With increased levels of ozone layer depletion caused by atmospheric pollution, attention has focused on the harmful effects of ultraviolet radiation (UV radiation) on the primary producers of the oceans which, by photosynthetic necessity, must be exposed. This is in light of new evidence which suggests that seawater is much more transparent to UV radiation than was previously believed (Smith and Baker, 1979). This could signify problems for the higher oceanic trophic levels which use phytoplankton as a food source.Ultraviolet radiation is a known inhibitor of photosynthesis (Gerber and Haeder, 1992; Smith et al., 1980). Gerber and Haeder (1992) found significant bleaching of photosynthetic pigments after prolonged exposure of a freshwater flagellate to UV radiation. They also predicted that physical damage was occurring within the chloroplasts since there was no energy transfer from the photosynthetic units to photosystem II. Helbling et al., (1992) also showed that UV radiation has deleterious effects on the photosynthetic rates of marine phytoplankton from both antarctic and tropical regions, with antarctic species showing the greatest sensitivity.

Ultraviolet radiation has been reported to have an effect on the growth rates of plants and algae. Behrenfeld et al. (1992) found that there was a decrease in the growth rates of a marine diatom species with an accompanying increase in the cell volume. Jokiel and York (1984) found that both UV-A and UV-B radiations were harmful to various species of microalgae. Among these, a symbiotic species of dinoflagellate (Symbiodinium microadriaticum) was exceptionally sensitive to the two types of UV radiation. This latter effect may have been a result of the removal of this dinoflagellate from the light filtering protection of its host's tissue.

Another damaging effect of UV radiation is its impairment of motility, phototaxis and gravitaxis. Haeder and Haeder (1991), working with a marine cryptoflagellate, found a reduction in the number of motile cells after a mere 20 minute exposure of the culture to a UV source. After 100 minutes there was a decrease in the velocity of the remaining motile cells. Blakefield and Calkins (1992) found that there were delays in positive phototaxis in Volvox aureus, a green phytoplankton. However, this inhibition could only be induced at high levels of artificial UV-B radiation. Similarly, Haeder and Liu (1990) found impaired gravitaxis(=geotaxis) in a freshwater dinoflagellate after exposure to an artificial UV-B radiation source.

In contrast to the extensive studies linking UV radiation to decreased growth and photosynthetic levels in algae, relatively little has been reported on the ultrastructural changes associated with UV radiation in plants and algae. Very few studies examined ultrastructural changes in microplankton associated with ultraviolet radiation. Michaels and Gibor(1973), working with Euglena found several qualitative ultrastructural changes associated with UV irradiation. After a five day exposure to an artificial UV source, the plastids of Euglena disappeared. Preceding this was a thickening of the thylakoids and a subsequent decrease in their number within the plastids. In addition, the plastids seemed to become more centrally located rather than their normal peripheral location in the cell. Finally, Mitochondria in UV irradiated Euglena appeared larger than their control counterparts. In another study, Lesser and Shick (1990) found that UV irradiated cells of appeared larger than their control counterparts. In another study, Lesser and Shick (1990) found that UV irradiated cells of a cultured zooxanthellae (Symbiodinium sp.) had higher mean values of accumulation bodies per cell. They described these bodies as possibly having autophagotrophic properties which led them to believe that there was increased cell damage in the treatment cells. Additionally, the volume fraction of chloroplasts decreased in the treatment cells relative to the controls. Zooxanthellae in situ (within their cnidarian host) showed a decrease in the volume fraction of thylakoid lamellae relative to the chloroplasts upon UV irradiation. This effect was not observed in the cultured zooxanthellae. In contrast to Michaels and Gibor (1973), there was no observable effect of UV on the mitochondria in the study by Lesser and Shick (1990).

The goal of this study was to examine the ultrastructural effects of ultraviolet radiation on the free living planktonic dinoflagellate Amphidinium carteri using qualitative transmission electron microscopy. There has been one purely ultrastructural study of Amphidinium carteri conducted by Dodge and Crawford (1969) and the current study has relied heavily on this study to interprett anatomical and ultrastructural features within the micrographs.

---

MATERIALS AND METHODS

CULTURES

One ml of a stock culture of Amphidinium carter was inoculated into each of nine 125 ml experimental glass pyrex flasks containing about 75 ml of sea water that had been filtered through Whatman GF/C glass mirofiber filters and enriched with f/2 algal culture medium (Guillard and Ryther, 1962). The stock culture is maintained in the laboratory in this same medium.

The cells were incubated at room temperature (26 oC during the day) under cool fluorescent white lights with an incident light intensity of 1.95 X 1015 quanta.sec-1.cm-2 as measured with a Biospherical light meter. Incubations in the light were for 24 hr per day for 9 days (i.e. there was no dark cycle). After 14 days I determined that the cells had reached the exponential phase of their growth cycle (as determined from cell counts).

EXPERIMENTAL DESIGN

Once the cells reached the exponential phase of their growth, the individual flasks were arbitrarily selected for treatments. There were two treatments and one control. The "24 hr" treatment consisted of a 24 hr/day incubation of cells in three replicate flasks at room temperature under a light intensity of 1.95 X 1015 Q.sec-1.cm-2 with an added source of ultraviolet light placed 30 cm overhead. The same conditions applied to the three replicate "12 hr" treatments except the daily exposure duration to UV was reduced to 12 hr/day followed by an incubation in the same light intensity as above without UV.

Controls consisted of three replicate flasks incubated for 24 hr/day under the same light intensity as above without UV. all treatment and control flasks were kept in a hood which was shielded from outside light sources with black plastic sheeting.

Experimental incubations lasted for five days. During this time the cells were dividing asexually by constriction (Bold and Wynne, 1985).

TRANSMISSION ELECTRON MICROSCOPY

At the end of the experiments, cells from each flask were centrifuged at 6800 rpm for 5 minutes. The supernatant was discarded and the pellet was re-suspended in 1% glutaraldehyde in single strength Sorenson's buffer to which NaCl was added (0.008 M Na2HPO4.7H2O; 0.0035 M KH2PO4; and 0.35 M NaCl). Buffer pH was adjusted to 7.4. After 2 hr the cells were rinsed three times (10 min ea.) in buffer without the glutaraldehyde and re- suspended in buffered 1% OsO4 for 2 hr. Cells were again rinsed three times (10 min ea.) in buffer, suspended in 2% buffered agar (Noble) [after it cooled down to 45oC] and dehydrated in a graded ethanol series (30% to 95% for 10 min each). The agar-cell complex was next incubated in 100% ethanol (four times, 15 min each) followed by two changes (10 min each) in propylene oxide. After this the agar-cell complex was incubated for eight hr in 2:1 propylene oxide:spurr epoxy resin followed by another eight hr incubation in a 1:2 mixture of the above. Finally, the agar- cell complex was incubated in two changes (four hr each) of 100% spurr and embedded in spurr resin for 16 hr under vacuum.

Ultrathin Sections were made with a Sorvall MT2-B ultramicrotome, picked up on 200 or 300 mesh copper grids and positively stained with 8 % uranyl acetate and 0.8% lead citrate. Micrographs were taken with a Jeol 1200 EX2 transmission electron microscope at 80 KeV using condenser 1.

CELL COUNTS

Cell counts were determined after initiation of treatments on days 0, 3, 6, 10 and 13 with a Coulter Multisizer (Coulter electronics). Counts were performed on one flask from each treatment and one from the controls.

---

RESULTS

GROWTH RATES

After five days, the 24 hr/day UV treatment cells did not achieve sufficient enough numbers to permit further processing for EM. Figure 1 shows the growth rates of the 12 hr/day treatment cells and the control cells. From table 1 it can be seen that the highest doublings/day for the treatment cells was 0.77 doublings/day (achieved between day 0 and day 3). For the controls, the highest doubling time was 1.0 doublings per day for the same period.

Table 1. Amphidinium carteri. Doubling times per day of treatment (12 hr/day) and controls over different time periods and overall.





period treatment (days)	0-3	0-6	0-10	overall (0-13)
12 hr/day	0.77	0.60	0.39	0.29
controls	1.00	0.29	0.74	0.49




TRANSMISSION ELECTRON MICROSCOPY

PLATE 1:



Figures 1-2 :(TEM Control Cells)

PLATE 2:



Figures 3-4 :(12 Hour UV treatment)

PLATE 3A:



Figure 5: Control chloroplast

PLATE 3B:



Figures 6-7: control and treatment chloroplast

PLATE 4A:



Figures 8-11: Treated plastids and thylakoids

PLATE 4B:



Figure 12: treatment chloroplast

PLATE 5:



Figures 13-14: Control mitochondria

PLATE 6A:



Figures 15-16: Control pyrenoids


PLATE 6B:



Figure 17: Control Golgi Body

PLATE 7:


Figures 18-19: Treatment nuclei

PLATE 8:



Figure 20: Control starch

PLATE 9:



Figures 21-22: Control trichocyst and body

---

DISCUSSION

• The rather low overall doubling times of the control cells (0.38 doublings per day), compared to previously unpublished results I obtained of a doubling time approaching 1.0 over extended periods for this same species, indicates the effect of the light regime employed in this experiment. Brand and Guillard (1981) found that continuous light resulted in a decrease in the reproductive rates of many different species of phytoplankton (including many dinoflagellates) and they concluded that many species of phytoplankton "need" a dark period. However, the observed differences between the doublings per day of treatment and control cells in this study can not be explained by the light regime alone, but rather, the effects of UV irradiation may play a part. This effect of UV irradiation is similar to that observed by Behrenfeld et al., (1992). Working with an estuarine diatom, they found that when UV-B was enhanced doubling times reached a minimum of 0.24 doublings/day. When UV-A was enhanced, the doublings per day reached a minimum of 0.33. When both UV-A and UV-B were excluded, the growth rate reached a minimum of 0.43 doublings/day. However, caution should be used when comparing this study with Behrenfeld et al., (1992) due to species specific differences.
  
  The most distinguishing difference between the control cells and the 12 hr/day treatment cells was in the size of the vacuolar space. This effect of UV radiation has not been reported in the literature. Since no other factors were different between the control cells and the treatment cells, I am led to conclude that this difference is due to the UV irradiation which the 12 hr/day cells received.
  
  Regarding the breaks in the thylakoid lamellae, I am in agreement with Lesser and Shick (1990) who reported similar effects of UV irradiation. This observation supports the prediction of Gerber and Haeder (1992) that structural damage must be occurring in the thylakoids in order to account for their observation of the impairment of energy transfer between accessory pigments and the reaction centers of photosystem II.
  
  The increased thickening of the thylakoid lamellae which was observed in some treatment cells resembles that which was reported by Michaels and Gibor (1973) and may indicate early stages of thylakoid deterioration and disappearance. Michaels and Gibor (1973) also noticed that UV irradiated plastids were more centrally located than in control cells. The observations in this study, of thylakoid lamellae which were not always parallel to the cell membranes, may be the precursor of plastid migration.
  
  It is difficult to account for the lack of a polysaccharide cap (Dodge and Crawford, 1968) which surrounds the pyrenoid or the rarity of starch or lipid granules which were abundant in the micrographs published by Dodge and Crawford (1968) because this phenomena occurred in both treatment and control cells. The nature of the light regime (24 hr/day exposure to light) may have played a part in this although the opposite effect would be expected (cells overflowing with starch!).
  
  The lack of flagella in this study can only be accounted for by the fixation technique which involved vigorous centrifugation. This may have dislodged the flagella from their transverse and longitudinal grooves. Experimental design and the methods employed in this experiment contain much room for improvement. First, it is probable that large amounts of UV-B (280-320 nm) were excluded from the experimental flasks because the latter were made of UV-B opaque glass, leaving the narrow opening of the flasks as the only accessible pathway to the cell culture. However, Helbling et al. (1992) found that pyrex glass was 20 % to 80 % transparent to wavelengths from 300 nm to 400 nm respectively. This is primarily UV-A (320-400 nm) which was responsible for over 50 % of the total inhibition of photosynthesis in natural phytoplankton assemblages in their study. It is possible that most (but not all) of the observed inhibition of growth and the ultrastructural effects observed in this study were due to UV-A.
  
  Second, in my efforts to achieve maximal growth rates in a short amount of time by employing a 24 hr/day light regime, I may have induced sub optimal conditions for growth. Any future studies of this nature should probably apply a 14:10 D:L light regime.
  
  Finally, a better method of achieving high densities of phytoplankton growth is to use large (1 L) open beakers equipped with a stirring device. If left for a sufficient enough time, the culture may reach sufficient densities to ensure that there will be more than enough cells per section rather than having to make countless sections in order to obtain reasonable results.
  
  In conclusion it is probable that UV-A (and smaller amounts of UV-B) had a significant effect on phytoplankton growth rates. These radiations may have also caused the observed differences in vacuolar space and the observed thickening and breakage of the thylakoid lamellae.

---

CITATIONS

Behrenfeld, M.J., J.T. Hardy and H. Lee II. (1992). Chronic effects of ultraviolet-B radiation on growth and cell volume of Phaeodactylum tricornutum (Bacillariophyceae). Journal of Phycology. 28:757-760.

Blakefield and Calkins (1992) Dodge, J.D. and R.M. Crawford. (1968). Fine structure of the dinoflagellate Amphidinium carteri Hulbert. Protistologica. T. IV, fasc. 2: 231-248.

Bold, H.C. and M.J. Wynne. (1985). Introduction to the algae, 2nd ed., Prentice-Hall, Englewood Cliffs.

Brand, L.E. and R.L. Guillard. (1981). The effects of continuous light and light intensity on the reproduction rates of twenty-two species of marine phytoplankton. Journal of Experimental Marine Biology and Ecology. 50:119-132.

Gerber, S. and D.P. Haeder. (1992). UV effects on photosynthesis, proteins and pigmentation in the flagellate Euglena gracilis: biochemical and spectroscopic observations. Biochemistry and Systems Ecology. 20: 485-492.

Guillard, R.R. and J.H. Ryther. (1962). Studies on marine planktonic diatoms. 1. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Canadian Journal of Microbiology. 8:229-239.

Haeder, D.P. and M. Haeder. (1991). Effects of solar and artificial UV radiation on motility and pigmentation in the marine cryptoflagellate Cryptomonas maculata. Environmental and Experimental Botany. 31: 33-41.

Haeder, D.P. and S.M. Liu. (1990). Effects of artificial and solar UV-B radiation on the gravitactic orientation of the dinoflagellate, Peridinium gatunense. FEMS Microbiological Ecology. 73: 331-338.

Helbling, E.W., V. Villafane, M. Ferrario and O. Holm-Hansen. (1992). Impact of natural ultraviolet radiation on rates of photosynthesis and on specific marine phytoplankton species. Marine Ecology Progress Series. 80:89-100.

Jokiel, P. and R. York, Jr. (1984). Importance of ultraviolet radiation in photoinhibition of microalgal growth. Limnology and Oceanography. 29: 192-199.

Lesser, M.P. and J.M. Shick. (1990). Effects of visible and ultraviolet radiation on the ultrastructure of zooxanthellae (Symbiodinium sp.) in culture and in situ. Cell and Tissue Research. 261: 501-508.

Michaels, A. and A. Gibor. (1973). Ultrastructural changes in Euglena After ultraviolet irradiation. Journal of cell science. 13:799-809.

Smith, R.C. and K.S. Baker. (1979). Penetration of UV-B and biologically effective dose-rates in natural waters. Photochemistry and Photobiology. 29:311-323.

Smith, R.C., K.S. Baker, O. Holm-Hansen and R.J. Olson. (1980). Photoinhibition of photosynthesis in natural waters. Photochemistry and Photobiology. 31:585-592.

List of Abbreviations Used