Polyploidy in a natural population of mussel, Mytilus trossulus.

We have analyzed natural polyploidy in a population of Mytilus trossulus from Vancouver Island (British Columbia, Canada) by means of cytogenetic techniques. Results obtained are the first reporting on this type of numerical chromosome aberrations in mussels.     

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca,Bivalvia)

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well‐organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5‐HT) immunocytochemical staining relative to the muscle ring differed significantly: 5‐HT‐immunioreactive cells were situated in the center of the striated muscle ring, while Phe‐Met‐Arg‐Phe‐NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co‐expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch‐contraction.     

Development of the muscle system and contractile activity in the mussel Mytilus trossulus (Mollusca, Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and florescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96 h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 microm in both mussle larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing videosystem. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult mollusks.     

Expression of several domains of twitchin and myorod in the ontogenesis of mussel Mytilus trossulus

The expression of MLCK- and PEVK-domains of twitchin, as well as the unique N-terminal domain of myorod in early development of the mussel Mytilus trossulus has been studied. The MLCK-domain of twitchin and the unique N-terminal domain of myorod appear at the early stages of development, whereas the PEVK-domain of twitchin is present only in muscles of adult mussel. The sizes of genes of the N-terminal domain of myorod, obtained at the blastula stage and from the adult animal are similar, but the proteins have significant differences in the amino acid sequences. Consequently, myorod and twitchin appear at early stages of larval mussels before the formation of "adult" muscles capable of catch contraction, and at these stages both proteins are isoforms, which differ from the isoforms of adult animals. It is possible that the MLCK-domain in the "larval" isoform of twitchin is necessary for regulating the formation of the contractile apparatus of molluscan smooth muscles, while the PEVK-domain is important for the regulation of the catch state in muscles of adult animals.     

A connective tissue tumor in the mussel Mytilus trossulus from a polluted region of Nakhodka Bay, the Sea of Japan

A tumor was found for the first time in a mussel Mytilus trossulus from a heavily polluted area of Nakhodka Bay, Sea of Japan. Tumor cells were found in the connective tissue of different organs and also in gill vessels and hemal sinuses of the visceral mass. They were both attached and diffuse. The tumor was at an advanced stage, replacing the normal connective tissue cells, and formed nodes. The tumor cells were polymorphic, with a high nucleocytoplasmic ratio, and had a prominent nucleolus. The size of their nuclei was three to five times that of the nuclei of agranular hemocytes. The mitotic activity of the tumor cells was more than an order of magnitude higher than in the normal cells: the mean mitotic index was 1.4 +/- 0.5%, ranging from 0.97 to 2.3% in different organs. The mitotic indices in the connective tissue cells of three normal mussels were 0, 0, and 0.12%. A significant proportion (up to 78%) of the mitotic cells were at metaphase. The frequency of abnormal mitoses was 17%. Metaphases with displaced (often multiple) chromosomes constituted 71% of abnormal mitoses; anaphases, 8%; and tri- and tetrapolar mitoses, 11%. The tumor described is similar to diffuse sarcomatoid diseases of mussels from other geographical regions.     

Nonhomologous Recombination Between the Large Unassigned Region of the Male and Female Mitochondrial Genomes in the Mussel, Mytilus trossulus

Doubly uniparental inheritance of mtDNA (DUI) is commonly observed in several genera of bivalves. Under DUI, female offspring inherit mtDNA from their mothers, while male offspring inherit mtDNA from both parents but preferentially transmit the paternally inherited mtDNA to their sons. Several studies have shown that the female- and male-specific mtDNA lineages in blue mussels, Mytilus spp., vary by upward of 20% at the nucleotide level. In addition to high levels of nucleotide substitution, the present study observed substantial gender-based length polymorphism in the presumptive mitochondrial control region (=large unassigned region; LUR) of North American M. trossulus. In this species, female lineage LUR haplotypes are over 2 kb larger than male lineage LUR haplotypes. Analysis of sequence data for these length variants indicates that the F LUR haplotypes of North American M. trossulus contain sequences similar to the F lineage control region in the congeners M. edulis and M. galloprovincialis. Relative to the F LUR in the latter two species, however, the F lineage LUR haplotypes in M. trossulus contain two large sequence insertions, each nearly 1 kb in size. One of these insertions has high sequence similarity to the male lineage LUR of M. trossulus. The tandem arrangement of F and M control region sequences in the F lineage LUR of M. trossulus is most likely the result of nonhomologous recombination between the male and the female mitochondrial genomes in M. trossulus, a finding that has important implications regarding the transmission and evolution of blue mussel mitochondrial genomes. [Reviewing Editor: Dr. Martin Kreitman]     

Within- and between-patch variability of predation intensity on the mussel Mytilus trossulus Gould on a rocky intertidal shore in Oregon, USA

Variability of predation intensity is an important cause of spatial differences of community structure and organization in the intertidal rocky shore. Field experiments were conducted to evaluate the within- and between-patch variability of the effects of different types of predators (small invertebrates and birds) on Mytilus trossulus Gould, which occupies an intermediate position in the competitive hierarchy among sessile organisms in disturbance patches within a California mussel (Mytilus californianus Conrad) bed community on the central Oregon Coast. Predation by birds did not significantly affect the mortality of M. trossulus. On the contrary, predation by small invertebrate whelks (Nucella spp.) had a significant effect on M. trossulus mortality. Predation by whelks also caused between- and within-patch variability of mortality of M. trossulus. Within patches, M. trossulus mortality at patch margins was significantly higher than at patch centers only when invertebrate predators were present. Wave exposure did not cause between-patch variability of predation intensity. Between-and within-patch variability of predation intensity may be caused by the variability of supply of whelks from the surrounding mussel mat. The movement of predators between patches and surrounding matrices may play an important role in the patch dynamics of these communities.     

Adaptivity of the bivalve Mytilus trossulus larvae to short-and long-term changes in water temperature and salinity

Adaptivity to short-term and long-term changes in water temperature and salinity was studied in larvae of the bivalve mollusk Mytilus trossulus. It was shown that water temperature of 4°C mostly suppressed growth and development of larvae. A temperature of 20°C promoted an enhanced larval growth and development. Though a temperature of 20°C caused enhanced larval growth, the temperature was not optimal, while its effect caused quality diversity of larval development, owing to the difference in their growth rates. Such diversity was not observed at moderate temperatures of 10 and 15°C. At 20°C, fast-growing mussel larvae were very sensitive to temperature drops. Growth of slowly-growing individuals did not depend on temperature in the range of 10 to 20°C. Daily temperature variations by 3–8°C did not markedly affect growth and development of the larvae. A continuous 24-h exposure to temperature drops by 3–8°C did not influence these very important physiological characteristics either. A salinity drop down to 8‰ exerted an adverse effect only on early larvae. Later on, the larvae showed their ability to adapt to such a strong desalination. The negative effect of reduced salinity (to 8‰) upon mussel larvae was increased at a temperature increase to 20°C.     

The relative sensitivity of sperm, eggs and embryos to copper in the blue mussel (Mytilus trossulus)

Copper, an essential element, is toxic at elevated concentrations, and as a result of anthropogenic activities is becoming increasingly prevalent in marine environments. In this study, we examined the effects of copper on early life stages of the blue mussel, Mytilus trossulus. We assessed the impacts of increasing copper concentrations on embryo development, egg viability, sperm fertilization capacity and, in particular, on sperm swimming speed using computer-assisted sperm analysis. Sensitivity to copper followed the pattern: embryos > sperm > eggs. A dramatic increase in abnormal embryo development was observed following exposure to copper concentrations exceeding 10 microg/L. Sperm swimming speeds decreased significantly when exposed to 100 microg/L of copper, but lower doses did not influence sperm swimming speed. Copper exposure (at any tested concentration) did not affect sperm flagellum length, or alter egg viability. Based on our results, we suggest that exposure of sperm to copper may interfere with mitochondrial activity, which reduces sperm swimming speed during the extended duration of sperm motility in blue mussel.     

放牧影响下不同盖度金露梅灌丛草本植物叶功能性状与土壤因子的关系

植物功能性状与环境因子的研究对认识不同环境梯度下植物群落的形成及对环境的适应机制具有重要意义。该研究以高寒金露梅(Potentilla fruticosa)灌丛草甸为研究对象,分析放牧影响下金露梅灌丛不同盖度梯度(金露梅灌丛盖度为0%、30%、60%和90%的灌草斑块)灌草斑块中草本植物叶功能性状与土壤因子的变化趋势及其相互关系。结果表明:(1)土壤容重(BD)、土壤pH和土壤养分在金露梅灌丛盖度梯度下差异显著,干柴滩金露梅灌丛具有明显的"沃岛效应";随着金露梅灌丛盖度的递增,土壤理化性质变化趋势各有分异。(2)随着金露梅灌丛盖度的增加,比叶面积(SLA)显著上升,叶片厚度(LT)和叶片磷含量(LPC)呈显著下降趋势(P0.05)。(3)比叶面积与叶片厚度呈负相关关系,与叶片氮磷含量(LNC、LPC)呈极显著负相关关系(P0.01),与叶片碳含量(LCC)呈极显著正相关关系(P0.01)。(4)RDA冗余分析表明,土壤因子对叶功能性状变异的平均解释变量达72.25%,影响植物功能性状的主导因子有土壤有机质(SOM)、土壤全氮(TN)、土壤有效氮(AN)、土壤有效钾(AK)和土壤容重(BD),但不同盖度金露梅灌丛群落中影响叶性状的主导因子存在差异。金露梅灌丛盖度较小(30%)时受SOM和TN、AN的影响较大,而金露梅灌丛盖度较大时(60%)时主要受AK和BD的影响。     

The Byssal Apparatus in the Pacific Mussel,Mytilus trossulus (Bivalvia,Mytilidae), from the Sea of Japan

Biology Bulletin - In the commercial Pacific mussel species, Mytilus trossulus, from the Sea of Japan, features of the morphological structure of the byssal apparatus, byssal threads, and pedal...     

Mytilus trossulus hsp70 as a biomarker for arsenic exposure in the marine environment: Laboratory and real-world results

Acute renal papillary necrosis (RPN) in animals is characterized by increased renal lipid accumulation. The excretion of renal lipids into urine has been determined to evaluate their possible use as sensitive early biomarkers for the diagnosis of RPN. This study investigates injury induced by two model nephrotoxins, mefenamic acid (MFA), a non-steroidal anti-inflammatory drug (NSAID), and its analogue N-phenylanthranilic acid (NPAA). Oral NPAA was given repeatedly at doses of 100, 250 and 500 mg kg^-1 daily for 5 days, followed by a 2 day respite over the weekend, and then four further daily doses. The same dosing procedure was used with MFA, but at doses of 75, 150 and 300 mgkg^-1. The control groups were given vehicle orally using the same volume given to the test groups. Urinary phospholipids (PLs), notably sphingomyelin (SPM), phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were measured and compared with other urinary parameters. Histopathological investigations were also performed to confirm the presence or absence of RPN. Following MFA treatment, PC, PI and PE were raised significantly (p < 0.001) on days 1 and 3 and for the remaining part of the experiment. After NPAA treatment, PI showed a transient elevation, and PC and PE levels were significantly increased from day 2 onwards. Both drugs caused a dose-related increase in PLs. There was no significant increase in the level of other urinary parameters. However, histopathological examination of the kidney on day 11 revealed lesions in the medulla and papilla following treatment with the two papillotoxins. These findings demonstrate the potential of urinary PLs as diagnostic non-invasive biomarkers for early renal injury associated with RPN, which may provide an important improvement in the approach to the therapeutic management of analgesic nephropathy.     

Quantitative analysis of sperm plane circular movement in the blue mussels Mytilus edulis, M. trossulus and their hybrids

Fertilization success of free spawning organisms such as Mytilus species depends on gamete interactions. Therefore, gamete traits such as sperm movement are important for determining fertilization success in free spawning organisms. Since little is known about sperm movement pattern in Mytilus species, the purpose of this study was to investigate sperm movement pattern of blue mussel M. edulis, M. trossulus and their hybrids using computer-assisted sperm movement video analysis. Sperm of all genotypes were found to conduct circular movement in a two-dimensional plane. Furthermore, new sperm movement parameters, real time radius (R), angle change rate (θ) and the center of circular track (O(t)) were developed to verify and quantitatively describe the plane circular movement pattern using software (Image-J) that may be widely applied to sperm movement study in other organisms. Angle change rate was positively correlated to fertilization success. However, no correlation between fertilization and real time radius was detected. Although no interspecific differences were found in the radius, the F1 (first generation) hybrid sperm had a lower angle change rate than M. edulis and M. trossulus. Published studies have shown that sperm circular movement is more prevalent in aquatic broadcast spawning species than in species with mating behavior or internal fertilization. Therefore, a two-dimensional circular movement pattern in sperm may represent a trait that increases fertilization success for broadcast spawning species by either increasing gamete interaction rate at a small scale and/or avoiding swimming further away from the eggs before sperm detects the chemoattractant gradient.     

周细胞覆盖率与胃癌侵袭相关性的分析

目的:探讨周细胞在胃癌血管周围的覆盖率是否可以作为判断胃癌预后的一个指标。方法:利用免疫组织化学双染的方法同时标记内皮细胞(抗CD34抗体)和周细胞(抗α-SMA抗体),检测48例胃癌石蜡标本当中微血管壁周细胞覆盖率(microvessel pericyte coverage index,MPI),应用SPSS11.5统计软件分析MPI与胃癌患者性别、年龄、肿瘤大小、浸润深度、淋巴结转移、临床分期、以及脉管内是否有癌栓等临床病理参数之间的关系。结果:MPI与胃癌的分化程度呈正相关(P=0.002),与脉管内是否有癌栓呈负相关(P=0.002),而与患者年龄、性别、肿瘤大小、浸润深度、临床分期、淋巴结转移等无关(P均0.05)。结论:利用免疫组织化学双染的方法可以清晰标记胃癌的微血管结构,血管的不成熟即周细胞覆盖率低成为胃癌患者预后差的重要因素。     

Study of two species of mussels, Mytilus trossulus and Mytilus galloprovincialis (Bivalvia, Mytilidae) and their hybrids, using PCR markers, in Peter the Great Bay of the Sea of Japan]

Bivalve mollusks of the genus Mytilus (M. trossulus and M. galloprovincialis) occurring in Peter the Great Bay of the Sea of Japan were first studied in Russia. A region of nonrepetitive sequences of the gene encoding the polyphenolic adhesive protein bissus was used as a species-specific genetic marker. After amplification using specific primers, a 126-bp fragment was found to amplify in all representatives of M. galloprovincialis collected from driftwood in the gulf Posset (the southwestern part of Peter the Great Bay). M. trossulus specimens from the same region were shown to have a 168-bp fragment. In Vostok Gulf (the eastern part of Peter the Great Bay), both artificially grown mussels and those from natural habitats contained a 168-bp fragment or two fragments (126- and 168-bp) that corresponded to a hybrid form between the above species. The possibility of using this genetic marker to identify closely related Mytilus strains and their hybrids in similar habitats, near the Primorye coast in particular, was demonstrated. The presence of approximately 9% of hybrid specimens confirms that a zone of hybridization between M. trossulus and M. galloprovincialis may exist in this region.     

Mytilus trossulus hsp70 as a biomarker for arsenic exposure in the marine environment: laboratory and real-world results.

The highly conserved heat shock protein 70 (hsp70) is induced by heat and chemical toxins, particularly heavy metals such as arsenic (As). The use of Mytilus trossulus (bay mussel) hsp70 as a 'screening' biomarker for marine heavy metals contamination was assessed. Some studies have found high hsp70 sensitivity to heavy metals, while others have found the opposite. Few studies have realistically used low heavy metals exposures, and fewer have used real-world contamination exposures. Clean sub-tidal mussels from the Puget Sound, Washington State (WA), USA, were acclimatized for 2 weeks and exposed for 24 h to As-spiked seawater (n=9) or to contaminated seawater from an arsenical pesticide plant in Tacoma, WA (n=10) followed by a Western blot for hsp70. Hsp70 inductions were insignificant at 10 microg l(-1) As(III), but were strong at 100 microg l(-1) (p<0.05) and 1000 microg l(-1) (p<0.01), with the induction threshold estimated at 30-50 microg l(-1) As(III). Hsp70 induction roughly correlated with arsenical toxicity, with As(III) > As(V) > (CH(3))(2)As(V). Altogether, the inter-individual variability of hsp70 levels tends to mask inductions at low As concentrations, making it a crude toxicity biomarker. In addressing this problem, the following options could prove promising: (1) pre- or post-stressing specimens for greater hsp70 sensitivity, (2) use of internal protein controls such as actin, (3) use of hsp70-reporter gene constructs, and (4) detection with hsp60, heme oxygenase-1, metallothionein, CYP450, MXR or GPx.     

Speciation and Distribution of Arsenic in the Nonhyperaccumulator Macrophyte Ceratophyllum demersum

Although arsenic (As) is a common pollutant worldwide, many questions about As metabolism in nonhyperaccumulator plants remain. Concentration- and tissue-dependent speciation and distribution of As was analyzed in the aquatic plant Ceratophyllum demersum to understand As metabolism in nonhyperaccumulator plants. Speciation was analyzed chromatographically (high-performance liquid chromatography-[inductively coupled plasma-mass spectrometry]-[electrospray ionization-mass spectrometry]) in whole-plant extracts and by tissue-resolution confocal x-ray absorption near-edge spectroscopy in intact shock-frozen hydrated leaves, which were also used for analyzing cellular element distribution through x-ray fluorescence. Chromatography revealed up to 20 As-containing species binding more than 60% of accumulated As. Of these, eight were identified as thiol-bound (phytochelatins [PCs], glutathione, and cysteine) species, including three newly identified complexes: Cys-As(III)-PC₂, Cys-As-(GS)₂, and GS-As(III)-desgly-PC₂. Confocal x-ray absorption near-edge spectroscopy showed arsenate, arsenite, As-(GS)₃, and As-PCs with varying ratios in various tissues. The epidermis of mature leaves contained the highest proportion of thiol (mostly PC)-bound As, while in younger leaves, a lower proportion of As was thiol bound. At higher As concentrations, the percentage of unbound arsenite increased in the vein and mesophyll of young mature leaves. At the same time, x-ray fluorescence showed an increase of total As in the vein and mesophyll but not in the epidermis of young mature leaves, while this was reversed for zinc distribution. Thus, As toxicity was correlated with a change in As distribution pattern and As species rather than a general increase in many tissues.Arsenic (As) is ubiquitously present, considered a nonessential metalloid for plants and animals, and poses serious health hazards to humans. High levels of As in soil and drinking water have been reported around the world, with the worst situations in south and southeast Asia, where millions of people are at risk of As poisoning through drinking water and food (Chowdhury et al., 2000; Smedley and Kinniburgh, 2002; Ohno et al., 2007). Recently, As induced yield loss; thus, a threat to the sustainability of food production has been recognized as the other side of the As calamity (Brammer and Ravenscroft, 2009; Panaullah et al., 2009). Considering the enormity of As contamination, phytoremediation or the development of crops that can be grown in contaminated environments without suffering from and accumulating As in edible parts seem to be the most appropriate strategies to counter the detrimental impacts of As. These strategies demand an understanding of the mechanistic details of As uptake, toxicity, and detoxification (Tripathi et al., 2007). The in planta distribution and speciation of As are important aspects in this direction.Inorganic arsenate [HAsO₄²⁻ or As(V)] and arsenite [H₂AsO₃⁻ or As(III)] are the most common forms of As in aquatic and terrestrial environments. Plants take up As(V) through phosphate transporters (Asher and Reay, 1979; Meharg and Macnair, 1990) and As(III) through nodulin26-like intrinsic aquaporins (Isayenkov and Maathuis, 2008; Ma et al., 2008). Inside the cell, As(V) is readily reduced to As(III) through As(V) reductase using reduced glutathione (GSH) as reductant (Duan et al., 2005; Bleeker et al., 2006). As(III) then gets complexed with thiol ligands via GSH and phytochelatins (PCs; Schmöger et al., 2000; Raab et al., 2004). Thus, complexation of As with PCs followed by sequestration of the complex in vacuoles has been suggested as the major mechanism of As detoxification in As-nonhyperaccumulator plants (Bleeker et al., 2006; Song et al., 2010). In contrast, in hyperaccumulator plants, PCs contribute only little (1%–3%) to As complexation (Zhao et al., 2003).An increased synthesis of PCs as well as GSH under As stress has been observed in hypertolerant (Hartley-Whitaker et al., 2001; Bleeker et al., 2003), hyperaccumulator (Zhao et al., 2003; Cai et al., 2004), as well as nonhyperaccumulator (Srivastava et al., 2007; Mishra et al., 2008, 2013) plants. However, the existence of As-PC complexation was concluded only indirectly through individual analysis of As and PCs in fractions after chromatographic separation (Sneller et al., 1999; Schmöger et al., 2000). Raab et al. (2004) for the first time showed complexes of PCs in plant extracts by using HPLC simultaneously coupled with element-specific (inductively coupled plasma-mass spectrometry [ICP-MS]) and molecule-specific (electrospray ionization-mass spectrometry [ESI-MS]) detectors. This method provides information about the diversity of ligands and As species present in plants. However, artifacts during sample preparation, such as ligand exchange for previously weakly bound metal(loids) due to the breakage of cells and subcellular compartments during the extraction of plants, cannot be excluded. In this respect, x-ray absorption spectroscopy of frozen-hydrated tissues has been proven to be a unique technique for the in situ investigation of chemical forms of metal(loids) in biological materials without much prepreparation of samples, thus minimizing the artifacts of sample preparation (Salt et al., 1995; Küpper et al., 2004). X-ray absorption near-edge spectroscopy (XANES) provides speciation of As in tissues typically by fingerprint-like comparison (if quantitative, linear combination fitting) with spectra of appropriate model compounds as standards (Pickering et al., 2000, 2006; Lombi et al., 2002, 2009; Meharg et al., 2008). Furthermore, high-resolution microscopic (µ) x-ray spectroscopy may reveal spatial distribution and speciation of As in intact biological samples (Hokura et al., 2006; Carey et al., 2010, 2011; Kopittke et al., 2012). However, species having almost identical absorption spectra cannot be distinguished by this technique, the use of inappropriate standards for fitting of experimental spectra may lead to misinterpretation, and quantification of minor contributions is always inaccurate in linear combination fitting. Studies regarding the distribution of As have been mostly done in the hyperaccumulator Pteris vittata, showing that the majority of As was accumulated in the pinnae, possibly in vacuoles (Lombi et al., 2002; Hokura et al., 2006; Pickering et al., 2006). In rice (Oryza sativa) grains, it was done with high-resolution techniques such as synchrotron-based microscopic x-ray fluorescence (µ-XRF) and secondary ion mass spectrometry by using either fractured or sectioned dry rice grains (Meharg et al., 2008; Lombi et al., 2009; Moore et al., 2010) or whole fresh rice grains (Carey et al., 2010, 2011). However, little information is available regarding the cellular or subcellular distribution of As in nonhyperaccumulator plants. Moore et al. (2011) investigated the subcellular distribution of As and silicon in rice roots through nanosecondary ion mass spectrometry. Recently, Kopittke et al. (2012) studied the spatial distribution of As in hydrated, fresh roots of cowpea (Vigna unguiculata) using µ-XRF including sequential computed tomography and found differences in As distribution in the plants exposed to As(V) or As(III). However, information about As speciation and distribution in leaf tissues and its relation to toxicity in nonhyperaccumulator plants is still lacking.In this study, we analyzed the speciation (through two complementary techniques) and distribution of As in leaves of the nonhyperaccumulator submerged aquatic plant Ceratophyllum demersum in environmentally relevant conditions. The accumulation and speciation of As were investigated in a concentration-dependent manner at the whole-plant level in fresh extracts through HPLC-(ICP-MS)-(ESI-MS). The in situ speciation at the differential tissue level in the leaf was performed by combining high-resolution µ-XANES and confocal optics on the detector side. Using µ-XRF with full quantification including the correction of x-ray absorption in thick samples, we furthermore investigated changes in the distribution pattern of As, copper (Cu), and zinc (Zn) under sublethal to lethal As exposure in the leaves of C. demersum. To our best knowledge, this is the first report of As speciation in different tissues through confocal µ-XANES and of As distribution in leaves of an As nonhyperaccumulator plant. We also report direct evidence of in situ As-PC complex formation by using µ-XANES spectra of As-(PC₂)₂ and As-PC₃ along with As-(GS)₃ standards and carefully correcting for self-absorption artifacts that distorted µ-x-ray absorption spectroscopy spectra in earlier studies. Since C. demersum has no roots, it takes up all nutrients directly via the leaves, allowing for studies of shoot effects without interference from root uptake and root toxicity. While it is not a crop species, it is still a flowering plant (belonging to the dicotyledons) and has been accepted as a good model for laboratory studies of shoot toxicity in higher plants (Xue et al., 2012). Mechanisms of metal toxicity found in C. demersum are similar to other plants (Küpper et al., 1996), and like other nonaccumulator plants including crops, it detoxifies As by PCs (Mishra et al., 2008). Furthermore, it is widespread in Asia, Europe, and North America, and it has been shown to be good for removing metal from low-concentration waste water (Keskinkan et al., 2004). Additionally, its has also been used successfully in tests of biological life support systems on space flights (Blüm et al., 1994; and many articles about the Aquarack/CEBAS (for Closed Equilibrated Biological Aquatic System) and OMEGAHAB systems). Finally, the structure of C. demersum leaves (round, less than 1 mm diameter) makes this species a suitable model for µ-XRF and µ-XANES measurement. It facilitates the recording of whole-leaf tomograms and tissue-specific µ-XANES without any disturbance such as mixing of intercellular or intracellular fluid while sectioning of the sample, thus avoiding artifacts during sample preparation.     

Revisiting Australian Ectocarpus subulatus (Phaeophyceae) From the Hopkins River: Distribution,Abiotic Environment,and Associated Microbiota

In 1995 a strain of Ectocarpus was isolated from Hopkins River Falls, Victoria, Australia, constituting one of few available freshwater or nearly freshwater brown algae, and the only one belonging to the genus Ectocarpus. It has since been used as a model to study acclimation and adaptation to low salinities and the role of its microbiota in these processes. To provide more background information on this model, we assessed if Ectocarpus was still present in the Hopkins river 22 years after the original finding, estimated its present distribution, described its abiotic environment, and determined its in situ microbial composition. We sampled for Ectocarpus at 15 sites along the Hopkins River as well as 10 neighboring sites and found individuals with ITS and cox1 sequences identical to the original isolate at three sites upstream of Hopkins River Falls. The salinity of the water at these sites ranged from 3.1 to 6.9, and it was rich in sulfate (1–5 mM). The diversity of bacteria associated with the algae in situ (1312 operational taxonomic units) was one order of magnitude higher than in previous studies of the original laboratory culture, and 95 alga-associated bacterial strains were isolated from algal filaments on site. In particular, species of Planctomycetes were abundant in situ but rare in laboratory cultures. Our results confirmed that Ectocarpus was still present in the Hopkins River, and the newly isolated algal and bacterial strains offer new possibilities to study the adaptation of Ectocarpus to low salinity and its interactions with its microbiome.     

Comparative Study of Microbial Communities from Cultured and Natural Populations of the Mussel Mytilus trossulus in Peter the Great Bay

The 525 strains of heterotrophic bacteria isolated from natural and cultured populations of the mussel Mytilus trossulus and the surrounding seawater were identified to a genus level on the basis of phenotypic analysis and the fatty acid composition of cell lipids. Gram-negative isolates were dominated by six genera of the family Enterobacteriaceae and by the genera Pseudoalteromonas, Vibrio, Photobacterium, Cytophaga/Flavobacterium/Bacteroides, Pseudomonas, and Moraxella. Gram-positive isolates were mainly represented by the genus Streptomyces. The taxonomic compositions of natural and cultured populations of the mussel M. trossulus in Peter the Great Bay were similar.