Free-living Forms Research Articles

Functional type 1 secretion system involved in Legionella pneumophila virulence.

Legionella pneumophila is a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence of L. pneumophila is now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle of L. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that the repeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation of L. pneumophila, via its T1SS, in its internalization into host cells.

Epibiont communities of loggerhead marine turtles (Caretta caretta) in the western Mediterranean: influence of geographic and ecological factors

This study reports for the first time on the whole epibiont fauna of loggerhead marine turtles,Caretta caretta, in the western Mediterranean, analysing the factors that account for the predictability and composition of the assemblage. A total of 104 loggerhead turtles stranded along the coasts of eastern Spain during 1995–2006 were surveyed for epibionts. A total of 39 epibiont taxa were identified, three of them being new records for loggerhead turtles:Bittiumsp.,Idotea metallicaandJassasp. The assemblage was composed of a group of 27 facultative taxa that use turtles as any inanimate buoyant substrate, and 12 taxa that have developed more specific associations to marine turtles, including six species that occur in marine turtles exclusively, two that dwell also on other hosts, and four that can also survive as free-living forms but have developed a strong association with marine turtles. Hierarchical clustering and Similarity Profile Analysis based on the occurrence of 166 epibiont taxa from nine available surveys indicated that the epibiont assemblages from loggerhead turtles in the western Mediterranean (WM) are similar to those from Central Mediterranean (CM), but significantly different from turtles surveyed in the eastern Mediterranean and the Atlantic. The subset of epibionts occurring on WM and CM turtles is defined by a combination of geographic factors (exclusive Mediterranean epibiont taxa) and ecological factors (relative absence of littoral-benthic taxa). Loggerhead turtles from WM and CM apparently exploit both pelagic and benthic habitats in similar fashion, representing a homogeneous unit for epibiont recruitment.

Phylogeny of Myzostomida (Annelida) and their relationships with echinoderm hosts.

BackgroundMyzostomids are marine annelids, nearly all of which live symbiotically on or inside echinoderms, chiefly crinoids, and to a lesser extent asteroids and ophiuroids. These symbionts possess a variety of adult body plans and lifestyles. Most described species live freely on the exterior of their hosts as adults (though starting life on the host inside cysts), while other taxa permanently reside in galls, cysts, or within the host’s mouth, digestive system, coelom, or gonads. Myzostomid lifestyles range from stealing incoming food from the host’s food grooves to consuming the host’s tissue directly. Previous molecular studies of myzostomids have had limited sampling with respect to assessing the evolutionary relationships within the group; therefore molecular data from 75 myzostomid taxa were analyzed using maximum likelihood and maximum parsimony methods. To compare relationships of myzostomids with their hosts, a phylogeny was inferred for 53 hosts and a tanglegram constructed with 88 associations.ResultsGall- and some cyst-dwellers were recovered as a clade, while cyst-to-free-living forms were found as a grade including two clades of internal host-eaters (one infecting crinoids and the other asteroids and ophiuroids), mouth/digestive system inhabitants, and other cyst-dwellers. Clades of myzostomids were recovered that associated with asteroids, ophiuroids, and stalked or feather star crinoids. Co-phylogenetic analyses rejected a null-hypothesis of random associations at the global level, but not for individual associations. Event-based analyses relied most upon host-switching and duplication events to reconcile the association history.ConclusionHypotheses were revised concerning the systematics and evolution of Myzostomida, as well their relationships to their hosts. We found two or three transitions between food-stealing and host-eating. Taxa that dwell within the mouth or digestive system and some cyst forms are arguably derived from cyst-to-free-living ancestors – possibly the result of a free-living form moving to the mouth and paedomorphic retention of the juvenile cyst. Phylogenetic conservatism in host use was observed among related myzostomid taxa. This finding suggests that myzostomids (which have a free-living planktonic stage) are limited to one or a few closely related hosts, despite most hosts co-occurring on the same reefs, many within physical contact of each other.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-014-0170-7) contains supplementary material, which is available to authorized users.

Ecological functioning of free-living marine nematodes in coastal wetlands: an overview

Nematodes are small multicellular, thread-like organisms, inhabiting almost all conceivable environments; among these, some 25 % are free-living marine forms with a population density of (1–12) × 106 inds m−2 in seabed sediment, reaching maximum values in muddy estuaries and salt marshes. A large quantity of carbon from the salt marsh plants enters the ecosystem via the detritus pathway, in which nematodes play an important role through their feeding and bioturbation activities. Vegetation influences the sedimentary environment and modifies the distribution pattern of nematode communities in coastal wetlands. Nematodes are coupled closely with bacteria/detritus in microbial food webs, stimulating bacterial growth and subsequent nutrient remineralization; they provide food sources for higher trophic levels and serve as a linkage between micro- and macro-fauna. Furthermore, nematodes have a potential to provide proxies that can be used in diagnosing environmental quality. In China, only a limited number of nematode data sets are available for the coastal wetlands across several different climatic zones. It is necessary to carry out additional investigations into the biology and ecology of nematodes in order to delineate their ecological functioning in coastal wetlands. On such a basis, the contribution made by nematodes to material cycling and the ecological functioning in coastal wetlands can be quantified. The assessment of their biological diversity should be a focus, which is fundamental in the study of wetland ecosystem dynamic mechanisms. In addition to laboratory and mesocosm experiments, mathematical models should be established to predict the responses of ecosystem to the environmental disturbance. Finally, it is necessary to improve the techniques for nematode analysis, to enhance the efficiency of data acquisition.

Rhodolith beds in the South-East Pacific

Rhodolith or maerl are the most common terms used for free living coralline red algae which live and produce sediments (Nelson 2009). They are common in the North Atlantic, Mediterranean, tropical West Atlantic, Gulf of California, Southern Japan, Western Australia and New Zealand (Foster 2001). Rhodolith beds reach the deepest section of the euphotic zone, their maximum is at approximately 286 m (Foster 2001). These beds provide habitat, refuge, settlement sites and nursery ground for a variety of marine life, but also information about past and present climate changes and represent an important economic resource used as fertilizer in agriculture (Foster 2001). Climate change, ocean acidification and fisheries have negative impacts on rhodolith beds, resulting in calls for conservation (Hall-Spencer et al. 2010). In the Eastern Pacific, rhodolith beds are known from the Gulf of California to Alaska and the Galapagos Islands, Costa Rica, and Panama. The knowledge on coralline algae along the Chilean coast is still scarce focusing on crustose and non free-living forms (e.g. Vidal et al. 2003). Here we give the first record of rhodolith beds in the South-East Pacific, based on scientific expeditions to Guarello Island (Madre de Dios Archipelago, c. 50°S 75°W), Melinka and Amita Island (Guaitecas, c. 43°S 73°W,), and Robinson Crusoe Island (Juan Fernandez Archipelago, c. 33°S 78°W). At three sites, Robinson Crusoe, Guarello and Amita, we found areas of average downward slope on rocky ground mixed with sediment spots. At depths from about 10–25 m many of these spots were covered with rhodolith beds showing a lumpy growth form in Robinson Crusoe (Fig. 1a) and fruticose habit in Guarello and Amita (Fig. 1b, c). Plants were approximately 5 cm in diameter and had one or two branches per cm in lumpy material and four to six branches per cm in fruticose material. In Melinka (Fig. 1d), rhodoliths with a lumpy growth form were found at intertidal pools. These records extend the distribution of rhodoliths and raises interesting questions about their distribution, taxonomy, evolution and ecology. Thus, rhodolith beds in the South-East Pacific need to be analyzed in detail in the future and their role in coastal processes evaluated.

Understanding Anaplasmataceae pathogenesis using “Omics” approaches

This paper examines how “Omics” approaches improve our understanding of Anaplasmataceae pathogenesis, through a global and integrative strategy to identify genes and proteins involved in biochemical pathways key for pathogen-host-vector interactions. The Anaplasmataceae family comprises obligate intracellular bacteria mainly transmitted by arthropods. These bacteria are responsible for major human and animal endemic and emerging infectious diseases with important economic and public health impacts. In order to improve disease control strategies, it is essential to better understand their pathogenesis. Our work focused on four Anaplasmataceae, which cause important animal, human and zoonotic diseases: Anaplasma marginale, A. phagocytophilum, Ehrlichia chaffeensis, and E. ruminantium. Wolbachia spp. an endosymbiont of arthropods was also included in this review as a model of a non-pathogenic Anaplasmataceae. A gap analysis on “Omics” approaches on Anaplasmataceae was performed, which highlighted a lack of studies on the genes and proteins involved in the infection of hosts and vectors. Furthermore, most of the studies have been done on the pathogen itself, mainly on infectious free-living forms and rarely on intracellular forms. In order to perform a transcriptomic analysis of the intracellular stage of development, researchers developed methods to enrich bacterial transcripts from infected cells. These methods are described in this paper. Bacterial genes encoding outer membrane proteins, post-translational modifications, eukaryotic repeated motif proteins, proteins involved in osmotic and oxidative stress and hypothetical proteins have been identified to play a key role in Anaplasmataceae pathogenesis. Further investigations on the function of these outer membrane proteins and hypothetical proteins will be essential to confirm their role in the pathogenesis. Our work underlines the need for further studies in this domain and on host and vector responses to infection.

TufA phylogeny and species boundaries in the green algal order Prasiolales (Trebouxiophyceae, Chlorophyta)

:The order Prasiolales is one of the most characteristic lineages of the green algal class Trebouxiophyceae. Although its phylogeny has been studied in detail in recent years, several important features of its systematics and taxonomy are still in need of clarification. We investigated the phylogeny of the tufA gene in the Prasiolales, a marker that has been recently proposed for DNA barcoding in green algae but for which the amount of sequence data available is still limited for the Trebouxiophyceae. The results produced a phylogeny that was in good agreement with previous studies based on other markers, and the concatenation of tufA, rbcL and psaB resulted in a substantial improvement in phylogenetic resolution. Prasiola meridionalis and Prasiola stipitata, the two most common marine species of Prasiola in the northern hemisphere, had identical tufA sequences; we interpreted this genetic similarity to be the result of a trans-Arctic radiation related to the climatic history of the northern Atlantic and northern Pacific Oceans in recent evolutionary times. Sequences of the lichenized species Prasiola borealis indicate that the free-living form of this alga has a cosmopolitan distribution in cold temperate zones. In combination with other studies, our results suggested that the species of Prasiola commonly involved in associations with fungi were probably P. furfuracea and P. delicata rather than P. crispa, as was formerly believed. Based on the level of phylogenetic resolution of tufA in the Prasiolales, this gene may be a valuable phylogenetic marker at lower taxonomic levels in the Trebouxiophyceae and Ulvophyceae.

Sea-surface temperature reconstruction from trace elements variations of tropical coralline red algae

We used laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) to obtain high-resolution variations of the Mg/Ca, Sr/Ca and Li/Ca composition of free-living forms (i.e. rhodoliths) of the coralline red algal species Sporolithon durum in order to test their potential to archive seawater temperature information. A monitoring experiment was conducted based on alizarin red S (ARS) staining of rhodoliths specimens collected in various locations across a ∼1 km2 rhodolith bed in the vicinity of Nouméa, New Caledonia, where in situ temperature (IST) variations were recorded for 22 months between November 2009 and August 2011. A >45-year comparison of Mg and trace elements with sea-surface temperature (SST) was established from the analysis of 5 different branches belonging to three of the largest (7.4–8.5 cm in diameter) rhodolith specimens observed at the site. Consistent mean Mg/Ca, Sr/Ca and Li/Ca concentrations and seasonal patterns are found for the rhodoliths' last living years (2009–2011) across 43 branches and for the full 1963–2008 period across the 5 branches. Average elemental concentrations (Mg/Ca: 0.31 ± 0.04 mol/mol; Sr/Ca: 3.5 ± 0.4 mmol/mol and Li/Ca: 0.08 ± 0.02 mmol/mol) fall within range of those found in the literature. Individual element variations show good reproducibility between records and Mg/Ca, Sr/Ca and Li/Ca co-vary systematically. Combined records of Mg/Ca, Sr/Ca and Li/Ca are highly correlated with the IST monthly pattern for the 2009–2011 period (0.82 < r < 0.91; p < 0.001) and with local variations of monthly SST for the 1963–2008 period (0.65 < r < 0.85; p < 0.001), with Mg/Ca systematically being the best fit to monthly seawater temperature variations. Inter-annual Mg/Ca anomalies show significant correlation with the Oceanic Nino Index (ONI), indicating that S. durum rhodoliths also have the capacity to record the regional climate pattern in the tropical Pacific. Finally, consistent variations between the combined Mg/Ca record in S. durum rhodoliths and one Sr/Ca record of a Porites sp. coral from the same site, as well as a similar relationship with local SST at both monthly and interannual scales, suggest that S. durum rhodoliths have the potential to compare favourably with corals in terms of SST reconstruction.

Allee Effects and the Evolution of Polymorphism in Cyclic Parthenogens

Cyclic parthenogens alternate asexual reproduction with periodic episodes of sexual reproduction. Sexually produced free-living forms are often their only way to survive unfavorable periods. When sexual reproduction requires the mating of two self-incompatible individuals, mating limitation may generate an Allee effect, which makes small populations particularly vulnerable to extinction; parthenogenetic reproduction can attenuate this effect. However, asexual reproduction likely trades off with sexual reproduction. To explore the evolutionary implications of such a trade-off, we included recurrent mating events associated with seasonal interruptions in a simple population dynamics model. Following an adaptive dynamics approach, we showed that positive density dependence associated with Allee effects in cyclic parthenogens promotes evolutionary divergence in the level of investment in asexual reproduction. Although polymorphism may be transient, morphs mostly investing into sexual reproduction may eventually exclude those predominantly reproducing in an asexual manner. Asexual morphs can be seen as making cooperative investments into the common pool of mates, while sexual morphs defect, survive better, and may eventually fix in the population. Our findings provide a novel hypothesis for the frequent coexistence of sexual and asexual lineages, notably in plant parasitic fungi.

赤潮过程中“藻-菌”关系的研究进展

微生物对促进海洋物质循环，维持水生环境的生态平衡具有重要作用。在赤潮事件中，基于微生物（尤其是细菌）的多样性和重要性，它们与藻类之间的相互关系成为了研究的热点。过去20年里，人们从不同角度对“藻-菌”间的关系进行了探索，包括物理学过程、生物学过程、环境过程以及化学过程。就化学过程而言，它作为一种较早出现的技术，在以往的研究中带给人们许多认识藻菌关系的方法。随着学科的渗透，化学法有了拓展与延伸，为人们认识藻菌关系带来了新的契机。从化学生态学领域来梳理“藻．菌”关系中涉及的现象和行为，包括菌对藻的有益面、菌对藻的有害面、以及藻类应答细菌行为的化学途径；并从信号语言（群体感应、化感作用）的角度来阐释两者之间的互生或克生关系。通过文献综述的方式来解读藻菌关系的互作过程和机理，为认识赤潮的发生和防控方法提供借鉴。

Divergent nod-containing Bradyrhizobium sp. DOA9 with a megaplasmid and its host range.

Bradyrhizobium sp. DOA9, a non-photosynthetic bacterial strain originally isolated from the root nodules of the legume Aeschynomene americana, is a divergent nod-containing strain. It exhibits a broad host range, being able to colonize and efficiently nodulate the roots of most plants from the Dalbergioid, Millettioid, and Robinioid tribes (7 species of Papilionoideae). In all cases, nodulation was determinate. The morphology and size of DOA9 bacteroids isolated from the nodules of various species of Papilionoideae were indistinguishable from the free-living form. However, they were spherical in Arachis hypogaea nodules. GusA-tagged DOA9 also colonized rice roots as endophytes. Since broad-host-range legume symbionts often carry multiple replicons in their genome, we analyzed the replicons for symbiosis genes by electrophoresis. DOA9 carried two replicons, a chromosome (cDOA9) and single megaplasmid (pDOA9) larger than 352 kb. The genes for nodulation (nodA, B, C) and nitrogen fixation (nifH) were localized on the megaplasmid. Southern blot hybridization revealed two copies of nodA on the megaplasmid, single copies of nodB and C on the megaplasmid, and one copy each of nifH on the chromosome and megaplasmid. These results suggested that Bradyrhizobium sp. DOA9 may have the unusual combination of a broad host range, bacteroid differentiation, and symbiosis-mediating replicons.

Emerging Electrophysiology of Facultative Bacterial Pathogens

In contrast to strictly marine microorganisms or obligatory parasites dwelling in a constant environment, facultative pathogens that spend part of their life inside the host and part as free-living forms possess exquisitely robust osmoregulation. Their survival and persistence in highly variable conditions outside of the host defines the pathways and efficacy of transmission. Giant spheroplasts as a system for patch-clamp recording, previously developed for E. coli, already permitted detailed characterization of osmoprotective tension-activated channels in this organism, but homologous channels from other bacteria have never been recorded in their native setting. We have recently developed giant spheroplast preparations for Vibrio cholerae and Pseudomonas aeruginosa. The first patch-clamp survey of both organisms revealed channels with conductances and gating phenotypes similar to MscS and MscL of E. coli, with comparable tension midpoints. Conductive responses to saturating tension are dominated by MscL-like channels in both species. Analysis of genomes pointed to single orthologs of MscL and MscS in V. cholereae, but P. aeruginosa, in addition to MscL, possesses two mscS-like channel genes. Homology models reveal differences from E. coli channels in the distributions of charged and aromatic residues, underlying potentially different interactions with lipids. MS channels in both species are modulated by ampthipathic autoinducers (CAI-1 and PAI-1) from respective bacterial species, which shift their activation curves to the right on the tension scale. This apparently indirect effect mediated by a distorted lateral pressure profile in the membrane suggests that autoinducers easily partition into the cytoplasmic membranes of these bacteria and may not require special transport systems for entry. Amenability to patch-clamp recording for the two new bacterial species opens broad opportunities for studies of solute permeability and electrogenic transport in their membranes.

The Pangaean origin of "candidatus liberibacter" species

SUMMARY Species of “Candidatus Liberibacter”, all vectored by psyllids, are generally recognized as the cause of four serious plant diseases, Huanglongbing, Zebra Chip, Psyllid Yellows and Yellows Decline, which currently threaten and destroy the citrus, potato, tomato and carrot industries, respectively. These are relatively recent diseases in plant crops, thereby inferring a modern evolutionary trajectory, but no overall hypothesis on their origins has been presented. Plate tectonic movements provide an explanation for the disjunction between modern geographic range and phylogeny. Northern species, on Laurasia, represent both modern European and North American ranges. Southern species, on Gondwana, indicate a further speciation event as ancient India, conjoint with Madagascar, separated from Africa, left Madagascar on its own, and produced the single heat tolerant species while drifting across the equator to its modern position. Phylogeny, epidemiology and geography of the modern species point backwards to a single original speciation event from a free-living form, associated opportunistically with angiosperm and psyllid insect ancestors. The original heat-sensitive species lived in the equatorial but cool climate of the Central Pangaean Mountains probably > 300 Ma. The obligately alternating insect/plant host lifestyle developed opportunistically with the evolution and spread of flowering plants and psyllids.

Physiological responses of three temperate coralline algae from contrasting habitats to near-future ocean acidification

Coralline algae are major calcifiers of significant ecological importance in marine habitats but are among the most sensitive calcifying organisms to ocean acidification. The elevated pCO2 effects were examined in three coralline algal species living in contrasting habitats from intertidal to subtidal zones on the north-western coast of Brittany, France: (i) Corallina elongata, a branched alga found in tidal rock pools, (ii) Lithophyllum incrustans, a crustose coralline alga from the low intertidal zone, and (iii) Lithothamnion corallioides (maerl), a free-living form inhabiting the subtidal zone. Metabolic rates were assessed on specimens grown for one month at varying pCO2: 380 (current pCO2), 550, 750 and 1000μatm (elevated pCO2). There was no pCO2 effect on gross production in C. elongata and L. incrustans but L. incrustans respiration strongly increased with elevated pCO2. L. corallioides gross production slightly increased at 1000μatm, while respiration remained unaffected. Calcification rates decreased with pCO2 in L. incrustans (both in the light and dark) and L. corallioides (only in the light), while C. elongata calcification was unaffected. This was consistent with the lower skeletal mMg/Ca ratio of C. elongata (0.17) relative to the two other species (0.20). L. incrustans had a higher occurrence of bleaching that increased with increasing pCO2. pCO2 could indirectly impact this coralline species physiology making them more sensitive to other stresses such as diseases or pathogens. These results underlined that the physiological response of coralline algae to near-future ocean acidification is species-specific and that species experiencing naturally strong pH variations were not necessarily more resistant to elevated pCO2 than species from more stable environment.

Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strains.

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.

Esculentin(1-21), an amphibian skin membrane-active peptide with potent activity on both planktonic and biofilm cells of the bacterial pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that forms sessile communities, named biofilms. The non-motile forms are very difficult to eradicate and are often associated with the establishment of persistent infections, especially in patients with cystic fibrosis. The resistance of P. aeruginosa to conventional antibiotics has become a growing health concern worldwide and has prompted the search for new anti-infective agents with new modes of action. Naturally occurring antimicrobial peptides (AMPs) represent promising future template candidates. Here we report on the potent activity and membrane-perturbing effects of the amphibian AMP esculentin(1-21), on both the free-living and sessile forms of P. aeruginosa, as a possible mechanism for biofilm disruption. Furthermore, the findings that esculentin(1-21) is able to prolong survival of animals in models of sepsis and pulmonary infection indicate that this peptide can be a promising template for the generation of new antibiotic formulations to advance care of infections caused by P. aeruginosa.

Pochonia chlamydosporia in the biological control of Fasciola hepatica in cattle in Southeastern Brazil

Biological control with the use of nematophagous fungi has been described very successfully by many authors and presents itself as a complementary control method, acting on the free-living forms of helminths. The efficacy of a formulation containing the fungus Pochonia chlamydosporia in controlling Fasciola hepatica eggs in faeces was evaluated in an experimental field assay. Two bovine groups (six animals each) were used: A (control) and B (treated with fungus). At 30days after deworming, the animals were separated into two similar paddocks with flooded areas and were given pellets containing 25% mycelial mass (group B) or no fungus (group A) at a dose of 1g/10kg body weight, twice a week, during 18months. Faecal samples were harvested fortnightly in the animals of groups A and B and they were submitted at examination of quantitative sedimentation. The mean count of F. hepatica eggs per grams of faeces was significantly higher in group A (1.19) compared with those from group B (0.82) (P < 0.01). After 18months, animals from group B had gained 42.33kg above (17.82% more by weight) (P < 0.01), compared with the control group (A). Every month, faecal samples from paddocks A and B were collected and they were incubated. P. chlamydosporia was identified only in sample source of the paddock B. It can be concluded that the application of this fungical formulation with P. chlamydosporia 25% mycelial mass was effective in reducing the availability of eggs in the environment and reinfections in calves in natural conditions.

Diversity of free-living and symbiotic Symbiodinium in the coral reefs of Sanya, South China Sea

Unicellular photosynthetic dinoflagellates from the genus Symbiodinium are found either free-living or in hospite with a wide variety of marine invertebrate hosts including scleractinian corals. The linkages between free-living and endosymbiotic Symbiodinium remain largely unstudied and constitute a major gap in knowledge. In this study, the diversity of free-living Symbiodinium spp. and those associated with scleractinian corals from coral reefs in Sanya CRNR, South China Sea were determined by Symbiodinium-specific primers for the hypervariable region of the chloroplast 23S domain V (cp23S-HVR). The results illustrated that the free-living Symbiodinium spp. were highly spatially heterogeneous with high diversity related to clades A, B, C, D, F, G and H. However, the reef corals mainly hosted only one symbiont type of either clades C or D, and few species could harbour both concurrently. Surprisingly, Symbiodinium spp. in scleractinian corals were totally different from free-living forms in adjacent waters. These results suggest that the corals did not recruit Symbiodinium spp. from adjacent waters and the exchange of Symbiodinium spp. between corals and waters is limited in this region.

RNAseq Analysis of the Parasitic Nematode Strongyloides stercoralis Reveals Divergent Regulation of Canonical Dauer Pathways

The infectious form of many parasitic nematodes, which afflict over one billion people globally, is a developmentally arrested third-stage larva (L3i). The parasitic nematode Strongyloides stercoralis differs from other nematode species that infect humans, in that its life cycle includes both parasitic and free-living forms, which can be leveraged to investigate the mechanisms of L3i arrest and activation. The free-living nematode Caenorhabditis elegans has a similar developmentally arrested larval form, the dauer, whose formation is controlled by four pathways: cyclic GMP (cGMP) signaling, insulin/IGF-1-like signaling (IIS), transforming growth factor β (TGFβ) signaling, and biosynthesis of dafachronic acid (DA) ligands that regulate a nuclear hormone receptor. We hypothesized that homologous pathways are present in S. stercoralis, have similar developmental regulation, and are involved in L3i arrest and activation. To test this, we undertook a deep-sequencing study of the polyadenylated transcriptome, generating over 2.3 billion paired-end reads from seven developmental stages. We constructed developmental expression profiles for S. stercoralis homologs of C. elegans dauer genes identified by BLAST searches of the S. stercoralis genome as well as de novo assembled transcripts. Intriguingly, genes encoding cGMP pathway components were coordinately up-regulated in L3i. In comparison to C. elegans, S. stercoralis has a paucity of genes encoding IIS ligands, several of which have abundance profiles suggesting involvement in L3i development. We also identified seven S. stercoralis genes encoding homologs of the single C. elegans dauer regulatory TGFβ ligand, three of which are only expressed in L3i. Putative DA biosynthetic genes did not appear to be coordinately regulated in L3i development. Our data suggest that while dauer pathway genes are present in S. stercoralis and may play a role in L3i development, there are significant differences between the two species. Understanding the mechanisms governing L3i development may lead to novel treatment and control strategies.

In vitro Antileishmanial, Trypanocidal and Mammalian Cell Activities of Diverse N,N'-Dihetaryl Substituted Diamines and Related Compounds

The leishmaniasis and Chagas diseases constitute a serious public health problem worldwide with few and ineffective treatment options. The search for new antiparasitic candidates at the initial steps of drug discovery and development is still necessary. The synthesis of 22 de novo synthetized N,N′-dihetaryl-alkyldiamine derivatives and in vitro antiparasitic activity were evaluated for the first time against intracellular and extracellular forms of Leishmania (Leishmania) infantum, L. (Viannia) panamensis, L. (Leishmania) amazonensis, and Trypanosoma cruzi. Additionally, the toxicity on mammalian cells was determined. Some of these substituted N,N′-diamines (25–35 % of the tested compounds) showed interesting results against free-living forms of parasites with activities at the inhibitory concentration (IC50) level of 1.96 to 28.83 μM for L. (L.) infantum promastigotes and IC50 of 0.02 to 5.31 μM for T. cruzi epimastigotes. No activity at the IC50 level on intracellular amastigotes of T. cruzi was observed. However, N1,N2-dibenzylethane-1,2-diamine 5a revealed an important activity against the intracellular amastigotes of L. infantum (IC50 25.42 μM ±0.33) and L. panamensis (IC50 58.20 μM ±3.23), while their analogue N1,N4-dibenzylbutane-1,4-diamine 5c resulted in activity only against L. panamensis (IC50 11.19 μM ±0.20) without toxicity on Vero and THP-1 mammalian cells. The active compounds against intracellular parasites with low toxicity in mammalian cells may be considered for future studies in experimental models.