The Effects of Long-term Molybdenum Exposure in Drinking Water on Molybdenum Metabolism and Production Performance of Beef Cattle Consuming a High Forage Diet.

Fifty-four multiparous beef cows with calves were used to evaluate the effects of Mo source (feed or water) on reproduction, mineral status, and performance over two cow-calf production cycles (553 days). Cows were stratified by age, body weight, liver Cu, and Mo status and were then randomly assigned to one of six treatment groups. Treatments were (1) negative control (NC; basal diet with no supplemental Mo or Cu), (2) positive control (NC + Cu; 3 mg of supplemental Cu/kg DM), (3) NC + 500 µg Mo/L from Na2MoO4·2H2O supplied in drinking water, (4) NC + 1000 µg Mo/L of Na2MoO4·2H2O supplied in drinking water, (5) NC + Mo 1000-water + 3 mg of supplemental Cu/kg DM, and (6) NC + 3.0 mg of supplemental Mo/kg diet DM from Na2MoO4·2H2O. Animals were allowed ad libitum access to both harvested grass hay (DM basis: 6.6% crude protein; 0.15% S, 6.7 mg Cu/kg, 2.4 mg Mo/kg) and water throughout the experiment. Calves were weaned at approximately 6 months of age each year. Dietary Cu concentration below 10.0 mg Cu/kg DM total diet reduced liver and plasma Cu concentrations to values indicative of a marginal Cu deficiency in beef cows. However, no production parameters measured in this experiment were affected by treatment. Results suggest that Mo supplemented in water or feed at the concentrations used in this experiment had minimal impact on Cu status and overall performance.

Impact of Cell Culture and Copper Dose on Gene Expression in Bovine Liver.

The objectives of these experiments were to investigate (1) the relative abundance of transcripts for Cu-responsive genes in whole bovine liver vs. cultured hepatocytes and (2) the influence of Cu dose on the relative abundance of transcripts for Cu-responsive genes in cultured bovine hepatocytes. Experiment 1: Liver samples were obtained immediately post-mortem from one healthy Angus steer. Half of the tissue samples were placed in RNAlater solution; the remaining half was used to isolate hepatocytes. Experiment 2: A subset of cultured hepatocytes was incubated in media containing: 0 mg/L, 0.10 mg/L, 1.0 mg/L, 10.0 mg/L, and 100 mg/L Cu for 1 h. Transcripts analyzed were aldehyde dehydrogenase (ALDH2), apolipoprotein A-1 (APOA1), antioxidant 1 (ATOX1), ATPase copper transporting alpha (ATP7A), ATPase copper transporting beta (ATP7B), betaine homocysteine methyltransferase (BHMT), flavin reductase (BLVRB), carbonic anhydrase II (CA2), copper chaperone for superoxide dismutase (CCS), cytochrome c oxidase copper chaperone (COX17), Cu transporter 1 (CTR1), glutamate dehydrogenase (GLUD1), glutathione synthetase (GSS), protein disulfide isomerase A3 (PDIA3), and superoxide dismutase (Cu-Zn) (SOD1). Β-Actin (ACTB) was selected as the endogenous control in both experiments. Experiment 1: Whole liver had greater (P < 0.01) relative abundance of mRNA for APOA1, ATOX1, ATP7A, ATP7B, COX17, CTR1, ALDH2, BHMT, BLVRB, CA2, GLUD1, and GSS when compared with cultured hepatocytes. Experiment 2: Copper dose impacted all identified transcripts. These results indicate that the relative abundance of Cu-responsive transcripts is different in whole vs. cultured hepatocytes and that the relative abundance of Cu-responsive genes is dependent on Cu dose in cultured hepatocytes.

Molybdenum Exposure in Drinking Water Vs Feed Impacts Apparent Absorption of Copper Differently in Beef Cattle Consuming a High-Forage Diet.

Twelve Angus steers were utilized to investigate the influence of molybdenum (Mo) in drinking water or feed on apparent absorption and retention of copper (Cu) and Mo. Steers were fed a low-quality grass hay diet for 14 days. Steers were then housed in individual metabolism stalls and blocked by body weight and dry matter intake (DMI) and randomly assigned within block to one of three treatments. Treatments consisted of (1) control (no supplemental Mo), (2) 5.0 mg Mo/kg DM from sodium molybdate dihydrate (Mo-diet), and (3) 1.5 mg Mo/L from sodium molybdate dihydrate delivered in the drinking water (Mo-water). Total fecal and urine output were then collected for 5 days. Dry matter, Cu and water intake, and DM digestibility were similar across treatments. Molybdenum intake was greater (P < 0.05) in Mo-water and Mo-diet steers when compared to controls but similar between Mo-water and Mo-diet steers. Apparent absorption and retention of Cu were greater (P < 0.05) in controls when compared to Mo-diet supplemented steers. Apparent absorption and retention of Cu in steers in the Mo-water treatment did not differ from controls or those receiving the Mo-diet. Molybdenum-diet and Mo-water supplemented steers had similar apparent absorption and retention of Cu. Apparent absorption and retention of Mo (% of Mo intake) was greater in controls when compared to Mo-supplemented steers. These data indicate that Mo consumed in water may impact Cu absorption and retention to a lesser extent than Mo supplemented in the diet.

CO2 induced seawater acidification impacts sea urchin larval development II: gene expression patterns in pluteus larvae.

Extensive use of fossil fuels is leading to increasing CO(2) concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO(2). As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO(2) 41 Pa e.g. 399 µatm) and CO(2) acidified seawater with pH of 7.7 (pCO(2) 134 Pa e.g. 1318 µatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10% reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (two-way ANOSIM: Global R=1) while acidification effects were less pronounced (Global R=0.518). Significant differences in gene expression patterns (ANOSIM R=0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO(2) treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO(2) effect. We found an up regulation of metabolic genes (between 10%and 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23% and 36% in msp130, SM30B, and SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na(+)/K(+)-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.

CO2 induced seawater acidification impacts sea urchin larval development I: elevated metabolic rates decrease scope for growth and induce developmental delay.

Anthropogenic CO(2) emissions are acidifying the world's oceans. A growing body of evidence is showing that ocean acidification impacts growth and developmental rates of marine invertebrates. Here we test the impact of elevated seawater pCO(2) (129 Pa, 1271 µatm) on early development, larval metabolic and feeding rates in a marine model organism, the sea urchin Strongylocentrotus purpuratus. Growth and development was assessed by measuring total body length, body rod length, postoral rod length and posterolateral rod length. Comparing these parameters between treatments suggests that larvae suffer from a developmental delay (by ca. 8%) rather than from the previously postulated reductions in size at comparable developmental stages. Further, we found maximum increases in respiration rates of +100% under elevated pCO(2), while body length corrected feeding rates did not differ between larvae from both treatments. Calculating scope for growth illustrates that larvae raised under high pCO(2) spent an average of 39 to 45% of the available energy for somatic growth, while control larvae could allocate between 78 and 80% of the available energy into growth processes. Our results highlight the importance of defining a standard frame of reference when comparing a given parameter between treatments, as observed differences can be easily due to comparison of different larval ages with their specific set of biological characters.

Impact of near-future ocean acidification on echinoderms.

As a consequence of increasing atmospheric CO(2), the world's oceans are warming and slowly becoming more acidic (ocean acidification, OA) and profound changes in marine ecosystems are certain. Calcification is one of the primary targets for studies of the impact of CO(2)-driven climate change in the oceans and one of the key marine groups most likely to be impacted by predicted climate change events are the echinoderms. Echinoderms are a vital component of the marine environment with representatives in virtually every ecosystem, where they are often keystone ecosystem engineers. This paper reviews and analyses what is known about the impact of near-future ocean acidification on echinoderms. A global analysis of the literature reveals that echinoderms are surprisingly robust to OA and that important differences in sensitivity to OA are observed between populations and species. However, this is modulated by parameters such as (1) exposure time with rare longer term experiments revealing negative impacts that are hidden in short or midterm ones; (2) bottlenecks in physiological processes and life-cycle such as stage-specific developmental phenomena that may drive the whole species responses; (3) ecological feedback transforming small scale sub lethal effects into important negative effects on fitness. We hypothesize that populations/species naturally exposed to variable environmental pH conditions may be pre-adapted to future OA highlighting the importance to understand and monitor environmental variations in order to be able to to predict sensitivity to future climate changes. More stress ecology research is needed at the frontier between ecotoxicology and ecology, going beyond standardized tests using model species in order to address multiple water quality factors (e.g. pH, temperature, toxicants) and organism health. However, available data allow us to conclude that near-future OA will have negative impact on echinoderm taxa with likely significant consequences at the ecosystem level.

A genomic view of the sea urchin nervous system.

The sequencing of the Strongylocentrotus purpuratus genome provides a unique opportunity to investigate the function and evolution of neural genes. The neurobiology of sea urchins is of particular interest because they have a close phylogenetic relationship with chordates, yet a distinctive pentaradiate body plan and unusual neural organization. Orthologues of transcription factors that regulate neurogenesis in other animals have been identified and several are expressed in neurogenic domains before gastrulation indicating that they may operate near the top of a conserved neural gene regulatory network. A family of genes encoding voltage-gated ion channels is present but, surprisingly, genes encoding gap junction proteins (connexins and pannexins) appear to be absent. Genes required for synapse formation and function have been identified and genes for synthesis and transport of neurotransmitters are present. There is a large family of G-protein-coupled receptors, including 874 rhodopsin-type receptors, 28 metabotropic glutamate-like receptors and a remarkably expanded group of 161 secretin receptor-like proteins. Absence of cannabinoid, lysophospholipid and melanocortin receptors indicates that this group may be unique to chordates. There are at least 37 putative G-protein-coupled peptide receptors and precursors for several neuropeptides and peptide hormones have been identified, including SALMFamides, NGFFFamide, a vasotocin-like peptide, glycoprotein hormones and insulin/insulin-like growth factors. Identification of a neurotrophin-like gene and Trk receptor in sea urchin indicates that this neural signaling system is not unique to chordates. Several hundred chemoreceptor genes have been predicted using several approaches, a number similar to that for other animals. Intriguingly, genes encoding homologues of rhodopsin, Pax6 and several other key mammalian retinal transcription factors are expressed in tube feet, suggesting tube feet function as photosensory organs. Analysis of the sea urchin genome presents a unique perspective on the evolutionary history of deuterostome nervous systems and reveals new approaches to investigate the development and neurobiology of sea urchins.

Afuni, a novel transforming growth factor-beta gene is involved in arm regeneration by the brittle star Amphiura filiformis.

The bone morphogenetic proteins (BMPs) are a family of the transforming growth factor-beta (TGF-beta) superfamily that perform multiple roles during vertebrate and invertebrate development. Here, we report the molecular cloning of a novel BMP from regenerating arms of the ophiuroid Amphiura filiformis. The theoretically translated amino acid sequence of this novel BMP has high similarity to that of the sea urchin BMP univin. This novel BMP has been named afuni. Whole-mount in situ hybridisation implicates afuni in arm regeneration. Expression occurs in distinct proximal and distal regions of late regenerates (3- and 5-week postablation). These sites are at different stages of regeneration, suggesting multiple roles for this gene in adult arm development. Cellular expression of this gene occurs in migratory cells within the radial water canal (RWC) of regenerating and nonregenerating arms. These migrating coelomocytes suggest a key role for the coelomic RWC as a source of the cellular material for use in arm regeneration by A. filiformis.

Anbmp2/4 is a new member of the transforming growth factor-beta superfamily isolated from a crinoid and involved in regeneration.

Invertebrates have frequently been used to help understand the complexities of regulatory gene function and evolution. The bone morphogenetic proteins (BMPs) are a highly conserved group of secreted regulatory factors that play an important part in early embryonic patterning. In the present study we have used the remarkable regenerative potential of crinoid echinoderms to explore the BMPs' site of expression in an adult developmental programme. Our results suggest that a crinoid BMP2/4 homologue is actively involved during the early stages of blastemal regeneration at a time when fundamental patterns are being established. This supports the idea of an evolutionary developmental programme where essential gene families are conserved throughout phylogeny in terms of both expression and function.

Expression of transforming growth factor beta-like molecules in normal and regenerating arms of the crinoid Antedon mediterranea: immunocytochemical and biochemical evidence.

The phylum Echinodermata is well known for its extensive regenerative capabilities. Although there are substantial data now available that describe the histological and cellular bases of this phenomenon, little is known about the regulatory molecules involved. Here, we use an immunochemical approach to explore the potential role played by putative members of the transforming growth factor-beta (TGF-beta) family of secreted proteins in the arm regeneration process of the crinoid Antedon mediterranea. We show that a TGF-beta-like molecule is present in normal and regenerating arms both in a propeptide form and in a mature form. During regeneration, the expression of the mature form is increased and appears to be accompanied by the appearance of an additional isoform. Immunocytochemistry indicates that TGF-beta-like molecules are normally present in the nervous tissue and are specifically localized in both neural elements and non-neural migratory cells, mainly at the level of the brachial nerve. This pattern increases during regeneration, when the blastemal cells show a particularly striking expression of this molecule. Our data indicate that a TGF-beta-like molecule (or molecules) is normally present in the adult nervous tissues of A. mediterranea and is upregulated significantly during regeneration. We suggest that it can play an important part in the regenerative process.

Abnormalities in sexual development of the amphipod Gammarus pulex (L.) found below sewage treatment works.

Increasing numbers of widely used industrial, agricultural, and natural chemicals are known to elicit endocrine-disrupting effects in a wide range of vertebrate and invertebrate species. The objective of this study was to determine whether the sexual development of the freshwater crustacean Gammarus pulex (L.) was affected below sewage treatment works (STW) previously known to contain endocrine-disrupting chemicals in their effluent. The gonadal structure, external sexual characteristics, and size of gammarids from exposed sites were compared to those of gammarids from a reference site. No significant difference was found in the gonadal structure of males collected below two STW. However, a highly significant number of females collected from a site known to elicit high estrogenic responses in vertebrates displayed an abnormal structure of oocytes in vitellogenesis. Body size was significantly shorter and male/female size differential was significantly reduced below one of the STW. Analysis of gnathopod and genital papillae length data suggests that different allometric relationships of these organs to body size exist between sample sites.

Development of serotonin-like and SALMFamide-like immunoreactivity in the nervous system of the sea urchin Psammechinus miliaris.

The present immunocytochemical study utilizes serotonin and SALMFamide antisera, together with confocal laser scanning microscopy, to provide new information about the development of the nervous system in the sea urchin Psammechinus miliaris (Echinodermata: Echinoidea). Special attention is paid to the extent of the nervous system in later larval stages (6-armed pluteus to metamorphic competency), a characteristic that has not been well described in this and other species of sea urchin. An extensive apical ganglion appears by the 6-armed pluteus stage, forming a complex of 10-20 cells and fibers, including discrete populations of both serotonin-like and SALMF-amide-like immunoreactive cells. At metamorphosis this complex is large, comprising at least 40 cells in distinct arrays. Serotonin-like immunoreactivity is also particularly apparent in the lower lip ganglion of 6- to 8-armed plutei; this ganglion consists of 15-18 cells that are distributed around the mouth. The ciliary nerves that lie beneath the ciliary bands in the larval arms, the esophagus, and a hitherto undescribed network associated with the pylorus all show SALMFamide-like immunoreactivity. The network of cells and fibers in the pyloric area develops later in larval life. It first appears as one cell body and fiber, then increases in size and complexity through the 8-armed pluteus stage to form a complex of cells that encircles the pylorus. SALMFamide-like, but not serotonin-like, immunoreactivity is seen in the vestibule wall, tube feet, and developing radial nerve fibers of the sea urchin adult rudiment as the larva gains metamorphic competency.

Regenerative response and endocrine disrupters in crinoid echinoderms: arm regeneration in Antedon mediterranea after experimental exposure to polychlorinated biphenyls.

Regenerative phenomena, which have the advantage of reproducing developmental processes in the adult organism, are very sensitive to environmental stress and represent stages that can be monitored for damage at the whole-organism, cellular and molecular levels. Some persistent and ubiquitous pollutants, which can affect the natural environment because of their bioaccumulation in organisms, exert their effects by acting as 'endocrine disrupters'. In this respect, they can cause dysfunction in steroid hormone production/metabolism and activity by their dramatic effects on gene expression, reproductive competence and growth. The aim of our present research was to assess the impact of such compounds on adult echinoderm reproductive physiology with particular reference to regeneration potential. It is known that vertebrate-type steroids are synthesized by echinoderms and play a role in the control of growth and reproduction. Our experimental model is the crinoid Antedon mediterranea, selected on the basis of its previously explored regenerative capabilities at the level of the arms. The regeneration response, analyzed at the tissue and cellular levels using both light and electron microscopy and immunocytochemistry, was employed to monitor the effects of exposure to persistent endocrine disrupter micropollutants such as polychlorinated biphenyls (PCBs) by means of laboratory tests performed under controlled conditions in terms of environmental variables and contamination levels. Our results indicate that exposure to endocrine disrupter compounds such as PCBs can induce anomalies in regeneration times, morphology and developmental mechanisms that can be interpreted in the light of significant dysfunctions in the endocrine mechanisms controlling regenerative development.

Growth factors, heat-shock proteins and regeneration in echinoderms.

The study of regeneration in armed echinoderm species, including crinoids, ophiuroids and asteroids, is attracting increasing attention. Recent interest has focused on the presence and potential role of growth factors, including members of the nerve growth factor (NGF) and transforming growth factor-beta (TGF-beta) families, in the regenerative process and their possible relationship to the normal developmental (ontogenetic) regulatory cascade. In addition, the expression patterns of the heat-shock family of stress proteins (Hsps) during regeneration are also important. Their role forms part of a normal stress response to the trauma of autotomy in combination with a putative function in tissue remodelling and associated protein turnover during regeneration. The temporal dynamics of the stress response may also be strongly indicative of environmentally adaptive pressures operating on these systems.

Changes in ubiquitin conjugates and Hsp72 levels during arm regeneration in echinoderms.

All organisms show a common defensive mechanism that results in the expression of conserved heat shock proteins (Hsps). These proteins function in a wide range of stressful conditions. We have monitored their levels in species of regenerating echinoderms with different mechanisms of regeneration and from different geographical locations. The effect of an artificial higher temperature on expression of Hsps was also studied. Two stress proteins (Hsp72 and ubiquitin) that are important in processes such as development and protein degradation were investigated. Using Western blot analysis and immunocytochemistry, we found significant changes in the level (Hsp72) and pattern of conjugation (ubiquitin) that corresponded with the repair phase (early regenerative stages) and with the later growth and regeneration of new tissues. Animals from the intertidal environment showed a distinctly sustained expression pattern of Hsp72 compared with benthic animals which suggests a functionally adaptative and dynamic stress response program.

Molecular approach to echinoderm regeneration.

Until very recently echinoderm regeneration research and indeed echinoderm research in general has suffered because of the lack of critical mass. In terms of molecular studies of regeneration, echinoderms in particular have lagged behind other groups in this respect. This is in sharp contrast to the major advances achieved with molecular and genetic techniques in the study of embryonic development in echinoderms. The aim of our studies has been to identify genes involved in the process of regeneration and in particular neural regeneration in different echinoderm species. Our survey included the asteroid Asterias rubens and provided evidence for the expression of Hox gene homologues in regenerating radial nerve cords. Present evidence suggests: 1) ArHox1 expression is maintained in intact radial nerve cord and may be upregulated during regeneration. 2) ArHox1 expression may contribute to the dedifferentiation and/or cell proliferation process during epimorphic regeneration. From the crinoid Antedon bifida, we have been successful in cloning a fragment of a BMP2/4 homologue (AnBMP2/4) and analysing its expression during arm regeneration. Here, we discuss the importance of this family of growth factors in several regulatory spheres, including maintaining the identity of pluripotent blastemal cells or as a classic skeletal morphogenic regulator. There is clearly substantial scope for future echinoderm research in the area of molecular biology and certain aspects are discussed in this review.

Identification of methyl farnesoate in the cypris larva of the barnacle, Balanus amphitrite, and its role as a juvenile hormone.

Previous investigations have shown that insect juvenile hormone (JH) and its analogues induce precocious metamorphosis of barnacle cypris larvae. In the present study, methyl farnesoate (MF; structurally identical to JH III, except for the absence of an epoxide group) has been shown to have a concentration-dependent effect on the development of cyprids of the barnacle Balanus amphitrite. Analysis of cypris extracts by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) confirmed the presence of endogenous MF. These data provide evidence that MF functions as a juvenilizing hormone in barnacle cyprids, an effect that hitherto has not been noted.

Preliminary observations on ascidian and echinoderm neurons and neural explants in vitro.

As part of a study on echinoderm and ascidian neural regeneration, attempts were made to develop a system for the maintenance of their neurons in vitro. It was found that neurons and neural tissue explants from the starfish, Asterias rubens, and the brittlestar, Ophiura ophiura, and explants from the brain of the ascidian, Ciona intestinalis, could be cultured for up to 6 weeks in a modified L15-based medium. Some cells extended axonal projections and produced growth cones under certain conditions. Attempts were made to stimulate neuron survival and outgrowth of echinoderm cultures with conditioned media containing growth factors or tissue extracts and with various substrates including extracellular matrix extracts from native tissue. Ascidian brain explants from both normal and regenerating animals were cultured in the standard conditions established for echinoderm tissue, with outgrowth being observed in 25% of explants. In these cultures labelling with bromodeoxyuridine suggested that regeneration continues in vitro, although results using substance P immunocytochemistry indicate neuronal differentiation may be impeded. These preliminary studies suggest it is possible to maintain adult echinoderm and ascidian neurons in vitro.

Cellular and molecular mechanisms of arm regeneration in crinoid echinoderms: the potential of arm explants.

Crinoid echinoderms can provide a valuable experimental model for studying all aspects of regenerative processes from molecular to macroscopic level. Recently we carried out a detailed study into the overall process of arm regeneration in the crinoid Antedon mediterranea and provided an interpretation of its basic mechanisms. However, the problem of the subsequent fate of the amputated arm segment (explant) once isolated from the animal body and of its possible regenerative potential have never been investigated before. The arm explant in fact represents a simplified and controlled regenerating system which may be very useful in regeneration experiments by providing a valuable test of our hypotheses in terms of mechanisms and processes. In the present study we carried out a comprehensive analysis of double-amputated arm explants (i.e. explants reamputated at their distal end immediately after the first proximal amputation) subjected to the same experimental conditions as the regenerating donor animals. Our results showed that the explants undergo similar regenerative processes but with some significant differences to those mechanisms described for normal regenerating arms. For example, whilst the proximal-distal axis of arm growth is maintained, there are differences in terms of the recruitment of cells which contribute to the regenerating tissue. As with normal regenerating arms, the present work focuses on (1) timing and modality of regeneration in the explant; (2) proliferation, migration and contribution of undifferentiated and/or dedifferentiated/transdifferentiated cells; (3) putative role of neural growth factors. These problems were addressed by employing a combination of conventional microscopy and immunocytochemistry. Comparison between arm explants and regenerating arms of normal donor adults indicates an extraordinary potential and regenerative autonomy of crinoid tissues and the cellular plasticity of the phenomenon.