Variation in the <Emphasis Type="Italic">COI</Emphasis> gene of the freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> from River Vuokkijoki

The freshwater pearl mussel Margaritifera margaritifera L. is one of the most endangered freshwater mussels in the world. Effective conservation of threatened species requires not only ecological, but also genetic information from the target species and populations. Since low genetic diversity can reduce the ability of a species to adapt to environmental changes, maintaining genetic diversity has been identified as one of the key elements in successful conservation programs. We examined genetic variation of the freshwater pearl mussel from the River Vuokkijoki, Karelia, Russia. We sequenced a fragment of the cytochrome c oxidase subunit I gene (COI) from 22 individuals and compared the data to 32 previously published COI sequences available in GenBank. We identified 10 different COI haplotypes in the sequenced samples, three of which had not been previously reported. Our results show that the River Vuokkijoki has high genetic diversity and suggest that the colonization of this northern freshwater pearl mussel population might have occurred from multiple and even distant refugia. Therefore, the freshwater pearl mussel population of the River Vuokkijoki is valuable for the conservation of the whole species.     

The relationship between the freshwater pearl mussel (<Emphasis Type="Italic">Margaritifera margaritifera</Emphasis>) and its hosts

Endangered freshwater pearl mussels Margaritifera margaritifera reveal a complex life cycle with an obligate host-dependent phase. Only two species, Atlantic salmon Salmo salar and brown trout S. trutta, are important hosts in Europe, indicating a high degree of specialization. Whilst freshwater pearl mussels with their filtering activity can provide important ecosystem services and indirectly improve the habitat quality for their salmonid hosts, their direct effects on physiological stress, reduced swimming performance, and increased mortality at high rates of infestation all support a parasitic character of the mussel during its hostdependent phase. From an evolutionary perspective, both the much greater generation time of the parasite compared to the host, as well as the great distribution ranges of M. margaritifera and its hosts should favour local adaptation patterns. The variable suitability of different salmonid strains and species as hosts for M. margaritifera and the resulting differences in the performance of larvae during the host-dependent phase indicate that host-management strategies should focus on maintaining high quality hosts at a regional scale to avoid selection or genetic drift effects which could erode the genetic and evolutionary potential for adaptation to changing environmental conditions.     

Some biochemical parameters of the transformation of xenobiotics in the freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis>

We determined the concentration of glutathione (GSH) and the activity of glutathione S-transferase (GST) in the gills and hepatopancreas of the freshwater pearl mussel Margaritifera margaritifera L. It was found that the concentration of GSH in the gills decreases with a decrease in temperature, which indicates that metabolic processes in these ectothermic animals are decelerated. It was also found that changes in the concentration of GSH and in the activity of GST do not depend on age; thus, these biochemical markers can be used as bioindicators regardless of age. No substrate-specific activity of GST was observed, which means that freshwater pearl mussels have limited ability to metabolize compounds with different chemical structures.     

Regional monitoring of freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> in the County of Norrbotten,Sweden

A total of 16 freshwater pearl mussel Margaritifera margaritifera rivers out of 64 known freshwater pearl mussel rivers are included in the Administrative County of Norrbotten regional monitoring program. First surveys were done in 1994 and the results show that only three rivers have viable populations according to national criteria despite little visible human impact and that juvenile mussels (<50 mm in length) have been found in all rivers at every occasion. The results also indicate that there might be “missing years” for juvenile recruitment suggesting that freshwater pearl mussel populations at the extreme end of the species range might be dependent on “golden moments” in order to keep up viable populations. Deeper knowledge and better tools for determining viable populations might be necessary in order to make the right decisions on how to manage the rivers and the freshwater pearl mussel populations.     

The freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L.: Metamorphosis,growth, and development dynamics of encysted glochidia

The growth and morphogenesis of glochidia of the freshwater pearl mussel Margaritifera margaritifera on the gills of the Atlantic salmon Salmo salar in the Syuskyuyanyoki River (Karelia) are studied. Comparative analysis of histological features of glochidia depending on the age of the cyst is made, and the results of studies of the relationship and the influence of seasonal changes in water temperature on growth and morphogenesis of glochidia, which is essential for adaptation of living organisms and ensuring the sustainability of the participants of the parasite–host relationship, are presented.     

The populations of the freshwater pearl mussel, <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> (Linnaeus, 1758), and the thick shelled river mussel, <Emphasis Type="Italic">Unio crassus</Emphasis> Philipsson, 1788, in Latvia

The populations of two endangered species—the freshwater pearl mussel Margaritifera margaritifera and the thick shelled river mussel Unio crassus in Latvia were studied. The specimens were counted, measured, population density and age structure were calculated. The possible host fish presence was found.     

Strong genetic differentiation and low genetic diversity of the freshwater pearl mussel (<Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L.) in the southwestern European distribution range

Genetic diversity of European freshwater pearl mussel, Margaritifera margaritifera (L.), appears exceptional with highest genetic variability found in the northernmost European populations of Scandinavia and lower genetic variability in central and southern Europe. The objective of this study was to investigate genetic diversity and differentiation of 14 southernmost populations on the Iberian Peninsula which greatly differ in terms of life span and habitat conditions from the rest of central and northern European populations. The analyses of ten microsatellite loci revealed a pronounced level of genetic divergence and very low genetic diversity. These results match the expectations of geographically peripheral populations with respect to their genetic composition. The life history strategy, the narrow ecological niche of the species, and anthropogenic habitat modifications have most likely shaped the genetic pattern of Iberian pearl mussel populations. The peripheral position with less optimal habitat conditions may increase the extinction risk of these populations and thus effective conservation strategies for the Iberian M. margaritifera are needed. The successful conservation of the species at its southwestern margin requires inclusion of genetically different conservation units which may reveal local adaptation.     

Ontogenetic morphometrics of individual freshwater pearl mussels (<Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> (L.)) reconstructed from geometric conchology and trigonometric sclerochronology

This study bridges two conchological approaches to model the growth characteristics of freshwater pearl mussel shell: size-at-age and sclerochronology. We demonstrate a simple numerical model that transfers sclerochronological data into realistic estimates of ontogenetic shell sizing. This model was constructed for a subset of shell growth data dealing with morphometrics and annual shell growth increments. Further, validation of the model was performed using a dataset that was withheld from the calibration. Both subsets of data showed significant correlations between the observed (measured by vernier callipers) and reconstructed size-at-age data, indicating a successful model. The practical applicability of the model was exemplified for the studied Finnish freshwater pearl mussel populations. In accordance with the previously set theory about the plasticity of life history traits of the species, the southern mussels showed higher growth rates than the northern mussels. Handling editor: K. Martens     

Using genetic monitoring to inform best practice in a captive breeding programme: inbreeding and potential genetic rescue in the freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis>

Freshwater pearl mussel (Margaritifera margaritifera) populations are declining in Northern Ireland to the extent that a captive breeding programme was established on the Upper Ballinderry river in 1998. Previous genetic analysis of the hatchery broodstock and their first cohort of offspring showed significant levels of inbreeding (F _IS  = 0.166). The broodstock, which currently numbers ca. 90 individuals, was supplemented with new individual mussels, whilst in 2013, a previously unknown population was discovered on the Lower Ballinderry river. The aim of the present study was to determine whether the rotation of the broodstock has led to a decrease in the levels of inbreeding in the second cohort of juveniles, and to determine whether the new population found in the Lower Ballinderry was genetically distinct from the captive bred population and populations from the Upper Ballinderry, which represent the source of the hatchery broodstock. Genotyping using eight microsatellite markers indicated that levels of inbreeding in the second cohort of captive-bred mussels were high, (F _IS  = 0.629), and were comparable to those sampled from the original cohort and the hatchery broodstock (F _IS  = 0.527 and 0.636 respectively). Bayesian analysis of population structure indicated that the newly discovered Lower Ballinderry population was genetically distinct from the broodstock and its source populations on the Upper Ballinderry. The observed differentiation was primarily due to differences in allele frequencies, and was most likely a result of genetic drift. The occurrence of ten alleles, albeit at low frequency, in the Lower Ballinderry population, including four private alleles, suggests that this new population could be incorporated into the broodstock with the aim of decreasing levels of inbreeding in the future.     

Threatened freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L. in NW Spain: low and very structured genetic variation in southern peripheral populations assessed using microsatellite markers

A genetic analysis of freshwater pearl mussel Margaritifera margaritifera populations from NW Spain, a peripheral area of its European distribution, was carried out using microsatellite markers. These populations were formerly reported as genetically differentiated on the basis of growth and longevity studies. Ten loci previously characterized in populations from central Europe were used to comparatively analyze the genetic variability at the southern edge of the species’ range. Iberian pearl mussel populations showed very low genetic variability and significant high genetic differentiation. Half of the total genetic diversity observed appeared to be distributed between populations, which suggested a highly structured adaptive potential in pearl mussel at the southern peripheral distribution of the species. Population distinctiveness was evidenced by assignment tests, which revealed a high accuracy of individual assignments to their population of origin. All data suggested low effective population size and major effects of genetic drift on population genetic structure. In order to avoid further loss of genetic variation in biologically distinctive populations from NW Spain, prioritization of genetic resources of this species is required for conservation and management.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

The zebra mussel (<Emphasis Type="Italic">Dreissena polymorpha</Emphasis>) impacts European bitterling (<Emphasis Type="Italic">Rhodeus amarus</Emphasis>) load in a host freshwater mussel (<Emphasis Type="Italic">Unio pictorum</Emphasis>)

The European bitterling, Rhodeus amarus, is a non-indigenous fish species in British fresh waters. It lays its eggs in unionid mussels which themselves are vulnerable to fouling by the non-indigenous zebra mussel, Dreissena polymorpha. Observations from an unmanipulated natural system showed that only 27% of zebra mussel-fouled Unio pictorum hosted bitterling, while 47% of unfouled U. pictorum hosted bitterling. We conducted a field experiment in the River Great Ouse catchment, Cambridgeshire, England in May–June 2007 and 2008 to quantify the impact of zebra mussels on bitterling load in host mussels. Zebra mussel-fouled unionids were significantly less likely to host bitterling than unfouled unionids. The number of unionids hosting bitterling did not differ significantly whether the zebra mussels fouling the unionid were alive or dead. Bitterling appeared to discriminate against zebra mussel-fouled unionids less as the 2007 breeding season advanced, potentially because preferred unfouled unionids had a higher bitterling load, and were therefore relatively lower quality hosts than at the start of the breeding season.     

Strategies for the conservation of endangered freshwater pearl mussels (<Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L.): a synthesis of Conservation Genetics and Ecology

Freshwater pearl mussels (Margartifera margaritifera L.) are among the most critically threatened freshwater bivalves worldwide. The pearl mussel simultaneously fulfils criteria of indicator, flagship, keystone and umbrella species and can thus be considered an ideal target species for the process conservation of aquatic ecosystem functioning. The development of conservation strategies for freshwater pearl mussels and for other bivalve species faces many challenges, including the selection of priority populations for conservation and strategic decisions on habitat restoration and/or captive breeding. This article summarises the current information about the species’ systematics and phylogeny, its distribution and status as well as about its life history strategy and genetic population structure. Based on this information, integrative conservation strategies for freshwater mollusc species which combine genetic and ecological information are discussed. Holistic conservation strategies for pearl mussels require the integration of Conservation Genetics and Conservation Ecology actions at various spatial scales, from the individual and population level to global biodiversity conservation strategies. The availability of high resolution genetic markers for the species and the knowledge of the critical stages in the life cycle, particularly of the most sensitive post-parasitic phase, are important prerequisites for conservation. Effective adaptive conservation management also requires an evaluation of previous actions and management decisions. As with other freshwater bivalves, an integrative conservation approach that identifies and sustains ecological processes and evolutionary lineages is urgently needed to protect and manage freshwater pearl mussel diversity. Such research is important for the conservation of free-living populations, as well as for artificial culturing and breeding techniques, which have recently been or which are currently being established for freshwater pearl mussels in several countries.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Genomewide analysis of <Emphasis Type="Italic">ABCB</Emphasis>s with a focus on <Emphasis Type="Italic">ABCB1</Emphasis> and <Emphasis Type="Italic">ABCB19</Emphasis> in <Emphasis Type="Italic">Malus domestica</Emphasis>

The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon–intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots of M9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">KRAS</Emphasis>, <Emphasis Type="Italic">BRAF</Emphasis>, <Emphasis Type="Italic">EGFR</Emphasis> and <Emphasis Type="Italic">HER2</Emphasis> gene status in a Spanish population of colorectal cancer

To evaluate the KRAS, BRAF, EGFR, and HER2 gene status in colorectal cancer by novel techniques and evaluate whether anti-HER2 therapies could be offered in the treatment of these patients. There are conflicting data on the prevalence of BRAF mutations and EGFR and HER2 gene amplification in colorectal KRAS wild type patients. In our study we tried to evaluate these expressions and their relationship to future treatment assays. Clinical–pathological data and paraffin-embedded specimens were collected from 186 patients who underwent colorectal resections at General Yagüe Hospital in Burgos, Spain. KRAS and BRAF status was analyzed by real-time PCR in all patients. EGFR and HER2/NEU gene amplification was detected using fluorescent in situ hybridisation technique (FISH) in 38 KRAS and BRAF wild type patients. KRAS mutations were present in 48% of the colorectal cancer patients. BRAF mutations were present in 6.25% of the KRAS wild type patients. EGFR and HER2 gene amplification was observed in 5.3% and 26.3%, respectively, of KRAS and BRAF wild type colorectal cancer patients. HER2, but not EGFR gene amplification, was frequently observed in KRAS and BRAF wild type colorectal cancer patients. These data indicate that HER2 amplification could be one of the genes to be considered in the therapeutic management of colorectal cancer.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.