BIOLOGY 468/568
PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY
Monday, 22-Apr-1996 15:33:05 PDT
STUDENT NAME:
Joseph A. Sisneros
PROJECT TITLE:
Effects of SDS on the Ultrastructure of the Gill Epithelia of
Littorina littorea.
Species Identification
Kingdom: Animalia
Phylum: Mollusca
Class: Gastropoda
Order: Prosobranchia
Family: Littorinacae
Genus: Littorina
Species: littorea
ABSTRACT
The epithelia of the gills of
Littorina littorea
were examined for changes in ultrastructure after exposure to 20 mg/l of sodium dodecylsulfate for 1 and 2 hrs. Transmission electron microscopy revealed four major changes induced in the ultrastructure of the gill epithelia: (1) noticeable loss of microvilli, (2) degeneration of the surface membrane of the gill epithelium, (3) an increase in the size and number of lysosomes in the gill epithelia, and (4) vacuolation of the nuclear envelope. The primary effect of SDS appears to be in the initial induction of ultrastructural changes in the apical surface of the gill epithelia. The secondary effects of SDS appear to induce internal ultrastructural changes in the epithelial cells.
Key Words:
Littorina littorea,
SDS, detergents, toxicity, TEM
INTRODUCTION
Present interest in this current study stems from the relatively recent finding that the anionic surfactant sodium dodecylsulfate (SDS) possesses a strong ability to repel sharks when administered into the buccal cavity (Gruber
et a
l. 1984, and Smith 1986). The major target tissue of SDS and its biochemical made of action in sharks is thought to be in the epithelial gill tissue (Moran
et a
l. 1984). However, testing the repellent properties of SDS and its effect on the gill tissue of sharks in either a laboratory or field situation can often present a challenge.
A more ideal situation in studying the effects of SDS on gill epithelial tissue would be to use a more convenient marine organism such as a mollusk. Using a mollusk instead of an elasmobranch for studying the sublethal effects of SDS on epithelial gill tissue has several advantages: (1) mollusks are organisms that are relatively abundant and readily available, (2) mollusks are much easier to manipulate in the laboratory than elasmobranchs, and (3) protocols have been developed for culturing mollusk tissue explants in vitro which can be used in controlled and reproducible chemical exposure studies (Flandre 1971). There currently exists a large body of literature on the lethal and sublethal effects of synthetic surfactants and oil dispersants on marine organisms. Previous pollution related studies have reported the effects of anionic surfactants and oil dispersants in both marine fishes and mollusks. Schmidt & Mann (1961) and Abel & Skidmore (1975) reported the general lethal effects of the anionic surfactants dodecylbenzene sulfate and sodium laurylsulfate on fish at the light microscope and the ultrastructural levels.
While other workers such as Nuwayhid and Davies (1980) have reported the ultrastructural effects of the oil dispersants BP 1100 X and BP 1100 WD on the mollusk
Patella vulgata
., there is still no information available on the sublethal effects of the anionic surfactant SDS on the gill epithelia of mollusks at the ultrastructural level. Therefore, the aim of this study will be to describe the normal ultrastructure epithelial gill tissue of the mollusk,
Littorina littorrea
, as well as describe the ultrastructural changes that occur following the exposure to the anionic surfactant SDS.
MATERIALS AND METHODS
A total of 10 periwinkles,
Littorina littorea
, were used in this study. The animals which were provided by the Department of Biology, California State University Long Beach were held in a 30 gallon fiberglass tank with circulating sea water for approximately six months before the onset of the study. The anionic surfactant used in this study, SDS, CH3(CH2)11OS03Na, was supplied by Sigma chemical company Co.
The gills of
Littorina
were studied by first dissecting them from the animal and then immediately placing them into the culture media, Delbecco Modified Eagle media (DME) + 20% fetal Bovine serum (FBS), and acclimating them for approximately 1 hour. After the gills had acclimated to the culture media, the gill explants were then separated into three groups. Four of the gill explants were used for the control group while the other six gill explants were used for the treatments groups. The 6 gills that were used in the two treatment groups were then exposed to 20 mg/l of SDS (20 ppm of SDS). The two treatment groups differed only in the amount of time exposure to SDS. In the treatment group three gill explants were exposed to SDS for 1 hr while in the other treatment group three gill explants were exposed to SDS for 2 hrs. The control group consisted of 4 gill tissue explants in vitro with the culture media without SDS exposure.
After the exposure procedures were carried out on the appropriate culture groups, the gill explants were collected from their respective cultures and fixed in 6% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 6.8-7.4) at room temperature for 1 hr. For transmission electron microscopy (TEM), the gills were washed in the buffer alone, after fixation, for 1 hr and then post-fixed in 2% osmium tetroxide with the buffer for approximately 1 hr. The gills were then washed in the sodium cacodylate buffer again for approximately 1 hr, dehydrated in alcohol, and embedded in Spur-Low-Viscosity embedding media. Sections were then cut on an LKB Ultramictome II. Thick sections were mounted on glass slides and stained with toludine blue for light microscopical examination. Ultrathin sections were mounted on copper grids and stained for approximately 30 minutes in uranyl acetate and 10 minutes in lead citrate. The stained grids were examined in a JEOL 1200 EX II electron microscope operated at 80 kV. For scanning electron microscopy (SEM), the gills were dehydrated in alcohol after fixation and dehydrated again in acetone. The gill tissue was then dried by the critical point drying method. Once the tissue had dried it was glued to a stub by silver dag, sputter coated with gold, and examined in a JEOL 35C scanning electron microscope operated at 15 kV.
RESULTS
Fig.l
A scanning electron micrograph of a normal gill of
Littorina littorea
. Scale bar = 1000 microns. 44X.
Fig.2.
A scanning electron micrograph of the surface of a normal gill of
Littorina littorea
. Ci, cilia; Gs, gill surface. Scale bar = 5 microns. 2100X.
Fig.3.
A scanning electron micrograph of the gill surface of
Littorina littorea
. Ci, cilia; Tm, tufts of microvilli. Scale bar = 1 micron. 14,000X.
Fig.4.
A light micrograph of a gill branch of
Littorina littorea
. Gb, gill branch; Nu, nucleus. Scale bar = 10 microns. 94OX.
Fig.5.
A light micrograph of gill epithelial cells from
Littorina littorea
. Bl, basal lamina; Ce, cuboidal epithelial cells; Nu, nucleus. Scale bar = 10 microns. 940X.
Fig.6.
A transmission electron micrograph showing the apical microvilli of a non-ciliated cell in
Littorina littorea
. Scale bar = 1 micron. 12,900X.
Fig.7.
A transmission electron micrograph of an animal exposed to 20 mg/l of SDS for 1 hr. Notice the loss of microvilli. Bd, bulbous distension of the apical membrane. Scale bar = 2 microns. 4900X.
Fig.8.
A transmission electron micrograph of an animal exposed to 20 mg/l of SDS for 1 hr. Notice the extrusion of the membrane. Bd, bulbous distension of the apical membrane; Ci, cilia; Ly, lysosome; Nu, nucleus; Vc, vacuole. Scale bar = 2 microns. 7200X.
Fig.9.
A transmission electron micrograph of an animal exposed to 20 mg/l of SDS for 1 hr. Bd, bulbous distension of the apical membrane; Ly, lysosome. Scale bar = 2 microns. 750OX.
Fig.10.
A transmission electron micrograph of an animal exposed to 20 mg/l of SDS for 2 hrs. Bd, bulbous distension of the apical membrane; Mv, microvilli; Nu, nucleus; Vc, vacuole. Scale bar = 2 microns. 7200X.
Fig.11.
A transmission electron micrograph of an epithelial cell of a normal animal. NU, nucleus. Scale bar = 1 micron. 11,400X.
Fig.12.
A transmission electron micrograph of an epithelial cell exposed to 20 mg/l of SDS for 1 hr. Nu, nucleus; Ve, vacuolated nuclear envelope. Scale bar = 1 micron. 35,500X.
Fig.13.
A transmission electron micrograph of an epithelial cell exposed to 20 mg/l of SDS for 2 hrs. Ly, lysosome; Nu, nucleus; Ve, vacuolated nuclear envelope. Scale bar = 1 micron. 21,100X.
Normal gill structure
Scanning electron micrographs of the whole gill (fig.1) reveal the general nature of the external gill structure of
Littorina littorea.
Higher magnifications of the gill reveal that the gill surface is covered with tufts of microvilli and cilia (figs.2 & 3).
Examination of the gill branches under the light microscope and TEM reveal that each gill branch is covered by a simple cuboidal layer of epithelial cells (figs. 4, 5,& 6). Beneath this epithelial layer lies a thick basal lamina which in turn covers a thin layer of collagenous connective tissue and muscle cells. Further TEM examination of the gill epithelium reveals that there are two types of epithelial cells: nonciliated and ciliated. Both of these two cell types can be found to be interspersed throughout the gill epithelium. The relatively large nonciliated cells possess densely packed microvilli that are found over the apical border of the epithelial membrane (fig.6). The ciliated cells also contain microvilli which can be found interspersed among the cilia in the apical membrane border as well. However, the ciliated cells appear to be smaller in size and less abundant in number when compared to the nonciliated cells. Nevertheless, both epithelial cell types possess a large nucleus and what appears to be randomly distributed mitochondria.
Changes induced by sodium dodecylsulfate
Gills of
Littorina
exposed to 20 mgll SDS for either 1 or 2 hours showed four main signs of damage.
(a) Microvilla Loss -
The loss of microvilli was observed in various sections of the epithelial membrane. Microvilli loss became quite apparent after just one hour of exposure to SDS (fig.7).
(b) Degeneration of the Cell Surface
- Perhaps the most notable changes that occurred in gill epithelia was in the microvillous membrane. The apical microvillous border of epithelial cells in many cases appeared to degenerate and protrude from the cell surface (figs.8,9 &10). The extreme form of degeneration of the cell surface appeared as bulbous distensions of the membrane (fig.8).
(c) Increase in Size and Number of Lysosomes
- Lysosomal activity appear to be greater in the treated groups than in the control groups. An increase in autolysis became apparent in the form of an increase in both the size and number of lysosomes in the epithelial cells for both exposure treatments (figs.8 &10).
(d) Vacuolation of the Nuclear Envelopes
- The nuclear envelopes of both nonciliated and ciliated cells in the treatment groups oftened appeared to be vacuolated when compared to the control groups (figs.11,12,&13). Greatest vacuolation appeared in the 2 hr treatment group (figs.10&13).
Gills of
Littorina
exposed for 2 hrs were generally similar to those animals that were exposed for 1 hr, though many of the 2 hr exposed sections showed damage in a more advanced state. It should also be noted that the expression of the above induced changes were very patchy. Every treated specimen had both affected and apparently unaffected areas.
DISCUSSION
This study was performed in hope of contributing to our limited knowledge of how surfactants act upon epithelial membranes of marine organisms as well as to determine, if any, the special properties of the shark-repelling surfactant SDS.
Very little work has been performed on studying the ultrastructural effects of synthetic surfactants on the epithelial cells in marine organisms. Abel & Skidmore (1975) have reported ultrastructural changes induce by sodium laurylsulfate in the gills of the rainbow trout, while Nuwayhid and Davies (1980) have documented the ultrastructural changes induced by oil dispersants in the gills of the mollusk
Patella
. In both of these studies, the researchers have suggested that the reactions of gill epithelia to the surfactants is nonspecific in terms of the site of damage to the gill epithelia.
Results from this study appear to be similar to the previous studies already mentioned. SDS appear to initially induced changes in the ultrastructure of the apical surface of the gill epithelia. These results also agree with Abel & Skidmore (1975) and Nuwayhid & Davies (1980) in that the damage to the gill appears to be non-site specific in the epithelial membrane. This appears to make sense with the actions of a detergent such SDS. Razin
et al
. (1965) have proposed that anionic detergents such as sodium laurylsulfate and sodium dodecylsulfate solubilize the lipid components of membranes. This proposed action would therefore account for the non-site specific epithelial damage observed in this study as well as the previous studies. Thus, in this respect SDS does not appear to have a unique chemical mode of action.
The results from this study also suggest that the secondary effects of SDS appear to be internally in the epithelial cells of the gill. The higher magnification micrographs of the gill epithelium show that is a marked difference in the internal damage of the treated gills versus the control gills (figs.6,8 &l0). This damage is seen in the form of an increase in lysosomal activity as well as a noticeable induced change in the nuclear envelopes (figs.10,12,&13). These findings were also reported by Nuwayhid and Davies (1980) in which they reported not only membranal surface damage such as the extrusion of the cytoplasm, but also increased lysosomal damage as well. This study has further demonstrated the primary and secondary effects of anionic surfactants that induce ultrastructural changes in the epithelial gill tissue.
This study has still not resolved the original question of what is responsible for inducing repellency in sharks. It maybe that the epithelial gill tissue may not be one of the organs response for the repellency behavior. Nevertheless, further studies need to be performed in studying the induced changes that surfactants elicit in membranal systems. Studying the gill response to other toxicants may reveal clues to how different poisons can affect marine organisms that are prone to the effects of chemical pollution.
CITATIONS
Abel, P.D., and J.F. Skidmore. (1975). Toxic effects of an anionic detergent on the gills of rainbow trout. Water Research 9: 759-765.
Gruber, S.H., E. Zlotkin, and D.R. Nelson. (1964). Shark Repellents: behavioral assays in laboratory and field. In: Eolis, L.J. Zadunaisky, and R. Giles, eds. Toxins, Drugs, and Pollutants in Marine Animals. Springer-Verlag, New York, NY. pp 26-42.
Flandre, 0. 1971. Cell cultures of mollusks. Invertebrate Tissue 1: 362-383.
Moran, A., Z. Korchak, N. Moran, and N. Primor. (1984). Surfactant and channel-forming activities of the Moses sole toxin. In Bolis, L.J. Zadunaisky, and R. Giles, eds. Toxins, Drugs, and Pollutants in Marine Animals. Springer-Verlag, New York, NY., pp 21-26.
Nuwayhid, M.A., and P.S. Davies. 1980. Changes in the ultrastructure of the gill epithelium of
Patella vulgata
after exposure to North sea crude oil and dispersants. Mar.Biol.Ass.U.K. 60: 439-448.
Razin, S., Morowitz, H.J., and T.M. Terry. 1965. Proceedings of the National Academy of Sciences of the United States of America 54 : 219 - 225.
Schmidt, O.J., and H. Mann. 1961. Action of a detergent (Dodecylbenzene sulfate) in the gills of trout. Nature 192:675.
Smith, L. 1986. Effectiveness of sodium lauryl sulfate and other chemicals as shark repellents in a laboratory swim-through test situation. Master Thesis, California State University, Long Beach 67p.
sisneros@roo.fit.edu