Journal of Experimental Marine Biology and Ecology
Temporal variability in, and impact of food availability on vanadium and iron concentrations in Ciona intestinalis tissues (Tunicata, Ascidiacea)
Introduction
Ascidians (Tunicata, Ascidiacea) of the suborder Phlebobranchia are unusual in the animal kingdom in that some accumulate high levels of vanadium in their tissues which is evidently non-toxic (Henze, 1911). The highest concentration of vanadium is found in a reduced form (VIII) in the vacuoles of hemocytes called signet ring cells (SRCs), so named because a single acidic vacuole containing the vanadium displaces the nucleus to the periphery of the cell. The concentration of vanadium is as high as 350 mM in the SRC vacuoles of some species (Michibata, 1991). Vanadium has also been localized in other hemocytes, and in lower concentrations in other tissues such as the branchial basket and tunic, probably reflecting infiltration of hemocytes into these tissues (Michibata et al., 1986).
Ascidians of the suborder Stolidobranchia contain detectable levels of vanadium but at levels several orders of magnitude lower than found in the Phlebobranchia (Michibata et al., 1986). Members of both suborders accumulate high amounts of iron in their tissues. The relationship, if any, between iron and vanadium, within and between these groups has received little study and the function of both are unknown. The functions suggested for vanadium include anti-fouling/anti-predator defense (Stoecker, 1980, Odate and Pawlik, 2007), anaerobic adaptation (Smith, 1989) and tunichrome/tunic formation (Taylor et al., 1997) but none have been supported with strong experimental evidence.
Previous work on metal accumulation by ascidians has focused primarily on the vanadium dynamics and to a large degree on the reduction of vanadium and the search for the true “vanadocyte.” The extent to which vanadium or iron levels in ascidian tissue vary, if at all, over physical or physiological variables is unknown. There is little known about the concentrations of these metals with respect to season, life span, changes in temperature and oxygen availability, food supply and so on. Understanding the parameters that coincide with patterns of variation will be essential in resolving the role these metals play and the mechanisms of regulation. For example, the study of the movement of vanadium or SRCs into storage or utilization sites would be aided by knowing about variability of vanadium levels in hemocytes and various tissues.
Seasonal variation is a logical starting point for studies of variation in the metal accumulation by benthic planktivores such as ascidians as in most ecosystems, food supply and environmental temperature vary over an annual cycle. As a result, physiological parameters such as water pumping and metabolic rates fluctuate temporally, which may in turn might contribute to seasonal differences in metal accumulation (Petersen et al., 1995, Ribes et al., 1998, Riisgard et al., 1998, Petersen et al., 1999, Coma et al., 2002). Water pumping rates by ascidians change with environmental particle (phytoplankton and other particles) concentrations, possibly resulting in changes in exposure to dissolved metals (Fiala-Medioni, 1978, Petersen and Riisgard, 1992, Ribes et al., 1998, Armsworthy et al., 2001). Although the importance of food derived vanadium sources is unknown, vanadium and iron are present in the phytoplankton food and during a spring bloom ascidians may be exposed to increased dietary metals (Unsal, 1982).
Given these factors, we hypothesized that vanadium and iron concentrations vary temporally, with higher concentrations during or following the spring bloom compared to the winter or fall. We further hypothesized that similar ecophysiological factors govern both vanadium and iron uptake resulting in similar seasonal pattern in concentrations for these two metals. A RP-HPLC technique was adapted, using pre-column chelation with 4-(2-pyridylazo)-resorcinol (PAR), for the determination of vanadium and iron concentration of several tissues of the ascidian Ciona intestinalis. This is a cosmopolitan phlebobranch ascidian accumulating vanadium to concentrations of 600 μM in its hemocytes, and for which putative vanadium binding proteins have been identified (Michibata et al., 1986, Trivedi et al., 2003). The animals were collected in six periods from summer to spring from Woods Hole, USA.
Michibata and colleagues (Michibata et al., 2003, Yoshihara et al., 2005, Yoshinaga et al., 2006, Ueki et al., 2007) hypothesize that vanadium (Vv) from seawater is taken up at the branchial basket, reduced to VIV, and transported into the SRCs by vanadium binding proteins or vanabins where it is further reduced to VIII. Although the exact mechanism of vanadium reduction is unknown, the presence of several enzymes of the pentose phosphate pathway in the SRCs (glucose-6-phosphate dehydrogenase, transkelotase, 6-phosphogluconate dehydrogenase), led to the hypothesis that the NADPH produced by these enzymes might participate in the reduction of vanadium (Uyama et al., 1998, Kanamori et al., 1999). We measured the activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) in the hemocytes to examine whether the activity of this enzyme co-varies with vanadium concentration. Finally, in a short feeding experiment, we examine whether vanadium and iron levels vary in response to particle concentration/food borne metals alone.
Section snippets
HPLC method
The HPLC method was adapted from several common methods including those of Tadayon et al. (1991), Tsai and Hsu, 1994, and Ming et al. (1992). The instrument used was a Waters® HPLC equipped with a dual absorbance detector and a temperature controlled autosampler. The column was a 25 mm long Symmetry C18 column (5 μm) equipped with a guard column. The mobile phase was 65 mM ammonium phosphate, 35% MeOH, and the flow rate was 0.8 ml min− 1.
Aliquots of biological samples and standards (typically 200 μL)
HPLC characteristics and validation
Fig. 1 presents typical chromatographs of standard injections of a 1.6 M HCl blank, standards and tissue PAR chelates. At pH 6.5, two PAR-metal complexes form for both vanadium and iron (Ming et al., 1992, Tsai and Hsu, 1994). The retention times under the conditions of this preparation are 4.7 and 8.1 min for vanadium [V(PAR) and V(PARH), respectively] and 27 and 32 min for iron [Fe(PARH) and Fe(PARH)2 respectively]. Total peak area was used for the calibration curves. The calibration curves were
HPLC quantification of metals in ascidian tissues
The HPLC method applied in this work was especially useful for routine quantification of metals in large numbers of biological samples, especially in the ascidian tissues which are relatively rich in vanadium and iron. Many of the techniques commonly used for the quantification of metals require equipment not held by the average laboratory and which are relatively expensive, making long term studies logistically and economically impractical. An additional advantage of the present method is that
Acknowledgements
This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (WRD). WRD holds the Canada Research Chair in Marine Bioscience. We thank the staff of the Marine Biological Laboratory, Woods Hole for the collection of specimens. [SS]
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