Miriam Blank

Molecular identification key based on PCR/RFLP for three polychaete sibling species of the genus Marenzelleria, and the species’ current distribution in the Baltic Sea

Studies of Marenzelleria species were often hampered by identification uncertainties when using morphological characters only. A newly developed PCR/RFLP protocol allows a more efficient discrimination of the three species Marenzelleria viridis, Marenzelleria neglecta and Marenzelleria arctia currently known for the Baltic Sea. The protocol is based on PCR amplification of two mitochondrial DNA gene segments (16S, COI) followed by digestion with restriction enzymes. As it is faster and cheaper than PCR/sequencing protocols used so far, the protocol is recommended for large-scale analyses. The markers allow an undoubted determination of species irrespective of life stage or condition of the worms in the samples. The protocol was validated on about 950 specimens sampled at more than 30 sites of the Baltic and the North Sea, and on specimens from populations of the North American east coast. Besides this test we used mitochondrial DNA sequences (16S, COI, Cytb) and starch gel electrophoresis to further investigate the distribution of the three Marenzelleria species in the Baltic Sea. The results show that M. viridis (formerly genetic type I or M. cf. wireni) occurred in the Öresund area, in the south western as well as in the eastern Baltic Sea, where it is found sympatric with M. neglecta. Allozyme electrophoresis indicated an introduction by range expansion from the North Sea. The second species, M. arctia, was only found in the northern Baltic Sea, where it sometimes occurred sympatric with M. neglecta or M. viridis. For Baltic M. arctia, the most probable way of introduction is by ship ballast water from the European Arctic. There is an urgent need for a new genetic analysis of all Marenzelleria populations of the Baltic Sea to unravel the current distribution of the three species.

Multiple Invasions – A Polychaete Genus Enters the Baltic Sea

Since 1985, the nonindigenous polychaete species Marenzelleria neglecta has been found in the Baltic Sea. The species, which was introduced by ship ballast water, spreads rapidly and dominates in many habitats today. Using three gene segments of the mitochondrial DNA (16S rDNA, Cytochrom oxidase I, Cytochrom b), we investigated four populations of the western and northern Baltic Sea in a preliminary survey and compared them with four other populations from the North Sea, the Baltic Sea and from the Arctic. First, we could demonstrate the applicability of the markers to discriminate the species with certainty. Second, with M. viridis and M. arctia, we could detect two more species of the same genus, which have recently been introduced into the Baltic Sea. One of these, M. arctia, was hitherto known as an exclusive arctic member of the genus. The impact of these two recently invaded Marenzelleria species onto the autochthonous fauna needs to be evaluated in the future. The Baltic Sea as a ‘natural aquarium’ now offers the possibility to investigate sibling species simultaneously. However, correct identification and denomination of Marenzelleria species are indispensable prerequisites for all future studies. Molecular markers allow the exact identification of all Marenzelleria species and must be used whenever a classical taxonomic identification is uncertain.

Widespread Occurrence of N-Terminal Acylation in Animal Globins and Possible Origin of Respiratory Globins from a Membrane-Bound Ancestor

Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or signaling.

A Membrane-Bound Vertebrate Globin

The family of vertebrate globins includes hemoglobin, myoglobin, and other O2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O2 binding with a variable affinity (P 50∼1.3–12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.

Oxygen Supply from the Bird's Eye Perspective GLOBIN E IS A RESPIRATORY PROTEIN IN THE CHICKEN RETINA

The visual process in the vertebrate eye requires high amounts of metabolic energy and thus oxygen. Oxygen supply of the avian retina is a challenging task because birds have large eyes, thick retinae, and high metabolic rates but neither deep retinal nor superficial capillaries. Respiratory proteins such as myoglobin may enhance oxygen supply to certain tissues, and thus the mammalian retina harbors high amounts of neuroglobin. Globin E (GbE) was recently identified as an eye-specific globin of chicken (Gallus gallus). Orthologous GbE genes were found in zebra finch and turkey genomes but appear to be absent in non-avian vertebrate classes. Analyses of globin phylogeny and gene synteny showed an ancient origin of GbE but did not help to assign it to any specific globin type. We show that the photoreceptor cells of the chicken retina have a high level of GbE protein, which accumulates to ∼10 μm in the total eye. Quantitative real-time RT-PCR revealed an ∼50,000-fold higher level of GbE mRNA in the eye than in the brain. Spectroscopic analysis and ligand binding kinetics of recombinant chicken GbE reveal a penta-coordinated globin with an oxygen affinity of P50 = 5.8 torrs at 25 °C and 15 torrs at 41 °C. Together these data suggest that GbE helps to sustain oxygen supply to the avian retina.

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.

A membrane-bound hemoglobin from gills of the green shore crab Carcinus maenas.

Most hemoglobins serve for the transport or storage of O2. Although hemoglobins are widespread in “entomostracan” Crustacea, malacostracans harbor the copper-containing hemocyanin in their hemolymph. Usually, only one type of respiratory protein occurs within a single species. Here, we report the identification of a hemoglobin of the shore crab Carcinus maenas (Malacostraca, Brachyura). In contrast to the dodecameric hemocyanin of this species, C. maenas hemoglobin does not reside in the hemolymph but is restricted to the gills. Immunofluorescence studies and cell fractioning showed that C. maenas hemoglobin resides in the membrane of the chief cells of the gill. To the best of our knowledge, this is the first time that a membrane-bound hemoglobin has been identified in eukaryotes. Bioinformatic evaluation suggests that C. maenas hemoglobin is anchored in the membrane by N-myristoylation. Recombinant C. maenas hemoglobin has a hexacoordinate binding scheme at the Fe2+ and an oxygen affinity of P50 = 0.5 Torr. A rapid autoxidation rate precludes a function as oxygen carrier. We rather speculate that, analogous to prokaryotic membrane-globins, C. maenas hemoglobin carries out enzymatic functions to protect the lipids in cell membrane from reactive oxygen species. Sequence comparisons and phylogenetic studies suggested that the ancestral arthropod hemoglobin was most likely an N-myristoylated protein that did not have an O2 supply function. True respiratory hemoglobins of arthropods, however, evolved independently in chironomid midges and branchiopod crustaceans.

Phylogeography, historical demography and postglacial colonization routes of two amphi-Atlantic distributed amphipods

To evaluate the influence of the ice ages on patterns of genetic diversity and to test generally accepted perspectives related to glacial refugia and proposed post-glacial colonization pathways in the North Atlantic area, we sequenced a portion of the mitochondrial cytochrome c oxidase subunit I of two amphi-Atlantic distributed amphipods, Gammarus duebeni (n = 418) and Gammarus oceanicus (n = 242). Both species showed several populations with a combination of highly reduced levels of genetic diversity and significant demographic expansion in previously glaciated sites, consistent with an expected extinction and recolonization scenario. Within G. oceanicus, two reciprocally monophyletic clades were found. One clade consisted of the specimens from populations of the American St. Lawrence River and two populations south of it. The other clade included specimens from populations of Europe, Greenland, Iceland and Svalbard, as well as from the Hudson Bay. For G. duebeni, we found no private American haplotypes, and post-glacial colonization from two founder populations is likely. A fixed genetic discontinuity was observed in both species between Iceland and Greenland on the one hand and Europe on the other hand. Our data hence indicate that the proposed postglacial colonization of America from Europe via the Faroe Islands, Iceland and Greenland could be more complex than previously thought and that parts of the North Atlantic region were not colonized from the Eastern Atlantic coasts. Unlike other studies, we speculate that there must be a Nordic glacial refugium for both species, which could be located in Greenland. The Greenlandic populations of both species exhibited private haplotypes, but signals of demographic expansion, conflicting with our theory. It is possible that the refugial character of the Greenlandic populations was erased by the LGM. Furthermore, we found evidence for glacial refugia along the Norwegian coast, the English Channel (G. duebeni) and on the Faroe Islands (G. oceanicus).

Phylogeny of the mud worm genus Marenzelleria (Polychaeta, Spionidae) inferred from mitochondrial DNA sequences

The taxonomy of the cryptic morphospecies of the mud worm genus Marenzelleria is particularly difficult and the phylogenetic relationship within the genus is unknown. Herein we reconstructed the phylogeny of all five species of this genus using sequence data of three mitochondrial genes (16SrDNA, cytochrome b, cytochrome oxidase subunit I) from 104 specimens out of 26 populations. For the three invasive species of the genus, Marenzelleria neglecta, M. viridis and M. arctia, individuals from native populations as well as from recently invaded populations were included. Nuclear 18S rDNA sequences were used to evaluate the appropriate outgroup taxon among several spionid polychaete species. The results supported the monophyly of Marenzelleria, and Malacoceros fuliginosus was found to be a suitable outgroup for the analysis of the mitochondrial gene segments. All phylogenetic reconstructions revealed a basal position of M. arctia and M. wireni, which have primarily Arctic distribution, with M. arctia obtaining the most basal position. Together with the present‐day distribution of the species, this indicates an origin of the genus in the Arctic region. The relationship of the species M. neglecta, M. viridis and M. bastropi could not be resolved sufficiently due to genealogical discordance that might reflect relatively young cladogenetic events.

Proteomic comparison of two invasive polychaete species and their naturally occurring F1-hybrids.

The mud worm genus Marenzelleria is highly invasive and is therefore studied intensively. In recently invaded habitats, sympatric populations of the sibling species Marenzelleria viridis and Marenzelleria neglecta are found. In these secondary contact zones, hybridization occurs frequently, revealing incomplete reproductive isolation between these recently diverged species. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometric methods were applied for a comparative analysis of these species and their F(1)-hybrids. Nineteen proteins were identified by cross-species identification strategies. A low degree of interindividual variability within either species allowed characterizing qualitative species-specific differences in 2-DE spot patterns as well as in peptide maps. Species-specific peptides were found in tryptic digests of various proteins, such as glyceraldehyde-3-phosphate dehydrogenase, troponin C, gelsolin-like protein, and peroxiredoxin-1. F(1)-hybrids of M. viridis and M. neglecta showed additivity of protein spot patterns, and the presence of both parental traits was confirmed by mass spectrometric data. This study is one of few dealing with global protein expression in polychaetes and is the first proteomic description of natural F(1)-hybrids in polychaetes. It furthermore indicates the feasibility of proteomic methods for analyses of speciation in Marenzelleria siblings as well as of hybridization events in secondary contact zones in general.