BIOLOGY 468/568
PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY
Monday, 22-Apr-1996 15:34:58 PDT
STUDENT NAME:
Alicia Staines
PROJECT TITLE:
Ultrastructural Study on the Accumulation of Mercury by
Littorina littorea
.
Species Identification
Kingdom: Animalia
Phylum: Mollusca
Class: Gastropoda
Order: Prosobranchia
Family: Littorinacae
Genus: Littorina
Species: littorea
ABSTRACT
Specimens of
Littorina littorea
have been exposed to 0.1, 1.0, 10, and 100 ppm mercury in seawater by adding mercuric chloride (HgCl2) to clean filtered seawater. After determining the highest concentration of mercury that did not cause 100% mortality, I examined tissues from the animals exposed to this concentration and compared them to control animals, exposed to seawater only using the JOEL 1200 EXII transmission electron microscope. In the treatment group, electron dense inclusions were obsterved in cytosomes, located in the epithelial cells, below a border of microvilli and cilia. Such inclusions were not found in the control tissues.
Key Words:
Mercury,
Littorina littorea
, ultrastructure.
INTRODUCTION
As a potential indicator of elevated levels of environmental pollution,
Littorina littorea
, the common winkle, has been extensively studied with respect to its accumulation of heavy metals. Metals in the marine environment have been found in many tissues of the marine mollusc,
Littorina littorea
, including the connective tissue, digestive gland, kidney, stomach and ctenidia (Mason
et. al
, 1984). Among the metals studied are Ca, Mg, K, and Mn by Mason
et. al.
(1984), Aq, As, Cd, Co, Cr. Cu, Fe, Hg, Mn, Ni, Pb, and Zn by Bryan et. al., (1983), by Bryan and Hummerstone (1977), Marigomez et. al. (1990), Nott and Langston (1989), Ireland and Wootton, 1977), and others. Bryan
et. al.
(1983) found a significant relationship between levels of mercury in the tissues of the winkle and
Fucus
, indicating that the diet of
L. littorea
was an important source of contamination. The concentrations of mercury in the tissues of
L. littorea
ranged from 0.14 to 1.90 pg Hg/g dry weight.
I was interested in the uptake of mercury by
Littorina littorea
directly from the water. This has been well studied in filter feeders such as
Mytilus californianus
(Eganhouse and Young, 1978),
Mytilus edulis
(Roesijadi, 1982), the quahog clam,
Mercenaria mercenaria
(Fowler et. al., 1975), and the oyster,
Ostrea edulis
(Wrench, 1978) and others. Uptake of dissolved mercury from seawater has been studied extensively in bivalves due to potential concentration of contaminants during filter feeding. Roesijadi (1982) found that dissolved mercury is initally accumulated by the gills and is then gradually redistributed to other organs. Although
Littorina littorea
, is not a filter feeder, its gills are in contact with seawater and could potentially take up contaminants from the water.
My goal was to determine if I could visualize the effects of mercury contamination using a transmission electron microscope.
MATERIALS AND METHODS
Determinina Lethal Dose Level :
For best visualization of mercury contamination, I chose the highest concentration of mercury that did not cause the death of all of the winkles. To determine this, I chose four concentrations: 0.1, 1, and 10 parts per million (ppm), used by Fowler et. al. (1975) on
Mercenaria mercenaria
, and because
L. littorea
does not concentrate contaminants by filter feeding, it may be able to withstand higher concentrations of pollutants in sea water than the bivalves. Therefore, I exposed one group to 100 ppm as well. I established these concentrations by adding mercuric chloride (HgCl2) to clean, filtered seawater. I then placed five randomly selected
L. littorea
(chosen from an aquarium containing winkles supplied by a commercial dealer) in each of four 500 ml erlemeyer flasks containing 0.1, 1, 10, 100 ppm HgCl2 and one 500 ml flask containing pure seawater as a control. In addition, I removed 10 winkles from their shells using a vice and placed two naked animals in each container since other researchers have observed
L. littorina
to completely withdraw into its shell when faced with unfavorable conditions, possibly preventing mercury uptake into the tissues. I supplied each flask with an airstone and placed a plug in each flask to prevent the animals from crawling out of the water and removing themselves from the treatments.
The animals remained in the water for six days, with one water change at the three day mark. I supplied them with no food to control for any effects of feeding on the accumulation of mercury. At the end of the six days, I removed animals from the seawater with the highest concentration of mercury that had survivors (1 ppm) and from the control.
Preparation for the TEM :
Using a vice, I removed the shells from the living winkles from the 1 ppm mercury concentration and from the control animals and dissected out the gill. This was done by cutting along the oesophageal gland running along the left side of the body, back to the kidney and across the dorsal side of the animal. I then folded back the mantle tissue to expose the gill. I excised the gill, removed the mantle edge and used a blade to cut the gill tissue into 1 mm cubes.
I fixed the tissue samples in 3% glutaraldehyde in a sodium cacodylate buffer at pH 7.4, adding NaCl to raise the osmolarity to 1100 mosmoles. I planned to visualize the mercury by looking for electron dense areas and as such, subsequently fixed only half of the tissue in 2% osmium tetroxide so as not to confuse mercury with osmium. I then dehydrated the tissue samples in a series of alcohols and embedded in low-viscosity Spurr's Resin (Mason
et. al.,
1984). I then cut semi-thin and ultra-thin sections on a Sorvell M2B microtome and viewed the ultra-thin sections on a JEOL 1200 EXII transmission electron microscope.
RESULTS
Lethal Dose Level
None of the naked winkles survived the experiment, including the controls, most likely due to damage caused by removing them from their shells. For the shelled animals, the lethal dose was between 1 ppm and 10 ppm HgCl2. Based on the mortality rates for the different treatments (Table 1), I used a treatment level of 1.0 ppm HgC2 for the TEM study, the highest concentration of mercury with any survivors.
Table 1: Mortality of shelled Littorina littorea in seawater treated with four levels of HgCl2.
n=5 per treatment
mortality counts
% Mortality
control
0
0
0. 1 ppm
1
20
1 ppm
4
80
10 ppm
5
100
100 ppm
5
100
TEM study
FIGURE 1:
Electron micrograph of ctenidal cell from control animal, no staining procedures, (X 18,000 magnification)
FIGURE 2:
Nucleus from treatment group (N) showing nucleus (N). exposed to 1.0 ppm HgCl2, no staining procedures (X 21,000)
FIGURE 3:
Control tissue showing mitochondria (M) and nucleus (N). (X 18,000)
FIGURE 4:
Tissue exposed to 1.0 ppm dose level showing dense inclusions in cytosome (Hg) with fibrillar margin (f), mitochondria (M), and cytoplasmic vessicles (V). (X 28,000)
FIGURE 5:
Close up of electron dense inclusion (Hg) in 1.0 ppm dose level tissue showing double cytosome membrane (CM). (X 140,000)
FIGURE 6:
Close up of electron dense areas in control cells and nearby vessicles (v). (X 51,000)
FIGURE 7:
Epithelial tissue from 1 ppm treated tissue with Microvilli (MV). (x 60,000)
FIGURE 8:
Close up of cell containing mercury inclusion found near microvilli (Hg) with small vesicles (v). (X 18,000)
FIGURE 9:
Close up of mercury inclusion (Hg). (X 30,000)
FIGURE 10:
Columnar epithelial cells in control tissue with microvilli (MV), cilia (C), mitochondria (m), and suspected protein (P). (X 9000)
FIGURE 11:
Close up of suspected protein (P) and mitochondria (M). (X 30,000)
FIGURE 12:
Cross section of cilia (C) at edge of epithelium. (X 90,000)
The completely unstained samples (unfixed in osmium tetroxide and grids unstained) showed no differences between the control and the 1 ppm treatment level. The only features visible were the nuclei, which appeared similar for both the control (Figure 1) and the treatment (Figure 2).
I then examined cells from the samples fixed with osmium tetroxide. Comparison of the control (Figure 3) with the 1 ppm treatment (Figure 4) showed both to have dense regions. Closer examination of these regions showed what I suspect to be mercury in the 1 ppm specimen (Figure 5).
This dense inclusion seems to be in a double membraned cytosome. The dense areas in the control speciment were much smaller and fewer in number than the inclusions from the treatment group, although this is inconclusive. The appearance of the dense areas in the control specimens were for the most part different from the inclusions in the treatment specimens (Figure 6) but in some cases could not be distinguished from the treatment inclusions.
Examination of the epithelial cells, which were columnar and possessed short mirovilli (Figure 7) and cilia, showed large, dense, globular areas in the treatment cells (Figures 8 and 9). Again, the dense inclusions, thought to be mercury, were located in the cytosomes. These cells were not present in the control cells (Figure 10). The dark areas in the control cells (Figure11) again have a very different appearance from what I hypothesize is mercury. Possibly they are proteins, which would also appear as dense areas. Figure 12 shows a cross section through what had appeared to be cilia in Figure 10, confirming their identification. I also observed many dense inclusions in cytosomes in the treatment group in a comparable region as that shown in Figure10. There were many inclusions along the base of the microvilli.
DISCUSSION
I was able to visualize a difference between the control group and the treatment group of 1 ppt HgCl2 for
Littorina littorea.
In the ctenidium, mercury was most abundant in cytosome inclusions below the microvilli in the epithelial cells. These results are identical to those obtained by Fowler
et. al.
(1975) for the quahog clam,
Mercenaria mercenaria
, for the mantle tentacle epithelial cells, although for the same dosage, 1 ppm, the inclusions were not as abundant for L. littorea as for M. mercenaria. This, however, is to be accepted, since the filtering of much larger concentrations of water by the bivalve would concentrate the mercury in its tissues.
The attempt at viewing unstained material was unsuccessful because there was not enough contrast to identify anything but the nuclei. As expected, there was no obvious difference between the two groups. I would not expect mercury to enter the nucleus, but instead be incorporated into the transport system of the animal. I would expect that, had I examined other tissues of the treatment animals, especially the kidneys, I would have found mercury as well, transported from the ctenidium throughout the body.
Another problem is that while I can identify dense inclusions in the tissues of
L. littorea
that I hypothesize are mercury, with the transmission electron microscope available to me, I cannot identify that those inclusions are indeed mercury. The next step would be to use an electron energy loss system or an energy dispersive system to identify the composition of the inclusions. I hypothesize that they are mercury obtained from the seawater. If it is possible to identify mercury in the tissues of
Littorina littorea
, it would show that this animal could be used as a possible indicator of mercury contamination. Mercury is a readily acquired contaminant that is often present in river runoff. For example, in 1979, an estimated 300,000 tons of mercury was delivered to the Southern California Bight from the Los Angeles Basin via runoff and another 2.1 million tons were present in municipal wastewater discharges (Young
et. al,
1980).
In conclusion, future work should include a qualitative analysis of the areas suspected of containing mercury. Using EDX, I would like to positively identify the mercury. I would also like to examine other tissues for the presence of mercury using methods similar to those used in this experiment and also a time series study to determine rates of both incorporation and elimination of mercury from the tissues of
Littorina littorea.
CITATION
Bryan, G.W. and L.G. Hummerstone. 1977. Indicators of heavy-metal contamination in the Looe Estuary (Cornwall) with particular regard to silver and lead. J. Mar. Biol. Ass. U.K. 57:75-92.
Bryan, G.W., W.J. Langston, L.G. Hummerstone, G.R. Burt, and Y.B. Ho. 1983. An assessment of the gastropod,
Littorina littorea
, as an indicator of heavy-metal contamination in United Kingdom estuaries. J. Mar. Biol. Ass. U.K. 63:327-345.
Eganhouse, R.P. and D.R. Young. 1978. in situ uptake of mercury by the intertidal mussel,
Mytilus californianus
. Marine Pollution Bulletin. 9:214-217.
Fowler, B.A., D.A. Wolfe, and W.F. Hettler. 1975. Mercury and iron uptake by cytosomes in mantle epithelial cells of quahog clams (
Mercenaria mercenaria
) exposed to mercury. J. Fish. Res. Board Can. 32:1767-1775.
Mariogomez, J.A., M.P. Cajaraville, and E. Angulo. 1990. Cellular cadmium distribution in the common winkle,
Littorina littorea
determined by X-ray microprobe analysis and histochemistry. Histochemistry. 94:191-199.
Mason, A.Z., K. Simkiss and K.P. Ryan. 1984. The ultrastructural localization of metals in specimens of
Littorina littorea
collected from clean and polluted sites. J. Mar. Biol. Ass. U.K. 64:699-720.
Nott, J.A. and W.J. Langston. 1989. Cadmium and the phosphate granules in
Littorina littorea
. J. Mar. Biol. Ass. U.K. 69:219-227.
Roesijadi, G. 1982. Uptake and incorporation of mercury into mercury-binding proteins of gills of
Mytilus edulis
as a function of time.
Wrench, J.J. 1978. Biochemical correlates of dissolved mercury uptake by the oyster,
Ostrea edulis.
Marine Biology. 47:79-86.
Young, D.R., T.-K. Jan, R.W. Gossett, and G.P. Hershelman. 1980. Trace pollutants in surface runoff. In Bascom, W. (ed.) Coastal Water Research Project, Biennial Report 1979-1980. pp. 163-169.
zedmason@csulb.edu