Article信州大学理学部附属諏訪臨湖実験所報告 9: 77-85(1995)departmental bulletin pape

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Myeong-seop,  BYEON

seung-IK,  CHOI

Tae-seok,  AHN

English

Shinshu University Institutional Repository

Rep. Su}ya l()'drobioL. 9: 77N85 (i99E)Preceedings of tite 7tfi Intentatiottal Sytnpasiutnon Riyer and Lake Environments, IP94, MatsumotoThe effects of the silver carp (Hypophthalmichthys molitrix)predation on the extracellular enzymatic activitiesSeung-Ik CHOI, Myeong-Seop BYEON and Tae-Seok AHNDept. of Environmental Science, Kangwon National University,Chunchon, 200-70l, Korea, Fax;+82-361-51-3991.INTRODUCTION   The ecological character of the aquatic environments and thequality of water are deterrained by the amount of the productionand the rate ofi organic materials decomposition. Particularly,in large reservoirs, water quality strongly depends on thestructure and the function (energy transfer efficiency) of £oodwebs. And these food webs are controlled by resource lindtation('ibottom-up") and by predation (Htop-down"). Solar energy,nutrient inputs and dynamics of an eeosystem set lts overalllevel of production, so to that extent the control may beenvi$aged as bottom-up. However, among those limits, sovae o£ theitcoarse-tuning'i and much of the "fine-tuningt' of strucicure aRdfunction re$ult fyova the cornplexity of top-down effects [12].ConsequeRtly, a deeper insight into that complexity of top-downprocesses i$ absoluteiy necessary xf the$e proce$ses should beused in biemanipulation as a new tool in water qualitymanagement [3]. By this time, rnost of the previous research onittop-down't proce$ses were focused on direct effects of predation.However, impacts of the mediation on the Rutrient regenerationand on the quality and quantity of food bases of differenttrophic levels have noic been so widely studied [3].   The aim of this study was to define a small but irnportantpart of tttop-down" process ; the effects of fi$h predation onphytoplankton, navaely, the predation induced changes of thephysiaochemical and extracellular enzyraes activities. We useda herbivorous fish (Hypophthalndchthys molitrix; silver carp)in the experiments.77MA[[iERIAL AND ME[rHODSCondition of reserach ponds   Two ponds, L 3m x W 3m x H 1.5m, were used. The experiinental78ponds were filled with water from Lake Paldang, and pho$phatebuffer soiution (for BOD dilution) was added as finalconcentration to be O.1 mg-P lfi for good phytoplankton growth.Twenty Hypophthalmichthys molitrix (30 to 35 cm) were releasedto the fi$h pond on 2 June, 1993. No fish pond was used as acontrol.Environmentai factors   Water samples were collected weekly ftom 4 June to 29 June,1993. Nutrient coRcentxations (total phosphorus, solubiereactive phosphate, total nitrogen, NH4), water temperature,dissolved oxygen and chlorophyll a were measured by standardraethods [1]. Bacterial ceils were counted on O.2 pvapolycarbonate membrane filters after staining with acridineorange [13].Enzyme activities   Phosphatase activity was determined by modified method ofHoppe [9], using the fluorogenic substrate, raethylumbelliferyl(r6UF)-phosphate (1mb). Cellobiohydrolase and B-glucosidaseactivities were aRalyzed with MUF-cel}obiopyranoside (5mlE) and)CUF-S-glucoside (5iaM), respectively [5, M].RESULT AND D:SCUSSXONEnvironmental factors   Variations of temperature, dissolved oxygen and pH are shownin Table 1. Temperature did not change greatly, and DO and pHincreased in fi$h pond and these might be caused by the increaseof phytoplankton biomass. The nutrients except ammonia betweentwo ponds had no difference (Table 2). This taay suggest thatsilver carp did not affect the nutrient concentrations. But theconcentration of ammonium ions showed a siight difference betweentwo ponds.Variations of totaX bacteria and chlorophyll a content   Variations of total bacterial nurabers and chlorophy)l acontents in the two ponds are shown in Fig. 1. Total numbers ofthe bacteria in the control pond and in the fish pond whenexperiment was started, were 3.o x lo6 ceils ml-i and s.3 x io679cells ial'i, respectively. This values were similiar to theresults which carried out in Lake Paldang by Ahn and Cho [2] inMay 1989. Duxing the experiment total number of the bacteria inthe control pond ranged 1.s x lo6 - 3.g x !o6 celis mi-i withlittle change. But the number was increased to 7.9 x 106 cellsml-i at 22th day and peaked to 1.1 x 107 cells ml-1 at 29th dayin fish pond. ChlorephyU a content was also not changed so asAODC in control pond, but it rapidly increased from 5.4 to 7.Smg m'3 after 8 days and 12.6 mg m-5 after 27 days in fish pond.The increase of total bacterial number and chlorophyll a contentsuggest that rapid mineralization of fish feces might supplyenough available nutrients.Phosphatase activity(PA)    Phosphatase is the enzytRe that hydrolyze the dissolvedorganic phosphorus and particulate organic phosphorus.Phosphatase activity is induced wheR inorganic phosphate in thewater is exhausted andlor content of cellular phosphate decreased[6]. So, phosphatase actvity indicates the concentxation ofavailable phosphate in water coluvan and the pho$phate de£icitof the phytoplankton cell [4]. The variation of PA of two pondsare shown in Fig. 2. At the first 8 days, no difference wasfiound in the Vmax of PA in the two ponds, the values wereconstaRt of 120 nM l-i hr-i. However, in the fish pond, a£ter13 days Vmax iRcreased to 251.2 nM 1-i hr-G, two fold of theinitial value. The hrkghest value was shown after 20 days, a$4s3.g nM l'i hr"i, and after then the values decreased gradually.Xn the other hand, in control pond, the Vmax peaked after 20 daysand the variation range was relativeiy smaller than that of thefish pond.   Phosphatase are usually secreted from phytoplankton [iO]. Inour experiments, the variation of Vmax of phosphatase activityshowed a same trend with the changes of the concentration ofchlorophyU a (Fig. 1). Therefore, we could assume that thephosphatase was released from the phytoplakton. Since Kt+Sn, thesum of substrate transfer constant and concentration of availablesubstrate rapidly decreased, so the turnover tine of substyatedicreased.80Cellobiohydrolase activity   The passage of organic molecules across the microbialcytoplasnic membrane is an active process requiring specifictransport enzymes (e.g. permease), and only srnaU and sif"plemolecules can be directly transferred from the environment intothe ceil [8]. In the lake ecosystem, the majority of organicsubstance is cellulose, ce21 wall component of phytoplankton andmacrophyte. Cellobiohydrolase is one of the enzyme thatcatalyzes the hydrolysis of blopolymers <[glucose]., n = 2 - 5).   The Vmax of cellobiohydrolase showed a distinct pattern oEactivity. The Vmax of cellobiohydrolase activity ranged 4.4 -7s.o nbc 1-i hr-i in both ponds. vmax was low (ca. 2o nM 1-i hr-i)during the first 8 days (Fig.3), and began to increase rnarkedlytill 13 days. After 20 days, the Vmax decreased in both ponds.In control pend, after 27 days, Vvaax decreased as 4.4 rpt i-3hr'3, one fifth of initial vaiue (2o nbE l"i hr-i). rn fish pond,the Vmax showed higher value than that of the controi pond. Xtmight be due to the higher bacterial number observed in fishpond.   Because concentration and composition of the dissolvedcarbohydrates of natural environments are highly variable andchanged rapidly [il], the substrates for cellobiohydrolase canbe as$uiged as very srttal± quantity. So the variation of Kt-"Sn canbe regayded as the vamatxon of enzyme affinity to substrate, Kmvalue [7]. The values of Kt+Sn ranged g.6 - 231.4 pm Yi inboth pond$ (Fig. 3>. At the early stage, substrate affinity wa$extremely dif£erenic between two pond$, highest in fish pond and!owest in centrol pond. Zn control pond, however, the affinitiesincreased until the end of experiment. But, in fish pond,affinlties were high during the early and iate, and low duringthe middle of experiment. It can be explained as foliows;Mainly the enzymatic affinity is a£fected by the temperature andpH conditlen. But duxing the experiraent, the difference of watertemperature and pH were less than 2 OC and ca. O.3 only,respectively. So the difference of aftinity was caused by thedifferences of carbohydrate composition, not by environraentalfactors. !n control pond, the biomass of phytoplankton is thernajor source for organic compounds, and in fish pend, beside thephytoplankton bioiRass, fish feces is another important source.81So, the affinity at control pond was slightly increasedthroughout the bacterial adaptation to organic materials producedby phytoplan3<ton within an enclosed $pace. While in flsh poRd,new substrates were released in the form of feces of sUvercarps, and zyiaogenous bacteria for these substrates were thedoiRinants, so the affinity wa$ extyemely low. After 8 days, thebacteia were adapted to the organic materials from phytoplankton.B-glucosidase activity   B-glucosidase is a broad--$pecificity enzyme that catalyze$ thehydorlysis of B-linked disacchairide of giucose, celluhexose, andcarboxymethylceUulose [3] aBd it is very iraportant andsignificant for glucose metabolism of mieroheterotrophs inaquatic ecosysteva [5].   The Vmax of B-glucosidase were ranged l6 - 252 nM 1'i hr-i inboth pends (Fig. 4>. !ts activities were genera21y higher thanthat of cellobiohydrolase. During the first 8 days, the Vmax wasnot different between two ponds. Howevenc, later in fish pond,Vmax increased last untii 27 days, while in control pond, Vmaxdecreased slightly after 13 days like cellobiohydroiase activity.Initial Km value in the fish pond was also lower than in thecontroi pond Uke ceUobiohydrolase activity. In both two ponds,the Km values slightly increased till the end of the experiment.Especiaily, in the fish pond, substrate affiRity decreasedsMghtly one hundred time at 15 days compared to initial value.According to Chr6st [7], the value of aftinity, was changed byvarious substrate, and the lowest affinity was £ound at glucosesupplemented sample. Mereover, addition of cellobiose stronglyinduced B-gXucosidase synthesis, which means that bacterSaproduced the enzyme with high specific activity and affinity.So, the lower affinities of the final days, in two ponds were dueto the diverse low molecule weight oecganic materials by bacterialdegradation. And in the fish pond, the bacteria was moreabundant, and the high Vmax could be sustained.ACKNOWLEGMENT    This study was partly supported from Research Center forMolecular Mlcrobiology, Seoui National University.82REFERENCES 1.APHA-AWWA-WPCF, 1989. $tandard method for the examinationof water and wastewater 17th ed., APHA. N.Y. 2.Ahn, T.S. and K.S. Cho, 1989. Bacteriai distributions andactivities in Lake Paldang. J. Kor. WPRC. 5:30-34. 3.Barman, T.E. 1969. Enzyme Handbook, vol. 2. Springer Verlag,Berlin. 928pp. 4.Choi, S.X., T.S. Ahn and B.C. Kiva, 1992. Degradation ratesof organic phosphate in Lake Soyang. Ko2r. J. Microbiol.3O:113--118. 5.Chr6st, R.J., 1989. Characterization and significance ofB-glucosidase act±vity in lake water. LinmoZ.Oceanogr.34:660-672. 6.Chr6st, R.J. and J. Overbeck, 1987. Kinetic$ of alkalinephosphatase activity and phosphorus availability fourphytoplankton and bacteriopiaRkton in lake Plussee(north Germaneutrophic lake). Microb. EcoZ., 13:229-248. 7.Chr6st, R.J., 1991. Environrnental control of the synthesisand activity of aquatic ectoenzymes. Xn R.J. Chr6st(ed.)Microbial enzymes in aguatic environment. pp. 29-59. SpringerVerlag, NY. 8.GeUer, A. 1985. Degradation and forvaation of refractoryDOM by bacteria during simultaneous growth on iabile substratesand persi$tent lake water contituentis. Swiss dr. Hydro2.47:27･-44. 9.Hoppe, H.G., 1983. Signiticance of exoenzymatac activitiesin the ecology of brackish water:measurenents by raeans ofmethylumbelliferyi-substrate. Mair. Eco2., 11:299-318.10.Jansson, M., H. Ols$on and K. Peterson, 1988. Phosphatase:Oxigin, characteristics and function in lakes. Hydrobio2ogia,17e:157･-176.il.Mtinster, U., 1991. Extxacelluar enzyme activity in eutirophicand poly huraic lakes. Xn R.J. Chr6st(ed.) Microbia2 enzyraes inaguatic environment. pp 96-122. Springer Verlag, NY.a2.Northcote, T.G., 1988. Fish in the structure and function offreshwater ecosysten: A "top-down" view. Can. J. Fish. Aguat.Sci. 45:361-379.13.Parsons, T.R., l984. A manual of chemical and biiologicalmethod fer seawater analysis. Pergamon Press.Table i. The envronmental conditions of research ponds watersParameter waterTemp.(℃) oo(ng, -1l )pHAquarium c F c F c FJune, 4 21 21 8.4 8.4 7.4 7.210 22 22 8.7 8.9 7.9 7.815 22 22 8.0 8.5 7.4 8.022 22 22 s.e 8.7 7.6 7.829 23 23 7.9 8.8 7.3 7.4   t    c:     F:   DO:Table 2.Control pond without Hlypophthalmichthys molitrixFish pond witli H>ipophthaimichtltys molitrixdissolyed oxygen The concentrations of nuuients of research pond watersNutrients TP(mgl) l-E) SRP(mgp l-') TN(rngN 1-t) NK4 (ingN l-`)Aquariutn C F C F c F c Fjune, 4 O.101 o.Iel O.06 O.06 1.79 1.79 O.394 O.39410 O.101 O.105 O.06 O.06 1.79 1.80 e.2go O.29415 o.!oo O.103 O.06 O.06 l.80 1.90 O.350 O.28022 O.103 O.I13 o.e6 O.(>6 l.88 1.90 O.300 O.28629 O.094 O.101 O.06 O.06 1.79 1.90 O.300 O.350tSaine as Table 1.TP:total phosplrms,TN:tota1 nitregen,SRP:soluble reactiveINff{4:ammoniaphosphate8384opAE oEv N >= g99so-vr-E-ct)"(i5 oooNrN.. ×voao<1412108642  g gR: g<,g> .. .-O            .--" o       ot --- K7-  ti."' W[(s. 'W'" -' -"-- nj.. w---- --N( O/@ -Nx @Fig.o               Days1 . The variatiens of bacterial number and chl. a  concentration at research ponds,  rC:Contro/ponct ftF7ShpondiTAtirtaEs >.rt :.2'Ilil ru o ca N w rus a o osat5004003002001OOe@ vmaxrc]o Vmaxav oZZ]Kt+sn(cJ[iSl:EgeKt+sntEJo o@×2 8 13Days20 2750004000300020001OOOoArr-Es =o+ rvYFig. 2. Kinetics of phosphatase activity ln research ponds.85   1OOvrAk'T.m- 80Es o 60 cosi9v. 40.so:ka- o 20 ¢oo@ vmaxrCJo Vmax(F]IZZj Kt+sn(clIIXKt+sn(FJo2 8 13Days20 272502001501OO50oA2e ¢o+ .l--tYFig. 3. Kinetics of cellobiohydrolase activity in research ponds.300k 2sor";wwE   200s ge 150p. co 8 ioo-= m ds 50'Iisos     o @ vinaxtc] O Vh7ax(rw[ZIIIii] Kt+snfc)[iSN Kt-i- snavo2502001501OO502 8 13 Days20 27oA'r-Ee =o+ 'l"-'MFig. 4. Kinetics ef beta-gluco$idase activity in research ponds.

The effects of the sliver carp (Hypophthalmichthys molitrix) predation on the extracellular enzymatic activities

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The effects of the sliver carp (Hypophthalmichthys molitrix) predation on the extracellular enzymatic activities

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