Algae (The Korean Journal of Phycology) ‘Volume 11(1): 23-34, 1996 Morphological Variations of the Marine Diatom Thalassiosira weissflogii under Culture Conditions Jae-Shin Kang!, Hyung-Sun Kim? and Jin Hwan Lee"* Department of Biology, Sangmyung University, Seoul 110-743, Korea °Department of Biotechnology, Koren Ocean Research & Development Institute, Ansan 425-170, Korea The diatom Thalasiosina weisslagi was cultured under different nitrate-nitrogen, phosphate-phosphorus and sii- ‘atesilica concentrations, salinity and water temperature. Specific growth rate was obtained for each group, and specimens were taken from each group while in the stationary phase and were observed with a scanning electron microscope to detect morphological variations, The result showed that the tendency for the average diameter to decrease with a higher specific growth rate was more distinc than the direct influence of nutrient concentation on, cell diameter. There were variations in the number of central strutted processes under limited N, P, and Si condi tions, The number of central strutted processes showed variations according to culture temperature showing from ‘no central strutted pracess at a low temperature of 10°C to one central strutted process at a high temperature of 30°C. A variation of number central strutted processes occurred at a low salinity (5-10%), also. Under limited phsphate-phosphorus conditions, a variation in which the central strutted process moved toward the margin ‘occurred. Also, seashell-shaped areolation was found. When the central strutted process shifted toward the margin, areolation changed to scallop-shaped, also, When silicate silica was limited, the girdle of the cells elongated 310 times their normal length. Under low temperature conditions, a variation of the areolation to the scallop-shaped form occurred, mostly in cells with no central strutted process. Key Words: diatom, nutrients, culture, morphology, variations INTRODUCTION Diatom morphology has been continuously studied during the past few centuries; therefore, the system of, classification has been formed thoroughly on the basis of the morphological pattern of diatoms (Simonsen, 1979; Round et a, 1990). Systematics has progressed with the developement of the microscope. Particularly, the elec~ tron microscope has made a great contribution to verify the fine structures of the diatom, and diatom taxonomy was more specific than classical taxonomy which only used a light microscope (Round, 1970). These studies, however, focused on differentiation among morphologi- cal patterns, and physiological phenotypes, such as aux- ‘ospore morphological variation, were ignored. Holmes and Reimann(1966) found a auxospore forms when Dedicated to Prof, In Kyu Lee on the occasion of his sixtieth birthday. “Address for reprint request ‘marine planktonic diatom Coscinadiscus was cultured in a different environmental condition for the first time. The shapes were considerably different from vegetative calls. (On the other hand, during the following decades, sev- eral other diatom species proved to be a much better, food for larval growth and for bivalve molluscas and shrimps (Guillard and Ryther, 1962; VanAlstyne, 1986) Physiological response when essential nutrition such as nitrogen, phosphorus or silicon were limited was experi- mented diversely in proportion to developing the cul- ture technique. For instance, Tilman et al. (1976) studied about phosphate and silicate growth and uptake kinetics of the diatoms Asterionella formosa and Cycloella menegh- iniana in batch and semicontinous culture. For limiting phosphate or silicate experiments, growth rate was observed at the minimum level and A. formosa was better able to grow at low POP concentrations than C. menegh- iniana. On the other hand, C. meneghiniana could better grow at low silicate concentrations than A. formosa Several studies ofthe growth rate of Skeletonema costatumel Se 24 Algae Vol. 11(1), 1996 as a function of nutrients have been made (Davis et al, 10973; Davis, 1976). In the case of S. costatum, functions of physical conditions, such as, light and temperature in cells, were studied (tts et al, 1964; Davis, 1976). Thalassiosita weissflogii was studied at various points of view because it grows easily at high temperatures, and low salinity-enriched media (Anderson and Morel, 1982). Fuhs (1969) studied about phosphorus content and rate of growth in diatom 7. weissflogii; as a tesult, while no growth occurs at a certain minimum content of, bound P, maximum growth rate is observed at 3 times the minimum level of P. Putt and Prezelin(1988) investi- gated diel patterns of photosynthesis and cell division in T. weissflogii. It was used when the effects of heavy metal were studied. For instance, several trace metals such as Fe, Cu, Mn, Mo, and Co are required in trace amounts for algal growth. Cadmium, a toxic metal, inhibits iron uptake and assimilation in 7. weissflogit (Harrison and Morel, 1983). That is, cadmium limited the growth rate of T. weissflogi by the deficiency of trace element metals. Also, T. weissflogii responds to iron limitation with deceeasing growth rate, decreasing quotas of cellular iron, and increasing rates of maximum short term uptake (Harrion and Morel, 1986). ‘A study on the morphological variation of the marine diatom cultured under different conditions was carried out with various species. Schmid (1982) studied wall morphogenesis in Thalassiosira eecentrica using an MT- inhi around the areolation, and there was some dislocation of the apiculis. Fryxelt et al. (1984) observed Thalassiosira fumnida from field and clonal cultures. The sesults, when they cultured 7: tumida, showed that it is morphological- ly Variable with heaviliy silicified valves and a lightly silicified valve occurring, in one chain. Also, their areola- tion had changed from a linear form, to radial form and the labiate process tended to be on long internal “necks”, Vitlareat and Fryxell (1990) observed Porosira glacialis from field and clonal cultures. The results showed a vari- ation of the cingulum pattern in culture species. This experiment examined the wall morphogenesis in T. weissfogii grown in a standard medium, media with dif- ferent nutrients, salinity and temperature. Comparisons will be made between field samples and laboratory cul- tures of T. weisflogii tor. In this case, most of the variation occured MATERIALS AND METHODS Thalas osira weisflogit (Grunow) Fryxell and Hasle were procured in Dec. 1989 from the Culture Collection of Algae and Protozoa (CCAP 1085/1), which was grown in an f/2-medium (Guillard and Ryther, 1962). ‘The cells were maintained as unialgal cultures in 30m! test tubes. The cultures were grown in a Low ‘Temperature Incubator at 20#1°C (Fisher, model 307), and ata light intensity of 3000Lux of White’ fluoroscent light illuminated from below. A light-dark cycle of 24hh = L:D was used On the nitrate-nitrogen, phosphate-phosphorus, sili- cate-silica concentration, the media was divided into four groups: zero, f/2, f, and 2f media. The media was made of nitrate-nitrogen concentrations using NaNO ranging from 0.98 mgN/I to 39.03 mgN/I. In the case of phosphate-phosphorus, NazHPOy12H;0 was used in concentrations ranging from 1.36 mgP/I to 20.64 mgP/ For silicate-silica, Na;SiO;-12H,O was used, and the range was fcom 3.12 mgSi/I to 157.61 mgSi/l. Each medium was given a pre-treatment before measuring concentration with @ spectrophotometer (Hech, DR/2000). Salinity effects were studied by culturing 7 groups, ranging from 5% to 35%. Temperature effects were also studied by culturing the samples at 10°C, 20°C and 30°C in an incubator. Light intensity and L:D cycle were made to coincide with the inocula conditions. Each medium was put into a 250 ml Erlenmyer flask and capped with a cotton plug before being sterilized at 121°C, 15 Ib, for 15 minutes. Each medium was cultured for 14 days after inoculation of 1% 10° cells/ml initially Each sample was counted daily with a Palmer-Maloney chamber and then a growth curve was drawn from the data obtained, In order to minimize experimental error, each sample was counted three times and the average number of cells was obtained. The specific growth rate was obtained using the Guillard (1973) method, the for- mula of which is as follows; Specific growth rate = 3.322 log(N./Ny) / (te—t) 3.322 : constant ty: initial times of exponential stage th rending times of exponential stage Nj :cell number of t N; :cell number of For the scaning electron microscope (SEM) study, both rinsed and cleaned material was prepared by modifying methods of Hasle and Fryxell (1970) and Simonsen (1974). Subsamples for the SEM study were dehydrated, ‘mounted and coated with gold, and then were observedusing a SEM (PHILIPS SEM 515) at the Korea Ocean, Research and Development Institute (KORDI). For the observations of morphological variations, LM and SEM pictures were taken of the present study. A specimens ‘were used in the stationary phase. Fach variation was given a test of significance using an ANOVA statistics, programme. Light intensity and salinity at every interval ‘were measured using a Quantum-meter (LI-190SA) and a salinometer (YSI model 33), respectively RESULTS 1. Growth rate of T: weissflogii The specific growth rate of T. weissflogii under normal culture conditions (20°C, 30%, f/2 media) was 0.28. The specific growth rate of each interval is given in Table 1 On nitrogen, each specific growth rate which was cul- tured in f/2 and f media (0.28, 0.29) was higher than those of groups 2f enriched media (0.23). And those growth rates were higher than sterilized seawater media (020). T. weissflogié was initiated on the 3rd day of the exponential phase and was finished on the 8th day in the same concentrations of nitrogen and sterilized seawater. But, in the case of f media, it was reached on the 12th day of the above phase after inoculation. And also the interval of siicate-silica and phosphate-phosphorus was similar to nitrogen concentration. Spec phosphate-phosphorus and silicate-silica sufficient cul- tures, like f/2 and f media, were higher than these of sterilized seawater. On the other hend, when they were cultured in the 2f extreme enriched media it became ower than in {/2 and f media. In salinity, the specific growth rate was the highest at 5%, which means that a specific growth rate increased in lower salinity. On tem perature interval, the specific growth rate was remark: ably low at 10°C, whereas it showed a rapid growth rate of 032 at 30°C growth rate of 2. The normal vegetative morphology of T. weissflogii The valve view of T. weissflogii was almost flat or slightly concave when it was grown in 20°C, 30% and £/2 medium (Figs 1-2). The areolae of the cell valve was poorly developed and in radial form. One labiate process, was located the ring of the marginal strutted processes, usally closer to one of them. Central strutted processes of the valve center were 2-11, Cells were drum-shape and length of girdle shorter than cell diameter. Diameters of valve were 5-32 jum, Number of areolae 30-40 in 10 jm on valve face. Cells have two rings of processes Kang etal: Morphological Variations of Thalossiosin weisflogit 25 ‘Table 1. Specific growth rate of T. weissflogit due to nutrient concentration, salinity and temperature Nutrients Media Specific growth rate NO,-N seawater 020 £2 0.28 ' 029 2F 023 PO,-P seawater oas 42 028 € 030 Fd 023 SiO -$i seawater 018 2 028 f 032 2 020 Ts Intervals Specific growth rate ‘Temperature (°C) 10 on 20 028 30 032 5 037 10 034 032 Salinity (%) 030 030 30 028 35 020 Marginal strutted process including one labiate process. Number of central strutted process was 2-28. Number of marginal strutted process was 9-16 in 10 um Comparisons of a specimen from the initial day, a specimen from the standard condition, and those of nat- ural seawater were 6-13 jm, 45-12 ym and 5-32 um in diameter range and 2-12, 2-13, 2-28 in the range of num- ber of central strutted process, respectively (Fig. 3). Valve surface in the above conditions showed that areo- lation was almost radial in form. Also, these specimens have two more central strutted processes, There was no ‘morphological variation other than the specimens from the initial day having a rather large average cell diame- ter, 10 uum 3. Morphological variations due to culturing conditions Nitrate-nitrogen: When T. weissflogii was grown in media of different nitrogen concentrations, the highest specific growth rate showed in the 18.45 mgN/1 group and the smallest cell diameter was found in this interval, (On the other hand, the sea water (0.98 mgN/l) group with the lowest specific growth rate showed the greatest26 Algae Vol. 11(1), 1996 Figs 1-2. Specimen of subsamples from initial day. Fig, 1. External view of valve and central strutted processes (arrow). Fig. 2 Internal view of valve and a marginal labiate process (arrow). Fig. 3. Extemal view of specimen of the cultured subsample from standard experimental group. Fig. 4. Specimen from limited Nitrate-nitrogen experimental group. showing the external view of valve and one central strutted process (arrow). Figs 5-6, Specimens from limited Phosphate-phosphorus experimental group. Fig 5. External view of valve having one central strutted process (arrow) and scallap-shaped arcolation (60" tilt). Fig, 6, External view of valve having two central strutted processes (arrow) and scalloped-shaped areolation. Seale bar=1 sum (Fig, 2, 4,6), and 2 en (Figs LKang etal: Morphological Variations of Thalassiosira weissfogit 27 ‘Table 2. Culture of T. weissfogi in the media of different nitrate-nitrogen concentrations (+: average of central. strutted process amber) 0.98 mgP/1 Average diameter (um) 839 Range of diameter (um) 4511 Range of central strutted 03, process number (+) (495) Variation of central 6 strutted process number ‘Number of 2 arcoletion variation Number of central - strutted process situation. variation 0.60 mgP/l 1845 mgP/I 39.05 mgP/1 683 579 oat 451 55.1 2.10 0.10 ss) (oa) : : 4 Table 3. Culture of T. weissfogii in the media of different phosphate-phosphorus concentrations (+ average of central. strutted process number) 1.36 mgP/1 Average diameter (um) 863 Range of diameter (um) 512 ‘Range of central strutted 03 process number (+) (401) Variation of central 10 strutted process number ‘Number of ” areolation variation Number of central “ strutted process situation variation average cell diameter (Table 2). The ranges of cell diameter showed few differences in each different interval group. The number of central strutted processes of specimens which were grown, under limited nitrogen conditions and enriched condi- tions varied from zero to one (Fig. 4). There were no dif- ferences in central strutted processes between natural specimens and standard cultured specimens. Also, the central strutted processes did not move toward the valve margin and there was a variation of areolation showing a scallop shape. Phosphate-phosphorus: When T. weissflogii was grown in a different phosphate-phosphorus concentra- tion, the highest specific growth rate in the 10.04 mgP/1 ‘group was 0.30, Coll diameter was the smallest in this interval of the 5.63 uum. On the contrary, the 20.64 mgP/1 S.16mgP/I 10.04mgP/1 20.64mgP/1 6.83 565 97 4512 an 613, 243 210 013 6.09) (492) 6.09) sample showed the biggest average cell diameter, 9.47 um (Table 3). There were no significant differences in the average diameter among the 4 groups. The average number of central strutted processes of the 1.36 mgP/1 group was rather smaller than those of the other 3 groups. When the concentration of phosphate-phospho- rus was excessive or insufficient, the central strutted processes showed a tendency to move toward the mar- ‘gin and showed that areolation was scallop-shaped (Figs, 5.6). In particular, there were frequent variations in the phosphate-phosphorus limited group (1.36 mgP/1). ‘When the central strutted processes moved toward the margin, areolation variation appeared to be scallop- shaped. Silicate Silicate-silica showed tendencies simi- lar to those of nitrogen and phosphate-phosphorus28 Algae Vol. 11(1),1996 Table 4. Culture of T. weisflagié in the media of different silicate-silca concentrations (+ ber) 3. mgsi/1 ‘Average diameter (4m) 867 Range of diameter (urn) 6514 Range of central strutted 09 process number (*) 4.73) Variation of central 0 strutted process number Number of| 2 areolation variation Number of central strutted process situation variation average of central. strutted process num- 157-61 mgSi/1 4250 mgSi/t 7882 mgSi/l 683 6.17 8.60 a 452 sar ssa 23 28 os 6) as) 63 ‘Table 5. A girdle variations of T,weisflagii due to silicate-silica,nitrate-nitrogen, and phosphate-phosphorus concentration 0.98mgN/l —1.36mgP/I Lengthened girdle 12% 9 3.12mgsi/ 4250mgSi/1 __78.82mgSi/1__ 157 61mgSi/ 365% 4 6% Table 6. Culture of T.awisslogt in the media of different salinities (+ : average of central strutted process number) Salinity (%) 10 Average diameter (m) 571 6.09 Range of diameter (m) 4512 4541 Range of central strutted 08 07 process number (+) 295) a6) Variation of central 28 8 strutted process number Number of 3 2 areolation variation Number of centeal - : strutted process situation variation (Table 4). The highest specific growth rate in the 78.82 mgSi/% group was 0.32. Cell diameters were the small- est in this interval of 6.17 jum. On the contrary, the 3.12 mgSi/% sample showed the biggest average cell diame- ter, 8.67 sm. There were no significant differences in the average diameter among the 4 groups. When the concen- tration of silicate-silica was limited, the variation showed that the number of central strutted processes is zero oF ‘one (10%). In contrast to groups with limited phosphate- phosphorus, when the concentration of silicate-silica was limited, the central strutted processes did not show a tendency to move toward the margin and showed a few 15 20 2B 30 35 607 7.09 642 6.83 826 4 5 4541 4512 5415 08 09 29 23 os «2 (496) 6a 6.09) (495) 5 2 : = 1 areolations which were scallop-shaped, Also, in the media with a different silicate concentration, the girdle of 36.5% cells in the seawater (3.12 mgSi/1) group had lengthened to 3-10 times that of normal cells (Figs 7-12, Table 5). This figure is almost negligible in comparison with those of the silicate-silica. It was observed 3-4 times more frequently in groups with limited silicate-silica than in groups with limited nitrate-nitrogen or phos- phate-phosphorus, Salinity: Among 7 different salinity intervals, 5% of the group showed the highest specific growth rate of 0.37, and the cell diameter was the smallest in this inter-Kang etal: Morphological Variations of Thalassiosira weissflogii 29 Figs 7-12. Specimens from limited Silicated-silica experimental group. Fig.7. Extemal view of valve having one central strutted process (arrow). Fig. 8. External view of lenthened girdle. Fig, 9. External view of enthened girdle. Fig. 10. Middle part of girdle of Fig. 9 specimen. Fig. 11. Upside valve of Fig, 9 specimen showing one irregularcentral strutted process located in near mar- sin. Fig. 12, Contrary valve of Fig. 9 specimen having irregular central strutted processes. Scale bars jum (Figs 10, 11), 2 mm. (Figs 7, 12),5 mm (Fig. 8) and 10 mm (Fig. 9).30. Algae Vol. 11(1), 1996 Figs 13-14. Specimens from low salinity experimental group. Fig. 13. External view of valve having one central strutted process (arrow). Fig. 14, External view of valve having no central strutted process. Fig, 15. External view of valve ofthe cultured speci mens from under 30°C temperature. Showing valve having no central strutted process. Fig. 16 Extemal view of valve ofthe cul- tured specimen from under 10°C temperature. Showing valve having one central strutted process without areolation variation. Figs 17-18. Auxospores. Fig. 17. External view of girdle of the cultured specimen under 5% salinity. Fig. 18. External view of whole cell of the cultured specimen under 10°C temperature, Scale bar=1 jem (Fig, 13-16, 18) and 5 ym (Fig. 17)‘Table 7. Culture of T. weissflogii under different temperatures (+ average of central strutted process number) Temperature (C) o 9 # Average diameter (um) 847683596 Range of diameter (um) on 452 4541 Range of central strutted os 198 process number 695) 60) C7) Variation ofcental ton 3 - 8 strutted processmumber one 12 = 5 Number of areolation woe . variation Number of central 6 - - strutted process situation val (571 jum). The average cell diameter showed a ten- dency to decrease with decreasing salinity (Table 6). The range of diameter showed few differences among differ- ent salinity intervals. The average number of central strutted processes in the 5% group was rather smaller that those of the other groups. When they were cultured in the lower salinity media, variations of central strutted processes were found at large quantities. 28% of the 5% group showed variations in the number of central strut- ted processes and 67% of that group showed a variation with no central strutted processes (Figs 13-14) Temperature Specifit growth rate increased as the temperature went up and the cell diameters decreased in the same case (Table 7). The lowest specific growth rate ‘was in the 10°C group and then the average cell diame- ter was the largest. The range of diameter showed that the minimum cell was longer than the other group (6 im). The average number of central strutted processes in the 10°C and 30°C groups was rather smaller than those of the other groups. The greatest number of areolation variations were in the 10°C group (17%). Among the 10°C group, 6% showed a variation in which the central strutted processes moved toward a margin. 15% and 19% of the cells of the 10°C group and the 30°C group, respectively, showed a variation in the number of central, strutted processes; 80% of the 10°C group showed a vari- ation with ro central strutted process, and 94.8% of the 30°C group showed a variation with one central strutted. process, That is, the 30°C group cells with one central strutted process (Fig, 15) and the 10°C group cells with no central strutted process were the majority (Fig. 16). Kang etal: Morphological Variations of Thalassiosira veissflgii 31 DISCUSSION ‘Temperature, salinity, nutrients, illuminance and light dark cycle have influenced phytoplankton growth and there are also other factors which cause changes in the specific growth rate (Rhee and Gotham, 1981a; Rhee and. Gotham, 1981b). Thalassiosira weissfogii, which was used. in this experiment, showed a different growth rate depending on the nutrients, temperature, and salinity. The effect of nitrogen concentration on the growth of diatom has been studied by Takeda (1970), Caperon and Meyer (1972), Malone et al, (1975), Collos and Lewin (1976) and others, and this effect is also evident in a vari ty of other species. In particular, the result ofthe study by Conover (1975) on the effect of nitrogen on the growth rate of 7. weissflagii coincides with that of this experiment. Among the results of this experiment, the effect of phosphorus on the growth of T. weissflogii ‘matches the results of the study done by Fuhs (1969) ‘using T. weisflogii, and the influence of the silicate-silica concentration has on the specific growth rate of T. wess- flogii coincides with the result of Chisholm et al (1978) using the same species. 7. weissflogii shows a rapid ‘growth tate in enriched nutrient groups (Takano, 1979). Under standard culture conditions, the specific growth rate of T.weissflogi is 0.28, similar to 0.23 of the results of Hur (1991). Also, T. weissflogii showed a high growth rate under low salinity (5-10%) and high temperature (20°C) conditions. This species is caused by a red-tide in the summer in a low salinity concentration of limited nutrients (Hasle, 1962; Takano, 1979). Inthe case of those cultured in enriched media in the laboratory, the same result was induced, This condition is identical to the optimum condition achieved by a preliminary experi- ment carried out during the periods from December 1991 to March 1992. Except for a slight decrease in the average diameter, a sample from the initial day and a sample from the stan dard group are very similar. The average diameter of T. ‘weissflogii in a natural seawater condition is 17-80 ym (Haste, 1962), longer than the initial day sample by 737 ym. The reason for this is, if the reduction in cell size results from repeated cell division, a diatom cell is able to restore its overall size by forming auxospores (Figs 17- 18). Consequently, a diatom in natural seawater is always able to maintain its regular size. But when they are grown in laboratory cultures, they show Poor aux ospore formation. Consequently, cells repeatedly divide32 Algae Vol. 111), 1996 without size restoration (Round et al., 1990). As a result, diatom cells go on decreasing in size. When samples from the initial day were compared with samples cul- tured on the 10th day in a standard form, the cell diame- ter of the sample of the initial day was longer than the others. This phenomenon can also be explained by the same reasoning. Also, because this experiment was car- ried. out by means of a stack culture, the quantity of dis- solved nutrients supplied is limited. For these reasons, the cell diameter of samples cultured for 10 days in stan- dard conditions was shorter than in samples of the initial day. The results were that, except for a slight decrease in the average diameter, there was no morphological varia- tion. Johansen and Theriot (1987) studied the relation- ship between valve diameter and the number of central, strutted processes in T. weissflogit. The results showed that the cultured cell size of T. weissflogit decreased and, the cell has fewer central strutted processes than the nat- ural form. When 7. weissflogii was grown in media of dif ferent nitrogen concentrations, the highest specific growth rate occurred in 18.56 mgN/ and the lowest spe- cific growth rate in 1.01 mgN/I. But, the average cell diameter was in an inverse proportion to the growth rate, that is, if only the average cell diameter was exam- ined. It appears that was a difference among the four {groups but, it could aot be called a ‘variation’, certainly. Because the average cell diameter was in inverse propor- tion to the growth rate, in the case of cells that divided vigorously, the average cell diameter decreased more sharply than those of cells that divided slowly. This phe- nomenon was the same in the case of phosphate-phos- phorus and silicate-silica, as well. That is, the difference in the cell diameter resulting from a culture-differing nutrient concentration condition could not actually be called a ‘variation’. In groups with limited nitrate-nitro- gen, phosphate-phosphorus and silicate-silica, there ‘were a number of cells with variations of the central strutted process number. Even in the preliminary experi- ‘ment, a variation in the central strutted process number was the most distinct in groups with a low salinity and little nitrate-nitrogen, phosphate-phosphorus. No clear explanation for this has been given, but one can assume that it has something to do with cell division taking place right after the uptake of nitrate-nitrogen, phos- phate-phosphorus and silicate-silica. On salinity, varia- tions in the number of the central strutted processes ‘were most evident in 5%. In the lower salinity groups, there were considerable variations in the central strutted processes which apeared when the cells were carefully observed. On temperature, variations in the central strut- ted processes number turned up at 10°C and 30°C, and 15% of the cells of 10°C showed a variation in the num- ber of central strutted processes. In addition, 80% of these variations showed a variation with no central strutted process at all. 19% of the cells at 30°C showed variation in the number of central strutted processes. Also 94.8% of these variations showed variations with one central strutted process. The results mean that at a high temperature, the cells have one central strutted process, and at a low temperature the cells have no cen- tral strutted processes, Villarreal and Fryxell (1983) stud~ ied temperature effects on the valve structure of the bipolar diatoms Thalassiosira antarctica, In the result, the variations showed that occluded processes and the exter- nal tube of the strutted processes disappeared at a low temperature. In the case of T. weissflogi, variations in the central strutted processes number appeared when nitrate-nitrogen, phosphate-phosphorus and silicate-sli- ca were limited, when salinity was low, or when temper- atures were low or high. When phosphorus was limited, 14% of the cells were observed as having their central strutted process shifted toward the margin. In comparison with result of prelimi- nary test that colchicin had an effect to make a morpho- logical variation in T. zeissflogii, in a sample grown in a standard medium treated with 1x 10% colchicin, 24% showed a variation in the position of the central strutted process and all of their areolation had changed from a radial form to a scallop-shaped one. Although a sample of the group treated with colchicin was similar to those cultured at low phosphorus concentrations, the cells grown in a medium treated with colchicin showed no variation in the number of central strutted processes. That is, because colchicin was used with the object of interrupting formation of the spindle fiber, it seems that a variation of the central strutted process number had nothing to do with the non-disjunction of chromosomes, and the variation of areolation has a corelation with it. In the results of a preliminary experiment, also, when phos- phate-phosphorus was limited, the cells were observed as having their central strutted process shifted toward the margin. And, in an additional low temperature con- dition a lot of cells were shown to have such a variation Regarding temperature, among the 10°C group, 16% showed a variation in which the central strutted process moved toward @ margin. A variation in the central strut- ted process number appreared at 10°C and 30°C, but a variation in the central strutted process positionappreared only at 30°C. That is, a variation in tempera- ture conditions incongruent to the central strutted process showed a change in the process number. But, variation of the central strutted process position appeared only under low temperatures. Also, there were the greatest number of areolation variations in the 10°C. ‘group (17%). 94% of the cells showed that a variation of areolation is a variation of the central strutted process that shifts toward the margin at 10°C. That is, when the central strutted processes shifts toward the margin, areo- lation becomes scallop-shaped as well. Unlike other groups in the limited silicate-silica group, thin cells with elongated girdles were observed in large numbers, Tilman et al. (1976) observed changes in the number of cells per colony number during the short, term culture of Asterionella formosa, and reported that cells that number, on the average, 7-8 per colony number from 20-21 under a limited silicate condition. The importance of salinity in the ecology of phyto- plankton is well known from previous studies done under natural marine conditions, and has been discussed extensively by maniy researchers in culture experiments (Fogg, 1965: Tokuda, 1968). Fasle (1962) reported that T. ‘weissflogit is a mes ohaloic species that causes blooming in 5-20% regions. Takano (1979) reported that it causes a red-tide in low salinity(0.2-10%), eutrophication regions. ‘The result of this experiment also showed a high specific growth rate under low salinity conditions (5-10%). In the lower salinity groups, variations in the central strutted processes apeared in a considerable number of cells. 25% of the 5% group showed a variation in the number of central strutted processes. On the other side, when limit- ed nutrients showed a low specific growth rate and vari- ations of various kinds in salinity when salinity is extremly low, a high specific growth rate and variations in the central strutted processes number were shown. ‘Variations in areolation and the central strutted process position almost did not apprear. Goldman (1977) studied the effect of temperature in phytoplankon culture, and in the case of T. weissflogii, the specific growth rate is high under high temperature conditions (Takano, 1979), This is because when temper- ature is optimal and nutrients are abundant, these two factors interact and cause the specific growth rate to rise (Rhee et a, 1981) Regarding the results of this experiment, variations in the central strutted processes number turned out to be statistically significant in the 5% group, 10°C and 30°C group, limited nitrate-nitrogen, phosphate-phosphorus, Kang ¢f al: Morphological Variations of Thalassosira weisslogif 33 and silicate-silica group. Also, variations in areolation and the central strutted process position was statistically significant. Fryxell et al. (1984) observed Thalassiosira tumida from fietd and clonal cultures. The results, when they cultured T. tumida, showed that it is morphologically variable with heaviliy silicified valves and lightly silicified valves occurring in one chain. Also, their areolation had changed from a linear form to a radial form and the labi- ate process tended to be on long internal “necks” Villareal etal, (1990) observed Porosira glacial from field and clonal cultures, The results showed a variation of the cingulum pattern in the culture species. In the case of experiments using T. weissflagii, morphological variation appeared, as well. The test groups used in this experi ‘ment represent conditions that are all possible in nature, and it-seems that the results could just as well be obtained from a natural marine environment. Although such variations have not yet been fourd, it deserves to be studied more. 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Accepted 15 March 1996.