Ecological functions of ciliated protozoa in marine

HIGH TECHNOLOGYLETTERS| Vol. 11 No. 1| March 2005 　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 101

Ecological functions of ciliated protozoa in marine ecosystem : ① effects on accumulation of ambient ammonia Xu Henglong ( 许恒龙)

3 33

3

, Song Weibo ②3 , Zhu Mingzhuang 3 , Wang Mei 3 , Ma Honggang 3 , Hu Xiaozhong 3

( The Laboratory of Protozoology , KLM , Ocean University of China , Qingdao 266003 , P. R. China) ( 33 Department of Biology , Liaocheng University , Liaocheng 252059 , P. R. China)

Abstract Effects of ciliated protozoa , Euplotes vannus and Uronema marinum , on accumulation of ammonia in marine waters are detected using experimental ecological method , in order to reveal the contributions and functions of ciliates to the marine ecosystem. During experiments , the concentrations of ammonia2N , and the densities of ciliates and bacteria are measured. The results reveal that ciliates can change the procedure of ammonia accumulation by their grazing activity , and maintain ambient ammonium at low lev2 els through interrupting the stationary phase of bacteria population growth and enhancing their growth and metabolism. The present work confirms that ciliates , as bacteria2predators , play positive roles in main2 taining and improving water quality in marine ecosystems , especially in intensive mariculture biotopes. Key words : Euplotes vannus , Uronema marinum , marine ciliates , ammonia , mariculture , experi2 mental ecology

0 　Introduction Ammonia are commonly excreted as the end product of protein catabolism , and may be toxic for organisms in waters if allowed to accumulate , because un2ionized am2 monia can pass through the cell membrane and are harm2 ful due to their fatsoluble property[123 ] . Ammonia toxicity is manifested by hyperactivity , convulsions , loss of equi2 librium , lethargy and coma. However , ammonia toxicity in aquaculture ponds is most likely expressed as the sub2 lethal reduction of fish growth or suppression of immuno2 competence , rather than as acute toxicity leading to mor2 tality. The mechanisms of ammonia toxicity have not yet been firmly clarified. However , a combination of plasma sodium depletion , biochemical effects of Krebs cycle sup2 pression by depletion of α2ketoglutarate resulting in re2 duced ammonia excretion , and other factors are implicat2 ed [4 ] . Moreover , ammonia can change into nitrite , an in2 termediate product and a potentially toxic nitrogenous compound , which may accumulate in aquaculture ponds during nitrification and denitrification. Many studies have demonstrated that these nitrogenous compounds can be2 come toxic and produce a detrimental effect on the growth , reproduction activities of animals or their larvae , or even result in death when reaching a certain level of concentration [1 ,5 ] . Recently , many investigations on the uptake and re2

generation of ammonia in microorganism communities have revealed that the ammonia uptake is mainly performed by picoplankton and nanoplankton , which involves pho2 totrophic ( e. g. unicellular alga with pigments ) , het2 orotrophic ( e. g. species of Zoomasigophorea and Cilio2 phora) and mixotrophic forms ( e. g. most species of Phy2 tomastigophorea) , though it has not come to an identical viewpoint on some important problems[6 ] . Ciliated protozoa , which commonly occur in all kinds of waters especially in eutrophic marine ecosystems , are considered , for a long time , as harmful microorgan2 isms for the cultured animals. Some new studies , howev2 er , have demonstrated that many periphytic forms are playing a positive role for water quality[7210 ] . In the present study , we focus on the effects of ma2 rine ciliates on decreasing and increasing of total ammonia in marine ecosystems with experimental ecological method employing Euplotes vannus Mü ller , 1786 and Uronema marinum Dujarding , 1841 , in order to reveal the contri2 bution of ciliates to the aquaculture systems. 1 　Materials and methods 1 . 1 　Culture and medium Mass cultures of the marine and Uornema marinum , which coastal farming pond ( salinity ca. lavae culturing in the suburb of

ciliate Euplotes vannus were isolated from a 19 ppt) used for prawn Qingdao , China , were

① Supported by the High Technology Research and Development Programme of China (No. 2002AA648010) and the National Natural Science Foundation of China (No. 40206021) .

② To whom correspondence should be addressed. E2mail : wsong @ouc. edu. cn Received on June 27 , 2003

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maintained routinely in bacterized marine water ( salinity 30 ppt ) at room temperature. The stock ciliates were in2 oculated into the medium with 0. 3 g/ l beef extract in or2 der to enrich bacteria as food for ciliates prior to experi2 ment . After 72 hours , cultures were transferred to the in2 cubator and maintained at 11 ℃. Two kinds of media were prepared with none2granu2 lar marine water ( salinity 27. 6 ppt ) , which was filtered through 0. 2 μm filter membrane ( Millipore , Bedford , MA) . Beef extract (BE) medium was prepared by adding 0. 3 ml of the 100 g/ L stock suspension of beef extract to 100 ml non2granular marine water , which was inoculated with bacterium Vibrio sp . at 12 hours before inoculating ciliates. Rice grain medium ( RM ) was prepared by adding 30 rice grains to 100 ml of the filtered marine wa2 ter , which was previously inoculated with Vibrio sp . as above. Stock cultures of Vibrio sp . , a dominant strain in marine water , which was collected from a farming pond for scallop culturing in Qingdao , was maintained at - 3 ℃ on slope in laboratory. Each slope was diluted to 1. 28 × 109 ind. / ml by adding filtered marine water prior to ex2 periment. 100 ml of medium was inoculated with 5 μl of bacteria suspension. 1 . 2 　Experimental procedure Experiments were designed as six treatments , con2 taining two controls. Each treatment was carried out as two independent replicates at 25 ℃ in incubator. BE2 treatment ( control I) , beef extract medium without cili2 ates ; R2treatment ( control II) , rice grain medium without ciliates ; BEM2treatment , beef extract medium inoculated with Uronema marinum ; BEV2treatment , beef extract medium inoculated with Euplotes vannus ; RM2treatment , rice grain medium inoculated with Uronema marinum ; RV2treatment , rice grain medium inoculated with Eu2 plotes vannus . In all treatments the initial densities of cili2 ates were controlled at 10 ind. / L. The beef extract treatments were monitored up to 336 hours ( 14 days) , while rice grain treatments were only examined up to 192 hours ( 8 days) . During experiment , the concentrations of ammonia , which were measured with DR/ 2010 Spectrophotometer ( Hach Company of Ameri2 ca) , were recorded every 12 hours , the densities of cili2 ates that were estimated by counting cells fixed with Lu2 gol’s solution under a stereomicroscope with 0. 1 ml counting glass and the densities of bacteria that were esti2 mated by FLB method were also measured every 12 hours[11 ] . 1 . 3 　Statistical analysis All data were subjected to one2way analysis of vari2

ance (ANOVA) . If significant differences were indicated at P < 0105 level , Duncan ’s Multiple Range test was then used to identify significant difference among treat2 ments. Two2way ANOVA was used to identify the signifi2 cant difference between controls and ciliate2treatments. All statistical significance tests were at P < 0105 level . 2 　Results 2 . 1 　Accumulation of ammonia All changing curves of ammonia concentrations are shown in Figs11 and 2. Statistical analysis revealed that there was significant difference in the changes of ammonia concentrations not only between the beef extract treatment and the rice grain treatment but also between ciliate2treat2 ment and the control , respectively. In all treatments , there was no significant difference between two species of ciliates ( Figs11 & 2) .

Fig. 1 　Accumulation of ammonia2N concentrations in all beef extract media up to 336 hours

Fig. 2 　Accumulation of ammonia2N concentra2 tions in all rice grain media up to 192 hours

In beef extract treatment , the ammonia concentra2 tions were distinctly higher than those in rice grain treat2 ment all through the experimental time , which remained



increasing till 168 hours after experiment and then turned into more or less stable status ( Fig11 ) . In ciliate2treat2 ment , the concentrations of ammonia remained evidently lower than those in controls after 240 hours ( second stage ) . Statistical analysis demonstrated that there was significant difference in changes of ammonia concentration between ciliate2treatment and their control at p < 0105 level ( F value = 5144 ; p = 0102 ) during the second stage. But there was not much difference between BEM2 and BEV2treatment . In rice grain treatments , however , the concentrations of ammonia remained at lower level than in beef extract treatment , which was decreasing gradually for 154 hours and then entered the stationary phase ( Fig12) . In ciliate2 treatments , the concentrations of ammonia were signifi2 cantly higher than that in controls in the first stage , which lasted up to 36 hours in RM2treatment and 144 hours in RV2treatment , and then dropped sharply into the second stage , in which the ammonia level was lower than that in controls. Statistical analysis revealed that there was sig2 nificant difference in changes of ammonia concentration between ciliate2treatment and control at the level of P < 0105 ( F value = 11. 87 ; P = 0. 001) during the second stage. 2 . 2 　Effects of ciliates on ammonia accumulation The accumulation rate ( mg ammonia2N l - 1 h - 1 ) of ammonia levels every 24 hours in all treatments were shown in Figs13 and 4. The changes in ciliate2treatments alternated in a larger range than in control . In all treat2 ments , including controls , the values decreased along with all cultures.

Fig. 3 　Accumulation rate of ammonia in all beef ex2 tract media up to 336 hours every 24 hours

Comparison between changes of ammonia concentra2 tion and the population growth of ciliates in all treatments is represented in Figs15～8. It was shown that the accu2 mulation rate reached the maximum during the logarithmic phase of ciliate growth , then decreased to a low level

when the abundance of ciliates reached maximum , and fi2 nally increased again to a peak level when the densities of ciliates dropped. It was assumed that in logarithmic phase of ciliate growth the bacteria Vibrio sp . was stimulated to the active reproduction status. The predation of ciliates may enhance the catabolism of bacteria and elevate accu2 mulation rate of ammonia in water. As ciliates reached a maximum of density and entered the stationary phase , the density of Vibrio sp . decreased sharply to the lowest level but their size turned to be small ( Figs19 ～ 14 ) . It was considered that uptake of ammonia was associated with the development of small2size bacteria , which was more active than old cells on anabolism. When the growth of ciliate turned to declining phase , the density of bacteria renewed to a high level because the predatory activity of ciliates was weakened and catabolism of bacteria was renewed.

Fig. 4 　Accumulation rate of ammonia in all rice grain media up to 192 hours every 24 hours

Fig. 5 　Comparison between accumulation rate of ammonia (BEV2NH3 ・H) and densities of Euplotes vannus (BEV2Ciliate) in beef ex2 tract treatment for 336 hours

Correlation analysis reveals that there was a low cor2 relation between accumulation rate and densities of cili2 ates , where r ( correlation coefficient ) < 014 except for RM2treatment , in which the correlation is negative , r =



- 0175. It appeared that the changes of ammonia were impacted by population growth of ciliate , but were not ev2 idently in proportion to the densities of ciliates.

2 . 3 　Effects of bacteria on ammonia accumulation Figs19～14 exhibit the comparison between accumu2 lation rate of ammonia and population growth of Vibrio sp . In all controls , the accumulation rate curves of ammonia showed a good synchronism with the growth curves of bac2 teria , and the changing values decreased slowly with the decreasing of density of bacteria ( Figs. 9～14) .

Fig. 6 　Comparison between accumulation rate of ammonia (BEM2NH3 ・H) and densities of Uronema marinum in beef extract treat2 ment (BEV2Ciliate) for 336 hours Fig. 9 　Comparison between accumulation rate of am2 monia ( BE2NH3 ・H) and densities of Vibrio sp . (BE2Vibrio) in control I

Fig. 7 　Comparison between accumulation rate of ammonia ( RV2NH3 ・H) and densities of Euplotes vannus ( RV2Ciliate ) in rice treatment for 192 hours

Fig. 8 　Comparison between accumulation rate of ammonia ( RM2NH3 ・H) and densities of Uronema marinum ( RM2Ciliate ) in rice treatment for 192 hours

Fig. 10 　Comparison between accumulation rate of am2 monia (BEV2NH3 ・H) and densities of Vibrio sp . (BEV2Vibrio) in beef extract treatment

Fig. 11 　Comparison between accumulation rate of am2 monia (BEM2NH3 ・H) and densities of Vibrio sp . (BEM2Vibrio) in beef extract treatment



teria.

Fig. 12 　Comparison between accumulation rate of ammonia ( R2NH3 ・H) and densities of Vibrio sp . (BEV2Vibrio) in control II

Analysis reveals that the correlation coefficients be2 tween changes of ammonia and densities of bacteria were 0. 42 , 0. 71 and 0. 21 in BE2 , BEV2 and BEM2treat2 ment , respectively. It was possible that bacteria played an important role in accumulation of ammonia in water though the correlation coefficient remained at low level in BEM2 treatment , which indicates that the anabolism of bacteria maintains at higher level than in BEV2treatment due to the higher natural growth rate of Uronema marinum . The curves of ammonia accumulation rate represent , however , another cases with the growth curves of Vibrio sp . in ciliate2treatment with rice grains , which may be due to shortage of nitrogenous source ( Figs112～14) . In the stationary phase of Euplotes vannus , the accumulation rate was elevated in RV2treatment . Catabolism of Vibrio sp . may be enhanced due to either their active develop2 ment or reproduction under the presence of ciliates with lower natural increasing rate of population growth. The population of Uronema marinum expanded more quickly than Euplotes vannus , which should enhance the uptake of ammonia in the anabolism of bacteria in RM2treatment . 3 　Discussion 3 . 1 　Positive role of ciliated protozoa in depurating water

Fig. 13 　Comparison between accumulation rate of am2 monia ( RV2NH3 ・H) and densities of Vibrio sp . ( RV2Vibrio) in rice treatment

Fig. 14 　Comparison between c accumulation rate of ammonia ( RM2NH3 ・H ) and densities of Vibrio sp . ( RM2Vibrio) in rice treatment

　　 In ciliate2treatment , the density of bacteria de2 creased sharply after inoculating ciliates , but the accumu2 lation rate did not drop down with it . It was likely that the ammonia catabolism of bacteria was enhanced by predation of ciliates. In the declining phase of ciliate , the accumu2 lation rate was elevated with the increasing density of bac2

quality

Recent studies have revealed that ammonium regen2 eration in micro2 and nano2plankton fractions was mainly associated with ciliates and in the picoplankton fraction with bacteria [9 ] . Macrozooplankton dynamics appears to regulate ammonium regeneration by ciliates and bacteria , which means that ciliated protozoa might play an important role in ammonium regeneration[9 ] . The present study has confirmed the previous assumption that ciliates decrease the ammonia accumulation. Analysis of the data has re2 vealed that ciliates could accelerate the metabolism of bacteria through interrupting the stationary phase of bacte2 ria growth. It is reasonable to believe that catabolism and anabolism of bacteria could be enhanced for their either active reproduction or development which might be acti2 vated by the grazing activities of ciliates , especially in logarithmic phase of ciliate growth. Generally , old bacte2 ria were dominant in stationary phase of their population growth , which need only a small amount of materials and energy for maintaining their living activities especially ni2 trogenous source , while puisne forms would obtain a large amount of structure materials and energy for its own growth. Therefore , the present work makes it clear that the ciliate protozoa make a great contribution to the de2 crease of ammonia in marine ecosystems through changing the procedure of ammonia accumulation.



3 . 2 　About the Accumulation of Ammonia Corresponding

( 2) Ciliated protozoa play important roles in acceler2 ating decontamination of water quality in marine waters.

to Different C : N Ratios

Most microorganisms oxidize carbohydrates to provide energy to the cell . Carbohydrate catabolism , the break2 down of carbohydrate molecules to produce energy , is therefore of great importance in cell metabolism. Glucose is the carbohydrate energy source used by cells though mi2 croorganisms can also catabolize various lipids and pro2 teins for energy production[12 ] . Proteins are too large to pass unaidedly through plas2 ma membranes. Microbes produce extracellular proteases and peptidases , which break down proteins into their component amino acids , which can cross the membrane. However , before amino acids can be catabolized , they must be converted to substances that can enter the Krebs cycle. In one such conversion , the amino group of an amino acid is removed and converted to an ammonium ion , which can be excreted from the cell . The remaining organic acid can enter the Krebs cycle [12 ] . In the present study , the medium with low C :N ratio ( beef extract medium) , and the medium with high C :N ratio ( rice grain medium) were used for all examinations. The results demonstrate that the accumulation of ammonia would reach a higher level in beef extract medium than in rice grain medium. Uptake and regeneration of ammonia were commonly concerned with the catabolism and an2 abolism of bacteria. In the medium with low C :N ratio , microorganisms can only obtain both materials and energy from catabolism of nitrogen2rich organic materials , e1g. beef extract and peptone. It is inevitable that the lower C :N ratio leads to excessive accumulation of ammonia. Therefore , it is very important in controlling the accumu2 lation of ammonia to make clear the property of organic pollutants in a marine ecosystem. 4 　Conclusion (1 ) The population growth of ciliated protozoa can change the procedure of ammonia accumulation and main2 tain ambient ammonium at low levels.

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Xu Henglong , born in 1964. PhD , associate pro2 fessor. His main research interests include ecology and toxicology of protists especially investigating protists in sewage treatment .
