BIOLOGY 468/568
PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY
STUDENT NAME:
Cory Pickering
PROJECT TITLE:
Ultrastructural effects of salinity on the Gills of the Goldfish
Carassius
.
Species Identification
Kingdom: Animalia
Phylum:
Class:
Order:
Family:
Genus:
Species:
Abstract
Fish are found in numerous environments from fresh water to salt water and anywhere in between. As a species migrates from one environment to another to spawn, it is subjected to massive change in salinity and osmotic stress. These stresses are most profound in the exposed respiratory surfaces in the gills. In this study,
Carassius
will be used to determine what, if any, ultrastuctural effects occur as salinity increases. After exposing a number of individuals to a 20% increase in salinity, differences were seen in electron micrographs of the gill filaments. Noticable increases in mitochondria were seen as well as a tonistic response in the chondrocytes. These results seem to confirm the findings of other reports.
Key Words:
Carassius
, hypersalinity, osmotic stress, gills, TEM.
INTRODUCTION
Gills form a highly characteristic feature in fishes. Their presence has a marked effect on the anatomy and function of the animal. The main functions of the gills are gas and ion transport. While in sea water, there is a net loss of water from the fish. But, when in fresh water the opposite occurs and there is a net gain of water (Evans, 1969). The gill structure plays a major role in this area. For example, a freshwater gill absorbs sodium and chloride from the external medium, therefore gaining water (De Renzis, 1973). Lauret (1994) found some distinct characteristics in the gill epithelium. They are laden with chloride cells, and within these cells higher mitochondrial concentrations are seen (Pisam, 1991). It has been seen that as salinity increases the chloride cells seem to go through a fundamental reorganization (Sardet, 1979).
In this study, goldfish (
Crassius
) will he subjected to an increase of salinity in the surrounding medium. Comparisons will be made between treated (increased salinity) and control (no added salt). Micrographs will he made with the use of an electron microscope (TEM). It is hoped, through the micrographs, to count the number and possibly see chloride cell re-organization. This project may add more insight into the cellular changes occurring as a fish migrates from the ocean to a lake (or vice versa).
Methods
Specimen
Large feeder goldfish (
Carassius
) were purchased at a local fish store. They were placed in two 2-gallon aquarium. One contained 3,000 mls of tapwater. The other contained 600 mls ofseawater in addition to the 3,000 mls of tapwater. Both tanks were filtered and maintained at 17oC. The experiment was run for one week, after which the gills were dissected out of 2 - 3 individuals.
Tissue Preparation
After dissection, tissue was fixed with 2% Glutaraldehyde in Sorensen's phosphate buffer for one hour. Following that were three 10 minute rinses with phosphate buffer. Post fixation was done with 1% Osmium tetroxide in phosphate buffer for one hour, under refrigeration. Tissues were again rinsed with phosphate buffer three times. The next step was dehydration using a series of cold graded ethanols (30-100%). Infiltration was done using propylene oxide and Spurr epoxy resin. Finally, tissue was embedded in 100% Spurr. (Note: all fixation solutions were made at a pH of 7.4).
Sectioning
Once the embedded blocks had been trimmed and faced, sectioning began. This was done using an LBK Ultramicrotome II with glass knives. Thick sections were cut and stained with toluidine blue for visualization under the light microscope. Ultra-thin sections were then made using a new knife. The ultra-thins were mounted on copper grids (200 mesh) and stained. Staining was done for 3.5 minutes in uranyl acetate followed by a gentle rinse with carbon dioxide free water. Lead citrate was also used, grids were left for 2.5 minutes. After another gentle rinse the grids were ready for viewing.
Viewing
Grids were viewed on a JEOL 1200II TEM at 40 kV and 80 kV. The third (largest) condenser lens and first (smallest) objective lens were used to provide the best contrast. When taking pictures an exposure time of 3.5 - 4.5 seconds was used, also to improve contrast. After development, micrographs were printed.
Results
From micrograph interpretation there appears to be a noticable effect caused by the increased salinity. The amount of mitochondria found associated within chloride cells increased as salinity increased. An effect was also seen in the chondrocytes.
Plate I
(a.)
Micrograph of a chloride cell taken from gill filament of
Carassius
under normal conditions (control).
Nu = nucleus, Mi = mitochondria EE = external environment, Er = endoplasmic reticulum. Bar 500 microns.
(b.)
Micrograph of chloride cell in a treated individual. Notice the higher number of mitochondria compared to (a.).
Nu = nucleus, Mi = mitochondria EE = external environment, Er = endoplasmic reticulum. Bar 500 microns.
Plate 2
(a.)
Another chloride cell taken from a control individual of
Carassius
.
Nu = nucleus, Mi = mitochondria, EE = external environment, > = cell
membrane, Er = endoplasmic reticulum. Bar 500 microns.
(b.)
Chloride cell found in gill filament of a treated individual again showing
increased numbers of mitochondria.
Bar 500 microns.
Plate 3
(a.)
Section through cartilagenous area showing chondrocytes under control
conditions.
Bar 1 micron.
(b.)
Section through cartliage showing chondrocyte of treated gill.
Nu = nucleus Mi = mitochondrion. Bar 1 micron.
Discussion
The results seem to follow several of the previous research found. Lubin (1989) studied Atlantic salmon and Rainbow trout, he found elongation and enlargement of the chloride cells occurring as salindty of the external medium increased. His results were based on a six month study. Although, here, the study was for only one week similar results were seen. Vickers (1961) on the other hand, found no change occurring in
Carassius
as salinity increased. In this study no increase in number of chloride cells was seen which is consistant with the Vickers work. There was, however, an increase in the number of mitochondria associated with the chloride cell. This was also found to occur in other studies. It would seem more mitochondria being present would allow the active transfer and movement of gases and ions to combat the osmotic shock.
An increase in mucus cells was thought to be found as a result of the salinity increase. This was never observed, but may have been due to the short amount of time the experiment ran for. Another factor influencing this "non-effect" may have been the low increase in salinity (20% seawater). In most other studies 100% sea water was used.
There are three aspects of the studt that could be improved. First, a long exposure time for the treated individuals. Second, a higher concentration of sea water to be used during treatment and finally the use of longer fixation times. This would allow better preservation and resolution of membranes within the tissue.
Overall, there does appear to be an effect at the cellular level as a fish confronts increases in environmental salinity. But, for some species of fish they must not be affected too badly or they would not be able to return to certain areas to spawn. The mechanisms behind these structural effects remains to be elucidated.
CITATIONS
Chavin, W. (1973). Responses of Fish to environmental changes. pp240-263, Charles C. Thomas Publishing.
DeRenzis, G. and Maetz, J. (1973). Studies on the mechanism of chloride absorption by the goldfish gill: Relation with acid-base regulation. J. Exp. Biol. 59, 339-35S.
Evans, David H. (1969) .Studies on the permiability to water of selected marine, freshwater and euryhaline teleosts. J. Exp. Biol. 50, 689-703.
Laurent, P. (1984). Gill internal morphology in: Fish Physiology, Vol X (Hoar, W. S. and Randall, D. J., eds.) pp. 73-183, New York: Academic Press.
Lubin, R. J. et al. (1989) Ultrastructural alterations in branchial chloride cells of Atlantic Salmon,
Salmo salar
. during parr-smolt transformation and early development in seawater. J. Fish Biol. 34, 259-272.
Perera, K. M. L. (1993) Ultrastructure of the primary gill lamellae of
Scomber austraiasicus
. J. Fish Biol. 43, 45-59.
Pisam, M. and Rambourg, A. (1991) Mitochondria-rich cells in the gill epithelium of teleost fishes: an ultrastructural approach. Int. Rev. Cytol. 130, 191 233.
0ardet, C., Fisam, M. & Maetz, J. (1979). The surface epithelium of teleostean fish gills, cellular and junctional adaptations of the chloride cell in relation to salt adaptation. J. Cell Biol. 80, 96-117.
Vickers, T. (1961) A study of the so called Chloride Secretory cells of the gills of teleosts. Q. J. Microsc. Soc. 102, 507 518.
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