The distribution and molecular diversity of the Eastern Atlantic and Mediterranean chthamalids (Crustacea, Cirripedia)

The three chthamalids Chthamalus stellatus , C. montagui and Euraphia depressa are common inhabitants of the intertidal zone in the Eastern Atlantic, Mediterranean Sea and Black Sea. In this study, we investigated the occurrence of these barnacles in a wide range of their distribution. Population divergences of these two species have been inferred using three molecular markers — internal transcribed spacer (ITS), elongation factor 1α (EF-1α) and cytochrome oxidase subunit I (COI). ITS sequences of C. stellatus were identical throughout the species range, whereas ITS sequences of C. montagui indicated that the Black Sea and Mediterranean populations are isolated from the Atlantic population. The COI and EF-1α sequences were the most variable and informative. They indicated a high genetic divergence between Atlantic, Mediterranean and Black Sea populations for C. montagui . In addition significant genetic structure was found among the populations of C. stellatus based on EF-1α but not COI. Interestingly, our molecular dating analysis correlated the pattern of diversification in C. montagui to major geological changes that occurred in the Mediterranean during the end of the Messinian and Pleiocene periods. We suggest that palaeohistory shaped the divergences between Chthamalus populations that have probably been maintained by current hydrographic conditions. Finally, COI phylogenetic analysis placed the genus Euraphia within the Chthamalus clade, suggesting the need for a taxonomic revision of Euraphia . This study represents the most detailed phylogeographical analysis of intertidal Mediterranean species to date, and shows that geological events have strongly shaped the current diversity pattern of this fauna.     

Replication-competent influenza A virus that encodes a split-green fluorescent protein-tagged PB2 polymerase subunit allows live-cell imaging of the virus life cycle

Studies on the intracellular trafficking of influenza virus ribonucleoproteins are currently limited by the lack of a method enabling their visualization during infection in single cells. This is largely due to the difficulty of encoding fluorescent fusion proteins within the viral genome. To circumvent this limitation, we used the split-green fluorescent protein (split-GFP) system (S. Cabantous, T. C. Terwilliger, and G. S. Waldo, Nat. Biotechnol. 23:102-107, 2005) to produce a quasi-wild-type recombinant A/WSN/33/influenza virus which allows expression of individually fluorescent PB2 polymerase subunits in infected cells. The viral PB2 proteins were fused to the 16 C-terminal amino acids of the GFP, whereas the large transcomplementing GFP fragment was supplied by transient or stable expression in cultured cells that were permissive to infection. This system was used to characterize the intranuclear dynamics of PB2 by fluorescence correlation spectroscopy and to visualize the trafficking of viral ribonucleoproteins (vRNPs) by dynamic light microscopy in live infected cells. Following nuclear export, vRNPs showed a transient pericentriolar accumulation and intermittent rapid (～1 μm/s), directional movements in the cytoplasm, dependent on both microtubules and actin filaments. Our data establish the potential of split-GFP-based recombinant viruses for the tracking of viral proteins during a quasi-wild-type infection. This new virus, or adaptations of it, will be of use in elucidating many aspects of influenza virus host cell interactions as well as in screening for new antiviral compounds. Furthermore, the existence of cell lines stably expressing the complementing GFP fragment will facilitate applications to many other viral and nonviral systems.     

Age-dependent genetic structure of arctic foxes in Svalbard

Arctic foxes are highly mobile arctic predators with a very weak population genetic structure over large parts of their range. Less is, however, known about the more local genetic structure within regions. Here, we analyze genotypes at 12 microsatellite loci for 561 arctic foxes trapped in the high-arctic archipelago Svalbard and investigate the genetic structure in three different age classes. Significant linkage disequilibrium, deficit of heterozygotes, genetic differentiation, and a decrease in relatedness with distance among animals trapped in their first winter suggested that some litter mates remain in proximity of each other during the first winter. This pattern was stronger for females than for males, indicating male-biased juvenile dispersal, and weaker for older animals. There was no genetic differentiation among adult foxes harvested in different hunting areas. The foxes from the protected area around Hornsund were however more differentiated than expected based on geographic distance alone, suggesting a possible disrupting effect of harvest on the spatial genetic structure in the rest of Svalbard. Our results also indicated a possible kin structure among adult females, suggesting natal philopatry, but further investigations will be needed to reach firm conclusions concerning kin structure in arctic foxes.     

Neurological and physiological disorders in Artemia harboring manipulative cestodes

There are many impressive examples of host manipulation by parasites, but mechanisms underlying these ethological changes, as well as their physiological consequences, are not well characterized. Here, we analyzed part of the cerebral proteome of brine shrimp Artemia infected by manipulative cestodes, using for the first time the ProteinChip Surface-Enhanced Laser Desorption Ionization and Time of Fly Mass Spectrometry (SELDI-TOF MS) system, which has been proposed as an excellent way to analyze the host genome during the host-parasite interaction processes. We found 2 peptides downregulated in individuals infected by the dilepidid, Anomotaenia tringae (4.5 kDa), and by the 2 hymenolepidids, Flamingolepis liguloides and Confluaria podicipina (3.9 kDa), which are potential candidates for involvement with the manipulation process. The identification of 2 head peptides (4.1 and 4.2 kDa) overexpressed in all the categories in brine shrimp living at the surface (both infected individuals and uninfected controls) suggests its association with the different environmental conditions experienced at the water surface. In parallel, brine shrimp infected by C. podicipina showed significant values of triglycerides, potentially augmenting their profitability and attractiveness for the predaceous definitive host (grebes). We discuss our findings in relationship with current ideas on the complexity of parasitically modified organisms.     

Synthetic riboswitches that induce gene expression in diverse bacterial species

We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.     

Cells under stress: a non-destructive evaluation of adhesion by ultrasounds

The adhesion process plays a major role in the development of osteoblastic cells on various substrates used in orthopaedic applications such as metals, bioceramics, or glass. High frequency and low power ultrasounds seem to be an appropriate tool for an evaluation of interface mechanical properties. Is it a non-destructive method? We investigated osteoblastic cell cultures, maintained in their medium with high frequency, bulk longitudinal waves. The influence of both acoustical frequency and acoustical power on cell adhesion is evaluated by cell detachment ratio and re-adhesion ratio. We demonstrate the existence of a power threshold depending on the frequency, allowing optimal cellular detachment and re-adhesion. Finally, a qualitative study of the detachment phenomena is performed by use of scanning electron microscopy (SEM), Confocal Laser Scanning Microscopy and cytochemical labelling.     

Dasyatispora levantinae gen. et sp. nov., a new microsporidian parasite from the common stingray Dasyatis pastinaca in the eastern Mediterranean

A new microsporidian infecting the Mediterranean common stingray Dasyatis pastinaca (Linnaeus, 1758) is described from Iskenderun Bay, Turkey. The parasite invades the disc muscles, producing slender, spindle-shaped subcutaneous swellings that develop into massive, elongated, tumor-like protuberances measuring up to 11 x 4 cm. Severity of the infection may vary from light (1 or 2 small lesions) to intense, with large parts of the dorsal surface covered with lumps and protrusions. These masses contained a yellowish-white caseous substance consisting of degraded host tissue and microsporidian sporophorous vesicles, which in turn contained developing sporonts, sporoblasts and spores. The ripe spore contained a uni-nucleate sporoplasm and large posterior vacuole, and measured 3.8-4.3 x 2.6-2.8 microm. Infection prevalence was 21% in a sample of 143 host individuals examined. All the infected stingray individuals were within the weight class of 300 to 800 g (200 to 305 mm disc width). Phylogenetic analyses of rDNA sequences indicate that this microsporidian belongs to the Pleistophoridae and clusters with species of the genera Ovipleistophora Pekkarinen, Lom & Nilsen, 2002 and Heterosporis Schubert, 1969. However, the morphology, development and host differ distinctly from all reported species, including those belonging to these 2 genera, and it is thus assigned to a newly erected genus and named Dasyatispora levantinae gen. et sp. nov. This is the first record of a microsporidian infection in a batoid. It is also the first microsporidian species to be formally described from an elasmobranch.