Vampyrellida
Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant prey-related differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.
flagellate community composition; freshwater; flagellate growth; food quality of bacteria; Limnohabitans; 454 pyrosequencing
Dementia is prevalent and costly, yet the predictors of inpatient hospitalization are not well understood. Logistic and negative binomial regressions were used to identify predictors of inpatient hospital utilization and the frequency of inpatient hospital utilization, respectively, among veterans. Variables significant at the P < 0.15 level were subsequently analyzed in a multivariate regression. This study of veterans with a diagnosis of dementia (n = 296) and their caregivers found marital status to predict hospitalization in the multivariate logistic model (B = 0.493, P = 0.029) and personal-care dependency to predict hospitalization and readmission in the multivariate logistic model and the multivariate negative binomial model (B = 1.048, P = 0.007, B = 0.040, and P = 0.035, resp.). Persons with dementia with personal-care dependency and spousal caregivers have more inpatient admissions; appropriate care environments should receive special care to reduce hospitalization. This study was part of a larger clinical trial; this trial is registered with ClinicalTrials.gov NCT00291161.
“Partners in Dementia Care” (PDC) tested the effectiveness of a care-coordination program integrating healthcare and community services and supporting veterans with dementia and their caregivers. Delivered via partnerships between Veterans Affairs medical centers and Alzheimer’s Association chapters, PDC targeted both patients and caregivers, distinguishing it from many non-pharmacological interventions. Hypotheses posited PDC would improve five veteran self-reported outcomes: 1) unmet need, 2) embarrassment about memory problems, 3) isolation, 4) relationship strain and 5) depression. Greater impact was expected for more impaired veterans. A unique feature was self-reported research data collected from veterans with dementia.
Methods and Findings
Five matched communities were study sites. Two randomly selected sites received PDC for 12 months; comparison sites received usual care. Three structured telephone interviews were completed every 6 months with veterans who could participate.
Of 508 consenting veterans, 333 (65.6%) completed baseline interviews. Among those who completed baseline interviews, 263 (79.0%) completed 6-month follow-ups and 194 (58.3%) completed 12-month follow-ups. Regression analyses showed PDC veterans had significantly less adverse outcomes than those receiving usual care, particularly for more impaired veterans after 6 months, including reduced relationship strain (B = −0.09; p = 0.05), depression (B = −0.10; p = 0.03), and unmet need (B = −0.28; p = 0.02; and B = −0.52; p = 0.08). PDC veterans also had less embarrassment about memory problems (B = −0.24; p = 0.08). At 12 months, more impaired veterans had further reductions in unmet need (B = −0.96; p < 0.01) and embarrassment (B = −0.05; p = 0.02). Limitations included use of matched comparison sites rather than within-site randomization and lack of consideration for variation within the PDC group in amounts and types of assistance provided.
Partnerships between community and health organizations have the potential to meet the dementia-related needs and improve the psychosocial functioning of persons with dementia.
Phytomyxids (plasmodiophorids and phagomyxids) are cosmopolitan, obligate biotrophic protist parasites of plants, diatoms, oomycetes and brown algae. Plasmodiophorids are best known as pathogens or vectors for viruses of arable crops (e.g. clubroot in brassicas, powdery potato scab, and rhizomania in sugar beet). Some phytomyxid parasites are of considerable economic and ecologic importance globally, and their hosts include important species in marine and terrestrial environments. However most phytomyxid diversity remains uncharacterised and knowledge of their relationships with host taxa is very fragmentary.
Our molecular and morphological analyses of phytomyxid isolates–including for the first time oomycete and sea-grass parasites–demonstrate two cross-kingdom host shifts between closely related parasite species: between angiosperms and oomycetes, and from diatoms/brown algae to angiosperms. Switching between such phylogenetically distant hosts is generally unknown in host-dependent eukaryote parasites. We reveal novel plasmodiophorid lineages in soils, suggesting a much higher diversity than previously known, and also present the most comprehensive phytomyxid phylogeny to date.
Such large-scale host shifts between closely related obligate biotrophic eukaryote parasites is to our knowledge unique to phytomyxids. Phytomyxids may readily adapt to a wide diversity of new hosts because they have retained the ability to covertly infect alternative hosts. A high cryptic diversity and ubiquitous distribution in agricultural and natural habitats implies that in a changing environment phytomyxids could threaten the productivity of key species in marine and terrestrial environments alike via host shift speciation.
Plasmodiophorid; Plant pathogen; Evolution; Taxonomy; Host range; Plasmodiophora; Polymyxa; Woronina; Sorosphaerula; Spongospora
We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected.
This revision of the classification of eukaryotes, which updates that of Adl et al. (2005), 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.
Algae; amoebae; biodiversity; ciliates; flagellates; fungi; parasites; protozoa; systematics; taxonomy
The basolateral complex of the amygdala (BLA) modulates memory for emotional events, and direct activation of the BLA following a learning session can enhance subsequent memory. Yet optimal enhancement of episodic memory during emotional events would likely require that BLA activation occur close in time to the event and to be brief enough to target specific memories if some events are to be remembered better than others. In the present study, rats were given a novel object recognition memory task in which initial encounters with some of the objects were immediately followed by brief electrical stimulation of the BLA, and these objects were remembered better one day later as compared to objects for which the initial encounter was not followed by stimulation. The results indicated that BLA stimulation can enhance memory for individual events, a necessary ability for the BLA to modulate episodic memory effectively.
basolateral amygdala; electrical stimulation; object recognition memory; memory enhancement; rat
We sequenced the 18S ribosomal RNA gene of seven isolates of the enigmatic marine amoeboflagellate Reticulamoeba Grell, which resolved into four genetically distinct Reticulamoeba lineages, two of which correspond to R. gemmipara Grell and R. minor Grell, another with a relatively large cell body forming lacunae, and another that has similarities to both R. minor and R. gemmipara but with a greater propensity to form cell clusters. These lineages together form a long-branched clade that branches within the cercozoan class Granofilosea (phylum Cercozoa), showing phylogenetic affinities with the genus Mesofila. The basic morphology of Reticulamoeba is a roundish or ovoid cell with a more or less irregular outline. Long and branched reticulopodia radiate from the cell. The reticulopodia bear granules that are bidirectionally motile. There is also a biflagellate dispersal stage. Reticulamoeba is frequently observed in coastal marine environmental samples. PCR primers specific to the Reticulamoeba clade confirm that it is a frequent member of benthic marine microbial communities, and is also found in brackish water sediments and freshwater biofilm. However, so far it has not been found in large molecular datasets such as the nucleotide database in NCBI GenBank, metagenomic datasets in Camera, and the marine microbial eukaryote sampling and sequencing consortium BioMarKs, although closely related lineages can be found in some of these datasets using a highly targeted approach. Therefore, although such datasets are very powerful tools in microbial ecology, they may, for several methodological reasons, fail to detect ecologically and evolutionary key lineages.
The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.
A group of protist experts proposes a two-step DNA barcoding approach, comprising a universal eukaryotic pre-barcode followed by group-specific barcodes, to unveil the hidden biodiversity of microbial eukaryotes.
The recently proposed new phylum name Cryptomycota phyl. nov. is validly published in order to facilitate its use in future discussions of the ecology, biology, and phylogenetic relationships of the constituent organisms. This name is preferred over the previously tentatively proposed “Rozellida” as new data suggest that the life-style and morphology of Rozella is not representative of the large radiation to which it and other Cryptomycota belong. Furthermore, taxa at higher ranks such as phylum are considered better not based on individual names of included genera, but rather on some special characteristics – in this case the cryptic nature of this group and that they were initially revealed by molecular methods rather than morphological discovery. If the group were later viewed as a member of a different kingdom, the name should be retained to indicate its fungal affinities, as is the practice for other fungal-like protist groups.
chitin; chytrid; Fungi; phylogeny; Rozella; Rozellida
Fungi are the principal degraders of biomass in most terrestrial ecosystems. In contrast to surface environments, deep-sea environmental gene libraries have suggested that fungi are rare and non-diverse in high-pressure marine environments. Here, we report the diversity of fungi from 11 deep-sea samples from around the world representing depths from 1500 to 4000 m (146–388 atm) and two shallower water column samples (250 and 500 m). We sequenced 239 clones from 10 fungal-specific 18S rRNA gene libraries constructed from these samples, from which we detected only 18 fungal 18S-types in deep-sea samples. Our phylogenetic analyses show that a total of only 32 fungal 18S-types have so far been recovered from deep-sea habitats, and our results suggest that fungi, in general, are relatively rare in the deep-sea habitats we sampled. The fungal diversity detected suggests that deep-sea environments host an evolutionarily diverse array of fungi dominated by groups of distantly related yeasts, although four putative filamentous fungal 18S-types were detected. The majority of our new sequences branch close to known fungi found in surface environments. This pattern contradicts the proposal that deep-sea and hydrothermal vent habitats represent ancient ecosystems, and demonstrates a history of frequent dispersal between terrestrial and deep-sea habitats.
life under huge barometric pressures; osmotrophy; environmental gene library; microbial diversity; SSU rDNA phylogeny
In the absence of activation signals, circulating human neutrophils and eosinophils undergo spontaneous apoptosis. The glucocorticoid Dexamethasone (Dex) accelerates apoptosis in inflammatory cells such as eosinophils, but uniquely delays neutrophil apoptosis. Corresponding to the opposite effects of Dex on granulocyte apoptosis, we demonstrate that in neutrophils and eosinophils Dex oppositely affects expression of the anti-apoptotic Bcl-2 family protein Mcl-1L. Mcl-1L expression declines over time in vitro; however, Dex maintains Mcl-1L expression in neutrophils. In contrast, Dex accelerates Mcl-1L protein loss in eosinophils. Neither Mcl-1S, a pro-apoptotic splice variant, nor Bax were affected. Dex treatment in the presence of a translation inhibitor stabilized existing Mcl-1L protein in neutrophils, while Mcl-1L stability in eosinophils was unaffected. Accordingly, delay of neutrophil apoptosis by Dex was prevented by antisense Mcl-1L siRNA. Our findings suggest that regulation of Mcl-1L degradation plays an important role in the opposite effects of Dex on granulocyte apoptosis.
Neutrophils; Eosinophils; Apoptosis; Mcl-1; Dexamethasone; Bax; Inflammation
It is much debated whether microbes are easily dispersed globally or whether they, like many macro-organisms, have historical biogeographies. The ubiquitous dispersal hypothesis states that microbes are so numerous and so easily dispersed worldwide that all should be globally distributed and found wherever growing conditions suit them. This has been broadly upheld for protists (microbial eukaryotes) by most morphological and some molecular analyses. However, morphology and most previously used evolutionary markers evolve too slowly to test this important hypothesis adequately.
Here we use a fast-evolving marker (ITS1 rDNA) to map global diversity and distribution of three different clades of cercomonad Protozoa (Eocercomonas and Paracercomonas: phylum Cercozoa) by sequencing multiple environmental gene libraries constructed from 47–80 globally-dispersed samples per group. Even with this enhanced resolution, identical ITS sequences (ITS-types) were retrieved from widely separated sites and on all continents for several genotypes, implying relatively rapid global dispersal. Some identical ITS-types were even recovered from both marine and non-marine samples, habitats that generally harbour significantly different protist communities. Conversely, other ITS-types had either patchy or restricted distributions.
Our results strongly suggest that geographic dispersal in macro-organisms and microbes is not fundamentally different: some taxa show restricted and/or patchy distributions while others are clearly cosmopolitan. These results are concordant with the 'moderate endemicity model' of microbial biogeography. Rare or continentally endemic microbes may be ecologically significant and potentially of conservational concern. We also demonstrate that strains with identical 18S but different ITS1 rDNA sequences can differ significantly in terms of morphological and important physiological characteristics, providing strong additional support for global protist biodiversity being significantly higher than previously thought.
Previous studies of the biochemistry and physiology of eosinophils have relied upon cells obtained from patients with eosinophilia (EE). It is unknown whether such cells might have been activated or partially exhausted by the pathological state causing eosinophilia. We examined cell surface charge, membrane transport of deoxyglucose, activation of lyso-somal acid phosphatase, and oxidative metabolism to provide a profile to compare EE with purified normal eosinophils (NE) and normal neutrophils.
Eosinophils or neutrophils were obtained in >95% purity from normal individuals and patients with eosinophilia of diverse etiologies. Cell surface charge was determined by electrophoretic mobility in micromoles per second per volt per centimeter. Normal eosinophils demonstrated a surface charge of 2.46±0.03. Stimulation of the cells by zymosan-activated serum (ZAS) reduced the surface charge to 1.82±0.02. In contrast, the charge of “resting” EE was already reduced (1.89±0.05) and was not altered by ZAS. Resting and stimulated neutrophils had a charge of 1.98±0.01 and 1.69±0.02, respectively.
Uptake of [3H]2-deoxyglucose has been shown to reflect carrier-facilitated hexose transport in granulocytes. Deoxyglucose uptake by resting NE and NE stimulated by ZAS was 2.40±0.40 and 5.44±0.39 (cpm × 10−3/2 × 105 eosinophils), respectively. Resting and stimulated EE demonstrated deoxyglucose uptake of 7.55±0.58 and 15.3±0.6, respectively.
Lysosomal acid phosphatase was determined by an electron microscopic cytochemical technique. In normal eosinophils and neutrophils, lysosomal acid phosphatase in mature cells is held in a latent form. Normal eosinophils demonstrated weakly positive acid phosphatase activity in 7.8±1.2% of the specific granules. Normal eosinophils, stimulated by opsonized staphylococci or the calcium ionophore A23187, develop rapid activation of acid phosphatase in ∼80% of the granules throughout the cells. Resting EE were usually already activated and demonstrated acid phosphatase in 48.6±8.6% of the granules (range, 2-95% granules positive; significant activation was observed in preparations in EE from 11 of 15 patients).
Oxidative metabolism was monitored by measurement of the hexose monophosphate shunt (HMPS) (metabolism of 1-[14C]glucose to 14CO2). Previous studies demonstrated that resting EE have an HMPS activity which is nearly that of stimulated neutrophils, yet EE remain capable of further 7-10-fold increase when stimulated by opsonized zymosan. In contrast, the HMPS of NE (resting and stimulated) was not significantly different from that of neutrophils.
Thus eosinophils obtained from patients with eosinophilia appear significantly activated when compared with normal eosinophils by the criteria of surface charge, activation of lysosomal acid phosphatase, membrane hexose transport, and hexose monophosphate shunt activities.
One distinctive aspect of the response to acute inflammation involves a rapid and persistent decrease in the numbers of circulating eosinophils, yet the mechanisms of this eosinopenia are undefined. One possibility is that the abrupt eosinopenia may be the result of release of small amounts of the chemotactic factors of acute inflammation into the circulation. These studies were designed to examine the numbers of circulating eosinophils after an intravenous injection of zymosan-activated serum, partially purified C5a or the synthetic peptide, N-formyl-methionyl-leucyl-phenylalanine. Each of these factors caused a virtual disappearance of circulating eosinophils within 1 min, a transient return of eosinophils to ∼50% of control levels after 10-90 min, and a subsequent decrease which persisted for 5 h. In contrast, the numbers of circulating heterophils, although dropping transiently, rapidly returned and rose to elevated levels for 6 h after injection. The response was not caused by adrenal mediation as it occurred normally in adrenalectomized rabbits. Two chemotaxins of allergic inflammation, histamine and the tetrapeptide valine-glycine-serine-glutamic acid, did not cause significant eosinopenia.
Circulating granulocytes of patients undergoing hemodialysis, which has been reported to activate complement, demonstrated similar eosinopenic and neutropenic-neutrophilic responses. Thus, in rabbits and in man, intravascular activation or injection of chemotactic factors (C5a or N-formyl-methionyl-leucyl-phenylalanine) causes a brief, nonspecific granulocytopenia followed by a prolonged eosinopenic-neutrophilic response analogous to that seen during acute infection.
Eosinophil and/or neutrophil leukocytes appear to have important roles in host defense against invasive, migratory helminth infestations, but the mechanisms of larval killing by leukocytes are uncertain. This study examines killing of newborn (migratory phase) larvae of Trichinella spiralis during incubation with granule preparations of human eosinophils or neutrophils and generators of hydrogen peroxide (glucose-glucose oxidase) (G-GO) or superoxide and hydrogen peroxide (xanthine-xanthine oxidase). Larvae were killed by either hydrogen peroxide-generating system in a concentration-dependent manner. Direct enumeration of surviving larvae after incubation in microtiter wells containing the appropriate reagents was used in assess larval killing. Verification of the microplate assay was demonstrated by complete loss of larval ability to incorporate [3H]deoxyglucose and loss of infectivity after incubation in comparable concentrations of G-GO. Larvae were highly sensitive to oxidative products; significant killing occurred after incubation with 0.12 mU glucose oxidase and complete killing occurred with 0.5 mU. Comparable killing of bacteria required over 60 mU glucose oxidase. At 5 mU glucose oxidase, killing was complete after 6 h of incubation. Killing by G-GO was inhibited by catalase but not by boiled catalase or superoxide dismutase and was enhanced by azide. Addition of peroxidase in granule pellet preparations of eosinophils or neutrophils did not enhance killing by G-GO. These data indicate a remarkable susceptibility of newborn larvae of T. spiralis to the hydrogen peroxide generated by neutrophil and eosinophil leukocytes.