Increasing densities of an invasive polychaete enhance bioturbation with variable effects on solute fluxes.

Bioturbation is a key process affecting nutrient cycling in soft sediments. The invasive polychaete genus Marenzelleria spp. has established successfully throughout the Baltic Sea increasing species and functional diversity with possible density-dependent effects on bioturbation and associated solute fluxes. We tested the effects of increasing density of M. arctia, M. viridis and M. neglecta on bioturbation and solute fluxes in a laboratory experiment. Benthic communities in intact sediment cores were manipulated by adding increasing numbers of Marenzelleria spp. The results showed that Marenzelleria spp. in general enhanced all bioturbation metrics, but the effects on solute fluxes varied depending on the solute, on the density and species identity of Marenzelleria, and on the species and functional composition of the surrounding community. M. viridis and M. neglecta were more important in predicting variation in phosphate and silicate fluxes, whereas M. arctia had a larger effect on nitrogen cycling. The complex direct and indirect pathways indicate the importance of considering the whole community and not just species in isolation in the experimental studies. Including these interactions provides a way forward regarding our understanding of the complex ecosystem effects of invasive species.

PCR survey of 50 introns in animals: cross-amplification of homologous EPIC loci in eight non-bilaterian, protostome and deuterostome phyla.

Exon Primed Intron Crossing (EPIC) markers provide molecular tools that are susceptible to be variable within species while remaining amplifiable by PCR using potentially universal primers. In this study we tested the possibility of obtaining PCR products from 50 EPIC markers on 23 species belonging to seven different phyla (Porifera, Cnidaria, Arthropoda, Nematoda, Mollusca, Annelida, Echinodermata) using 70 new primer pairs. A previous study had identified and tested those loci in a dozen species, including another phylum, Urochordata (Chenuil et al., 2010). Results were contrasted among species. The best results were achieved with the oyster (Mollusca) where 28 loci provided amplicons susceptible to contain an intron according to their size. This was however not the case with the other mollusk Crepidula fornicata, which seems to have undergone a reduction in intron number or intron size. In the Porifera, 13 loci appeared susceptible to contain an intron, a surprisingly high number for this phylum considering its phylogenetic distance with genomic data used to design the primers. For two cnidarian species, numerous loci (24) were obtained. Ecdysozoan phyla (arthropods and nematodes) proved less successful than others as expected considering reports of their rapid rate of genome evolution and the worst results were obtained for several arthropods. Some general patterns among phyla arose, and we discuss how the results of this EPIC survey may give new insights into genome evolution of the study species. This work confirms that this set of EPIC loci provides an easy-to-use toolbox to identify genetic markers potentially useful for population genetics, phylogeography or phylogenetic studies for a large panel of metazoan species. We then argue that obtaining diploid sequence genotypes for these loci became simple and affordable owing to Next-Generation Sequencing development. Species surveyed in this study belong to several genera (Acanthaster, Alvinocaris, Aplysina, Aurelia, Crepidula, Eunicella, Hediste, Hemimysis, Litoditis, Lophelia, Mesopodopsis, Mya, Ophiocten, Ophioderma, Ostrea, Pelagia, Platynereis, Rhizostoma, Rimicaris), two of them, belonging to the family Vesicomydae and Eunicidae, could not be determined at the genus level.

Molecular characterisation of the Microsporidia of the amphipod Gammarus duebeni across its natural range revealed hidden diversity, wide-ranging prevalence and potential for co-evolution.

Microsporidia comprise an unusual group of intracellular, eukaryotic parasites that exhibit ubiquitous distribution throughout the animal kingdom. We analysed the small subunit ribosomal gene (SSUrDNA) using PCR and sequencing and screened 894 Gammarus duebeni (Crustacea, Amphipoda) specimens from 35 European marine and freshwater populations. We discovered considerable hidden microsporidian diversity. Blast searches, sequence analysis and phylogenetic analysis revealed intraspecific sequence variants of known species Dictyocoela duebenum,Dictyocoela muelleri, Pleistophora mulleri and Nosema granulosis. For seven SSUrDNA sequences, we did not find corresponding GenBank entries; they likely represent new species, provisionally classified within the genus Microsporidium. Phylogenetic reconstructions revealed their position as polyphyletic, thereby lending support to the hypothesis of an early microsporidian radiation within this host group. Nevertheless, four of the presumptive novel species formed a discrete and well-supported subclade in the phylogenetic analysis. The respective host specimens were collected from disjunct freshwater sites in Wales, Ireland and Brittany (France) and may represent a new, G. duebeni-specific, microsporidian genus. At the population level, our screening showed that parasitism through Microsporidia is the rule rather than the exception in G. duebeni. We found Microsporidia in 91% of sampled G. duebeni populations. This finding may have considerable consequences for the interpretation of results from ecological, behavioural, physiological and evolutionary studies of the host, as parasitism can significantly influence these traits. Because the host G. duebeni has a complex phylogeography and evolutionary history, the studied host-parasite system may have potential as a model system for investigating processes of co-evolution.

Glacial-driven vicariance in the amphipod Gammarus duebeni.

We have examined the genetic diversity using mitochondrial COI and ND2 sequence data from 306 specimens of the amphi-Atlantic-distributed amphipod Gammarus duebeni. Marine populations from the Atlantic Ocean, the Baltic and North Sea, as well as freshwater populations from Ireland, Cornwall and Brittany were analysed. G. duebeni is a complex of five allopatric lineages. Freshwater populations result from multiple invasions of marine ancestors, represented by distinct lineages. We interpret the recent distribution of lineages as the outcome of a series of spatio-temporal vicariant events caused by Pleistocene glaciations and sea level changes. The freshwater lineages are therefore regarded as 'glacial relicts'. Furthermore, inter-specific competition with, for example, Gammarus pulex (which is absent in Ireland and western Brittany) may be another important determinant in the distribution of freshwater G. duebeni. In Ireland and Brittany, three freshwater refugia are suggested. The significantly limited gene flow detected among marine populations is more likely due to inter-specific competition than to salinity. The G. duebeni-complex represents a model system for the study of allopatric speciation accompanied by major habitat shifts. The pattern of spatio-temporal origins of the freshwater entities we describe here provides an excellent system for investigating evolutionary adaptations to the freshwater environment. Our data did not confirm the presently used subspecies classification but are only preliminary in the absence of nuclear genetic analyses.

Stress protein response in two sibling species of Marenzelleria (Polychaeta: Spionidae): is there an influence of acclimation salinity?

The induction and synthesis of stress proteins in the polychaete sibling species Marenzelleria viridis and M. neglecta was investigated at two different acclimation salinities (10 and 25 ppt). By in vitro labeling of dissected metameres with (35)S-methionine/cysteine and electrophoretic separation, four size classes of heat shock proteins (Hsps) were detected corresponding to 86, 78, 75 and 27 kDa. All Hsps, with the exception of Hsp86, represent a complex of multiple isoforms. The sibling species differed in three aspects of their heat shock response: (1) the induction temperature for Hsp75 synthesis was 25 degrees C and 30 degrees C in M. viridis and M. neglecta, respectively; (2) the relative level of synthesis of Hsp75 was higher in M. viridis; (3) the heat shock response was inactivated at a higher temperature in M. neglecta compared to M. viridis. The results showed that acclimation salinity had no explicit effect on Hsp synthesis in either species and that M. viridis was thermally more sensitive than its sibling species. We proposed that temperature, alone or in combination with other abiotic factors, plays a far greater role in the biogeographic distribution in Marenzelleria spp. than has been estimated so far.

Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas.

Nineteen specimens from primary human malignant mesotheliomas obtained from 19 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 by combined RFLP-PCR/SSCP analysis. In addition, all tumours were screened for deletions and point mutations in the tumour suppressor genes p53, p16INK4a (CDKN2A) and p14ARF (exon-1beta) by combined multiplex-PCR/SSCP analysis. No mutations were found in N-ras, p53 and CDK4. Three tumours displayed homozygous deletion (co-deletion of exons 1, 2 and 3) of p16INK4a. One of them displayed additional homozygous deletion of p14ARF (exon-1beta). Two silent point mutations and 2 polymorphisms were found in p16INK4a in 3 tumours. Our preliminary data indicate that disarrangement of the Rb1 pathway may be involved in mesothelioma formation.

A new molecular phylogenetic hypothesis for the evolution of freshwater eels.

Phylogenetic analysis of a segment of the mitochondrial 16S rDNA of eight Anguilla species from the Indo-Pacific region and from the North Atlantic revealed that the genus Anguilla appears to be surprisingly young, based upon the small observed maximum genetic distance of 4.8% and the high degree of morphological similarity among the species. The placement of A. marmorata as the most ancestral lineage suggests that the genus is likely to have originated in the Indo-Malayian region, from which it quickly spread. Two Pacific species, A. obscura and A. japonica, branched next. A. japonica was placed as sister group to all remaining species, which formed three clades: the first comprising A. australis, the second A. reinhardti and A. mossambica, and the third A. anguilla and A. rostrata. All analyzed specimens of A. rostrata originating from southern New Jersey to Nova Scotia had identical mitotypes, while five mitochondrial genotypes were found in Europe differing by zero to two substitutions. The two Atlantic eel species are very closely related; all surveyed specimens of A. anguilla differ by three to five substitutions from their American allies, corroborating the existence of two distinct biological species. This was also confirmed by restriction analysis of a 350-bp segment of the cytochrome b, in which American specimens were distinct in sharing a single diagnostic restriction site of HinfI. Our results suggest little to no gene flow between the two nominal Atlantic eel species.

Mutational analysis of the N-ras, p53, p16INK4a, CDK4, and MC1R genes in human congenital melanocytic naevi.

Eighteen human congenital melanocytic naevi (CMN) from 17 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 and for sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis. In addition, all lesions were screened for deletions and point mutations in the tumour suppressor genes p53 and p16INK4a (CDKN2A) by combined multiplex PCR/SSCP analysis. Positive screening data were specified by sequencing of the corresponding PCR product. Activating point mutations in the N-ras gene (nine CAA (Gln) to AAA (Lys) transversions and one CAA (Gln) to CGA (Arg) transition at codon 61) were detected at high frequency (56%). Furthermore, three missense mutations (V92M) and two silent mutations (CGA (Arg) to CGG (Arg), codon 213, exon 6) were found in the MC1R and p53 genes, respectively. No mutations were found in p16 or CDK4. The activated N-ras oncogene, which is also found in human cutaneous melanomas, may constitute a potential risk factor for melanoma formation within CMN.

Cryptic species in marine polychaete and their independent introduction from North America to Europe.

The vast body of ballast water carried across oceans by freight ships represents a major source for the introduction of foreign species into marine ecosystems. The worm Marenzelleria viridis, originally found only in North America, appeared in estuaries of the North Sea in 1979 and 6 years later also in the Baltic, where it has developed into a major faunal element. Two competing hypotheses are discussed here: either both populations owe their presence to a single introductory event in the North Sea, or each population originated from a separate introduction. Our phylogeographic analysis of Baltic, North Sea and American Marenzelleria, based on mitochondrial 16S rDNA sequences (326-bp segment) of 98 individuals from 17 localities on the North American, North Sea, and Baltic coasts not only favors the two-event hypothesis, but also separates the locations of origin for the introductions. Eighteen mitochondrial genotypes were identified altogether. In agreement with allozyme data, three lineages were identified: genotypes assigned to the same lineage differed from each other by up to 5 point mutations, and those assigned to different lineages differed by up to 17. The existence of three morphologically indistinguishable, and thus cryptic, species is therefore suggested. The individuals from the Baltic Sea probably originated from the Atlantic coast of the United States between Chesapeake Bay and Georgia, and the North Sea populations may stem from the U.S. coast region north of Chesapeake Bay to Nova Scotia. Despite their similar morphologies, the two European Marenzelleria species may differ ecologically with respect to their preference for habitat salinity. Assuming that transport via ballast water occurs quite frequently, we hypothesize that both European cryptic species of Marenzelleria may originally have been introduced to both the North Sea and the Baltic Sea but that neither of them was able to proliferate in both water bodies owing to their differential physiological performances at high and low salinities.

Biochemical parameters as a measure of food availability and growth in immature rainbow trout (Oncorhynchus mykiss).

1. Rainbow trout held in brackish water (15 parts per thousand) were starved or fed different amounts of food. 2. A significant correlation was found between the growth rates of the different animals and the feed rates. 3. The RNA:DNA ratio in the white epaxial muscle is lowest in starved fish and increases in proportion to the feed rate and individual specific growth rate. The correlations are significant at the P less than 0.01 level. 4. Liver metabolism varies according to food availability. 5. The protein synthesis capacity of the liver (RNA:DNA ratio) and liver somatic index increase as the feeding rate increases. It also correlates significantly with the specific growth rates of the different animals. 6. The intermediary metabolism of the central metabolic organ, the liver, varies in the same way. 7. The activities of the NADPH producing liver enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (IDH) and malic enzyme (ME) increase as the feed rate (and therefore the specific growth rate) increases. 8. G6PDH and IDH activity in the kidney is influenced to a much lower degree by food intake. 9. Summarizing, it can be stated that biochemical parameters can be used to describe comprehensively the metabolic status and growth of rainbow trout.

The role of elongation factors in protein synthesis rate variation in white teleost muscle.

Protein synthesis-stimulating activity was assayed in the cytosolic fraction of white muscle from teleost fish (rainbow trout, carp) and of rat liver. In vitro protein synthesis-stimulating activity in the cytosolic fraction is reduced by food deprivation. The addition of elongation factors EF1, EF2, or EF1 + EF2 compensates for the starvation-induced loss of protein synthesis-stimulating activity in trout muscle cytosol. The action of EF2 is stronger than that of EF1 in this respect. However, EF1 enhances in vitro protein synthesis-stimulating activity in rat liver cytosol more than EF2. The EF2 concentration in the cytosolic fraction of white muscle from starved trout is significantly lower than in fed specimens.

Effects of salinity on vitamin B6 deficiency in rainbow trout (Salmo gairdneri Richardson).

1. The link between vitamin B6 deficiency and sea water adaptation in rainbow trout was investigated. 2. A plasma-hyperosmotic salinity (20%) exacerbates the effect of vitamin B6 deficiency in rainbow trout. 3. Na/K-ATPase activity in the gill and kidney of the rainbow trout is not affected by vitamin B6 deficiency.