Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

Redescription of Dicyemennea eledones (Wagener, 1857) (Phylum Dicyemida) from Eledone cirrhosa (Lamarck, 1798) (Mollusca: Cephalopoda: Octopoda).

Dicyemids are common parasites found in the kidneys of many cephalopods. Species identification previously relied on old species descriptions containing considerable confusions, casting doubt on taxonomy and identification. Detailed morphological description and genotyping of all developmental stages are required for an exact taxonomy. To this end, we undertook the redescription of the dicyemid Dicyemennea eledones (Wagener, 1857), infecting the cephalopod Eledone cirrhosa (Lamarck). Samples were collected off Concarneau in the Bay of Biscay, France, and off La Goulette in the Gulf of Tunis, Tunisia. Dicyemennea eledones is a large species, with adults reaching c.7,000 µm in length. The vermiform stages are characterised by having 23 peripheral cells, a conical calotte and an axial cell that extends to the base of the propolar cells. An anterior abortive axial cell is present in vermiform embryos. Infusoriform embryos consist of 37 cells; a single nucleus is present in each urn cell and the refringent bodies, which were not always seen, are possibly liquid. For the first time, an 18S rDNA sequence is generated for D. eledones, illustrating genetic differences with the other dicyemid 18S rDNA sequences available in databases. This sequence can now be used for D. eledones barcoding, making the identification of the species easier and more reliable.

Diversity of apostome ciliates, Chromidina spp. (Oligohymenophorea, Opalinopsidae), parasites of cephalopods of the Mediterranean Sea.

Chromidina spp. are enigmatic apostome ciliates (Oligohymenophorea, Opalinopsidae) that parasitise the renal and pancreatic appendages of cephalopods. Only four species have been described, among which only three have been formally named. No DNA sequence has been reported so far. To investigate Chromidina spp. diversity, we sampled cephalopods in the Mediterranean Sea off Tunis, Tunisia, and identified two distinct Chromidina spp. in two different host species: Loligo vulgaris and Sepia officinalis. From haematoxylin-stained slides, we described morphological traits for these parasitic species and compared them to previous descriptions. We also re-described the morphology of Chromidina elegans (Foettinger, 1881) from Chatton and Lwoff's original materials and designated a neohapantotype and paraneohapantotypes for this species. We describe a new species, Chromidina chattoni Souidenne, Florent and Grellier n. sp., found in L. vulgaris off Tunisia, and evidence for a probable novel species, found in S. officinalis off Tunisia, although this latter species presents similarities to some morphological stages previously described for Chromidina cortezi Hochberg, 1971. We amplified, for the first time, an 18S rDNA marker for these two Chromidina species. Phylogenetic analysis supports the association of Chromidina within apostome ciliates. Genetic distance analysis between 18S rDNA sequences of representative apostomes indicates Pseudocollinia as the most closely related genus to Chromidina.

Haplomyxa saranae gen. nov. et sp. nov., a new naked freshwater foraminifer.

A new naked foraminifer, Haplomyxa saranae gen. nov. sp. nov., is described from an established cell line made from a single cell isolated from a freshwater garden pond. The new species was morphologically close to Reticulomyxa filosa, the only valid naked freshwater foraminifer species. However the two species differed when it came to the morphology of the cell body, the number of cysts, and the nutrition. The 18S rRNA gene had one of the longest sequences to date (4863 nucleotides), and it contained many insertions that are typical of Foraminifera. The size of this gene was 45% longer than the one of R. filosa due to the elongation of A+T rich regions, but molecular phylogeny based on conserved regions of the 3'-end placed the new species in the same morphological clade K. This report includes both morphological and genetic data which undoubtedly show that the new species is a new naked freshwater foraminifer and the second species of the clade K.

Ciliate Nassula sp. grazing on a microcystin-producing cyanobacterium (Planktothrix agardhii): impact on cell growth and in the microcystin fractions.

The proliferation of microcystins (MCs)-producing cyanobacteria (MCs) can have detrimental effects on the food chain in aquatic environments. Until recently, few studies had focused on the fate of MCs in exposed organisms, such as primary consumers of cyanobacteria. In this study, we investigate the impact of an MC-producing strain of the cyanobacterium Planktothrix agardhii on the growth and physiology of a Nassula sp. ciliate isolated from a non-toxic cyanobacterial bloom. We show that this Nassula sp. strain was able to consume and grow while feeding exclusively on an MC-producing cyanobacterium over a prolonged period of time (8 months). In short-term exposure experiments (8 days), ciliates consuming an MC-producing cyanobacterial strain displayed slower growth rate and higher levels of antioxidant enzymes than ciliates feeding on two non-MC-producing strains. Three high-performance methods (LC/MS, LC/MS-MS and ELISA) were used to quantify the free and bound MCs in the culture medium and in the cells. We show that ciliate grazing led to a marked decrease in free MCs (methanol extractable) in cells, the MCs were therefore no longer found in the surrounding culture medium. These findings suggest that MCs may have undergone redistribution (free vs bound MCs) or chemical degradation within the ciliates.

Mechanistic issues of the interaction of the hairpin-forming domain of tBid with mitochondrial cardiolipin.

BACKGROUND: The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated. PRINCIPAL FINDINGS: The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix alphaH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, alphaH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix alphaH6 to efficiently induce cytochrome c release and apoptosis. CONCLUSIONS: Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix alphaH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix alphaH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated.

Anti-Plasmodium activity of ceramide analogs.

BACKGROUND: Sphingolipids are key molecules regulating many essential functions in eukaryotic cells and ceramide plays a central role in sphingolipid metabolism. A sphingolipid metabolism occurs in the intraerythrocytic stages of Plasmodium falciparum and is associated with essential biological processes. It constitutes an attractive and potential target for the development of new antimalarial drugs. METHODS: The anti-Plasmodium activity of a series of ceramide analogs containing different linkages (amide, methylene or thiourea linkages) between the fatty acid part of ceramide and the sphingoid core was investigated in culture and compared to the sphingolipid analog PPMP (d,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). This analog is known to inhibit the parasite sphingomyelin synthase activity and block parasite development by preventing the formation of the tubovesicular network that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite. RESULTS: Analogs containing methylene linkage showed a considerably higher anti-Plasmodium activity (IC50 in the low nanomolar range) than PPMP and their counterparts with a natural amide linkage (IC50 in the micromolar range). The methylene analogs blocked irreversibly P. falciparum development leading to parasite eradication in contrast to PPMP whose effect is cytostatic. A high sensitivity of action towards the parasite was observed when compared to their effect on the human MRC-5 cell growth. The toxicity towards parasites did not correlate with the inhibition by methylene analogs of the parasite sphingomyelin synthase activity and the tubovesicular network formation, indicating that this enzyme is not their primary target. CONCLUSIONS: It has been shown that ceramide analogs were potent inhibitors of P. falciparum growth in culture. Interestingly, the nature of the linkage between the fatty acid part and the sphingoid core considerably influences the antiplasmodial activity and the selectivity of analogs when compared to their cytotoxicity on mammalian cells. By comparison with their inhibitory effect on cancer cell growth, the ceramide analogs might inhibit P. falciparum growth through modulation of the endogenous ceramide level.