I Int. Revueges. Hydrobiol.

75

1990

71-78

EVAPIP arid KLAUS PHILIPP

Department of Biology and Department of Chemistry, University of Winnipeg, Winnipeg, bfanitoba, Canada

Seasonal Changes in the Chemical Composition of Ceratophyllum demersum L. in a, Small Pond

key words: Cerntophgllum, carbohydrate, protein, fatty acids, alkaloids, periphyton

Abstract
Seasonal content of tntal soluble carbohydrate, total soluble protein, free fatty acids and alkaloids were examined in A population of Ceratophyllum demersum growing in a pond on the 8011thwestern edge of the Canadian Shield. Total soluble carbohydrate and total soluble protein were inversely correlated. Free fatty acids were dominated by (16:O k16:1), 18:2 and 18:3. Relative proportions of the different free fatty acids remained roughly constant throughout the season. Alkaloid concentrations showed large fhictuetions, with the highest values found during the first half of the season. Carbohydrate and protein contents of associated periphyton were not correlated with host macrophyte metabolism.

1. Introduct,ion
CerutvphyllurrL demersum I , . is a very coninion rriacrophyte in central North America, where it occupies a wide range of habitats (PIP, 1979, 1984). It lacks roots and is often anchored to the bottom by older portions of stern buried in sediment. This niacrophyte can grow a t a variety of depths, ranging from just below the surface to 14 m (PIPand SIMMONS, 1986). Despite the great contribution which C. demersum makes to the priniary production of many aquatic ecosystems (e.g. BEST, 1976, 1979; PIP and SUTHERLAND-GUY, 1987), little information is available regarding the chemical coniposition of this species over the course of a growing season, particularly wit,h regard t o free fatty acids. The objective of the present study was to examine the seasonal flux of total soluble carbohydrate, soluble protein, free fatty acids and alkaloids in a conmiunity of G. denzersum in a pond located in southeastern Manitoba, Canada. Soluble carbohydrate and protein contents of the periphyton found on the plants were also examined for conlparison.

2. The Study Site

The study was conducted during May to August, 1982 in an undisturbed pond approxiinately 2 ha in area located 18.2 kin west of Rennie, Manitoba on Highway No. 44 (49"54'40"N, 95'47'17" W). The bottom of the pond, with a maximuni spring water depth of 3 111, consisted of a layer of peat and sunken logs overlying a clay base. Subinerged niacrophytes covered niost of the bottom and consisted of

72

E. PIP and K. PHILIPP

Potantoyeton zosteriformis FERN., P. pusillus I,. and ChurcE sp. The nonrootcd species Ceratophyllurn demersum L. and Utricularia vulgaris L. fornied inobile niats which floated immediately below the water surface. Periphyton on all riiacrophyt cs was dominated by diatoms throughout the season (e.g. Epithemia, !Pabellaria, Gonrphonettrcc, Cymbella, Rhopalodia, Navicula, Xynedra, Cyclotella, Cocconeis). Water chemistry during the sanipiing season showed nutrient-rich conditions, with the following ranges of values: p l i 6.7-7.3, tots1 dissolved solids 211-305 rrig 1-1, total alkalinity 162-230 nig 1-1 CaC'O,, chloride 0-3 mg 1-1, sulphate 0-0.4 nig 1-1, total inorganic nitrogen 0.24-0.34 itig 1-1, total niolybdenuni reactive phosphorus 0.58-3.08 ]rig 1-1. Water temperature a t the iimnediate plant collection site ranged from 13.5-33.5 ('. during the saiiipling season.

3. Mi~terinls and Methods

Green C. denierauni was collected from the floating mats just below the wat,cr surface elt.vrn times during the Alay-Aizgnst season. Each sample consisted of shoots from sevcrol different) plants; sample size increi~sed in dry weight from a n initial mean per shoot of 0.3 g t o i i m;ixinium of 2.1 g by t.he end of the season. While the earlier samples consisted of ent.irc plants, in later collections only representative shoots were taken duo t o the interwoven niiture of the C:erotophyllum mats. The shoots were washed vigorously in plastic bags t o remove as much peripliytoii as possible. The plant,s and the periphyton which had been removed were placed separately o n ice in darkened containers, end transported t o the laboratory within 5 h whore they were frozen and subsequently frwze-dried. Shoots from the different plants collected on each of the sampling days were combined to give composite samples for analysis. The ratio of dry weight of the periphyton recovered relittive t.o the dry weight of the host CerutopliyZZum averaged 0.06 for the snmpling season :is ii whole. Total soluble carbohydrntc in plants was determined by homogenizing a 0.5 g sample of freezedried tissue with 40 n i l of 80 yo ethanol in a blcndcr. The homogcnate was filtored throiigh 4 layers of cheesecloth ;md centrifuged a t 5000 x g for 5 minutes. Carbohydrate content of tlirce aliquots of the supernatant was determined using ROE'S(1955) anthrone method. For C. deniersum, this method was found to have a n extraction efficiency of 53 1-2 yo for the total sugtirs; efficiency was determined by reextraction of the pellet with successive 20 ml volumes of hot ethanol until no additional sugars could be obtained. Iiidividual sugars were ident)ified by fractionating the supernatant on ion-exchange columns, and separating t>hccomponcnt,s for tho snger fraction by paper chromatography (PIPand STEWART, 1976). Preliminiiry identifications were mnde by comparing the positions and colors of the spots obtained by developing with aniline diphenylamine phosphate (BACON and DICKINSON, 1!)67 ; SMITH, 1960) iqpinst those of known markers. Identifications mere confirmed by nuclew magnetic rcsonanee analysis ( 1 3 4 a t 75.47 Mhz and 1-H at 300 Mhz) of individual sugars c l u t d from undeveloped chromatograms and dissolved in deuterium oxide. Total solublc protcin was determined by homogenizing il 0.5 g sample as above, but using 0.1 I % NaOH. The homogenate was filtered and centrifuged, and protein content, in three aliquots of the supernatant was determined using the Coomassie Blue method of BRADNORD (1976). Total soluble carbohydrate and protein in pcriphyton were detjcrmined hy homogcnizing 0.01-0.12 g of freeze-dried periphyton wit.h 5 ml 80 yo cthanol or 0.1 N NnOH respectively using a n clcctric Pottar-Elvcjham t,issiie homogenizer equipped with a teflon pestle. The l ~ o n ~ o g e n ~ i t r wiis centrifuged for 5 min in a clinicol centrifuge. Carbohydrate arid protein were dct>crmincd for three aliquots of eiwh supernat'ant using the above methods. For extraction of free fatty acids from the plant material, 2 g of freeze-dried sample were homogenized in i i blcndcr with 50 ml of cold isopropanol (HARBORNE, 1973). The homogcn;ite was partially evsporstod under nitrogen gas ovcrnight in a n ice-wiitcr bath. Subsequent extractioii wiis carried out for 8 h > a t 60 C. under nitrogen with 250 ml of 2 :1 chloroform : methanol. Aiiot,hor 250 nil of the chloroform-methnnol solvent were added a t the end of this period, and extract,ion was continued under nitrogen overnight. The homogenate was filtered through Whatman No. 50 filter paper, and t,he volume was reduced by evaporation t o 10 nil by passing nitrogen gas over

Cheniicnl Composition of Ceratophyllum

73

it. Lipids were extracted from the filtrate with petroleuni ether. The ether fraction was eviiportitcd
to dryness under nitrogen and stored a t - 20 C.

l h e separation, identification and quantification of the free fatty acids in the extracts wcrc accomplished in onc step by packed-column gas chromatography on t i Pye Series 104 Model 64 gas chromatograph with FTD. Column packing was 12 yo Carbowax 20 M/tercphthdic acid (Mkrndel Co., Rockwood, Ontario) on Chromosorb H P (100jl20 mcsh) (Chromatographic Specinlt,ies Inc., Brockville, Ontario); colnmn length was 2.13 m. The carrier gas was nitrogen cLt ii flow rate of 70 mlimin; hydrogen and air flow rates were 25 mlimin and 300 rnlimin rcpectively. Ovcn temperature was hcld constant a t 250 C., and the detcctor oven tempcraturc was 300 C. The column was preptired a s follows: 1.37 g of t,he finely ground Carbowax liquid phasc. was transferred t o a 500 ml roundbottom flask and dissolved in 37 ml of chloroform. Tcn g of Chronio~ by drying a t 130 C for 1 h, and adding while still hot t.o the above solution. sorb HP w : prepctred Thc solvent was renioved in a rotary evaporator with the flask immersed in a 60 C water btith. Thc dry packing wiis resicveci; 7.3 g of the latter were used to pack a 2.13 m, 6 nini diameter glass column. The column was conditioned overnight i t t 200 C. and a nitrogen flow rate of 70 nd/ niin; resulting gaps were removed by recompacting. il stock standard solut,ion was prepared containing thc free fntty acids 6 : 0, 8 : 0, 9 : 0, 10 : 0. 12 : 0, 1 3 : 0 , 14 : 0 , 14 : 1 ; 16 : 0, 16 : 1, 17 : 0, 18 : 0, 18 : 2 and 18 : 3. The amountsiricreasedfrom 3.2 mg of caproic iicid to 28.6 rng of linolcic and 19.6 mg of linolenic acid. The acids were tiiken up in 1.5 g of chloroform. The stock solution was subsequently diluted t o concentrations ranging from 0.4-4 nigiml. A volunic of 0.4 p1 of the dilute standard solution was injected. The system separated ti : 0, 8 : 0, 9 : 0, 10 : 0, 12 : 0 and 13 : 0 esscntially completely. Myristic and rnyristolcic acids were completely resolved from tridecanoic and pulmitic acids, but only partially from each other; i~ similar degree of resolution was obtained for palmitic and palmitoleic acids relative to their ticwrest neighbors a n d t,o ciich other. The 18-carbon acids were largely, though not completcly, resolved from each othcr. Pliint sample unknowns were preparod by dissolving approximately 10 mg of the dried extract in chloroform t o give a concentration of approximately 0.1 g of sampIe per nil of solution; 0.4 to 1.0 pl of sample solution was injected. Duc to drifting baselines, quantification was accomplished by measuring peak areas wit'h i~ revcrsing planimrter. Slightly overlapping peaks were delineated by extrapolation to the baseline. The mcthod yielded good correlation between wpights of free fatty acids and peak areas ( r =0.99, p 0.001, .n = 7). Whcre overlap was greatcr, separate curves were fitt.ed t o tho composite peaks sucb t h a t the sum of the individual areas was equal to the area of the unresolved peak; this proced i m yieldcd morc accurate results than did simple extrapolation. Alkaloids were extractrd hy homogenizing 2 g of freeze-dried tissue in a blender with 40 nil of 10 yo glacial acetic wid in ethanol. The homogenate was incubated a t room temperat'ure for 4 11 (HARBORNE, 197R), arid centrifuged a t 3,000 g for 5 min. The supernatant was made alkaline to pH 10.0 with concentratcd ammonium hydroxide, then centrifuged a t 13,000 g for 15 min. The pellet was suspendcd in 20 ml 1 96 ammonium hydroxide. An equal volume of chloroform was shaken with the suspension and allowed to stand 15 min (e.g. CRONWELL,1955). The chloroform fraction was collected. The oqueoos layer was made slightly acid with concentrated hydrochloric acid, thcn alkaline wit.h concentrated ammonium hydroxide. This was extracsted with ii 5 : 1 v : v mixture of chloroform :isopropanol, which was then pooled with the original chloroform fraction. The pooled fractions were evaporated t o dryness, t'hen taken up in 1 ml chloroform arid 0.1 ml 1 yo ammonium hydroxide. The concentrate was spotted on activated silica gel G thinlayer chromatographic glass plates and run in a 200 : 3 mixture of methanol : concentrated nmmonium hydroxide solvent until the solvent front was a t least 15 cm from the origin. Thc platcs were examined for fluorescence under ultraviolet light. Fluorescent spots werc rcsuspended in 3 nil 95 "/o ethanol and spectra wcre scanned using a Beckman DU-7 spectrophotometer. Blanks consisted of the above steps, minus the plant material.

r,

71

E. PIP m d K. PHILIPP

4.Result's
Total soluble carbohydrate showed three peaks during the saiiipling season (Fig. 1). The sugars consisted primarily of glucose, fructose and sucrose, but stachyose, raff inose and irielibiose were identified as well. Total soluble protein showed the highest values

i
CARBOHYDRATE PROTEIN

12 2025 2 8
MAY JUNE

22

20

11 31
AUGUST

JULY

Figure I. Total soluble Carbohydrate and total soluble protein concentrations in C . demersui~t during the 1982 season. Carbohydrate values have been corrccted for extraction efficiency. Standard errors of replicates are within the areas encompassed by the points.

a t the beginning of the season (Fig. I ) , followed by a series of fluctuations. Total soluble protein was significantly inversely correlated with total soluble carbohydrate ( T = -0.62, I, =0.02, n= 11). The concentrations of soluble protein and carbohydrate in periphyton (Fig. 2 ) were not significantly correlated with host iriacrophyte protein or carbohydrate levels, nor with seasonal water temperature pattern or with any of the water chemistry parameters examined.
2u

1
I

CARBOHYDRATE PROTEIN

MAY

JUNE

JULY

AUGUSl

Figure 2. Total soluble carbohydrate and total soluble protein concentrations in periphyton of C . demersum during the 1982 season. Standard errors of replicates are within the areas encompassed by the points.

Stepwise niultiple regression analysis was performed with protein content>as the dependent variable and water chemistry and teniperature as the variables in the regression block. The procedure entered two variables above the p = 0.05 level (n= 11):log transforrrietl total alkalinity (Bz=0.53, p = 0.011) and temperature (untransfornied) (R2=0.85, p=O.OOl after step 2 ) , both of which were inversely related to protein content. Stepwise riiiiltiple regression with soluble carbohydrate as the dependent variable failed to admit any of the environmental parameters into the equation,

Chemical Composition of Ceratophyllum

75

even though carbohydrate and protein were inversely related. Carbohydrate was significantly correlated only with p H (r=0.60, p =0.026, n = 11). All of the extracts contained the same free fatty acids (Table 1). Heptanoic acid (7 :O), not present in the standard solution, was identified by means of a straightiine plot of the logarithm of the adjusted retention volume of standard saturated fatty acids versus carbon atoni number. Besides the fatty acids which could he identiTable 1. Concentrations of free fatty acids in C. dernersum extracts during the 1982 season. Values represent mglg dry plant tissue.
Sampling time
7:O

8:0
0.17 0.02 0.03 0.05 0.03
0.01

9:0
0.11 0.02 0.04 0.04 0.03 0.01 0.02 0.02

Free fatty acids 1O:O 12:O

0.30 0.06 0.16 0.22 0.14 0.03 0.06 0.14
0.06

16:0+16:1

18:2 0.94 0.41 0.78 0.52 0.60 0.29 0.39 0.37

18:R

June 2 June 8 June 22 July 6 July 20 August 3 August 17 August 31

0.02 0.06 0.06 0.03 0.02 0.04 0.02

0.02 0.02

0.02 0.03 0.06 0.03 0.01 0.06 0.02

1.42 0.65 1.20 0.88 0.75 0.50 0.84 0.64

0.76 0.46 0.00 0.64 0.45 0.29 0.44 0.36

fied, all samples yielded a partly-resolved quadruplet peak located on both sides of tridecanoic acid but none of these peaks corresponded to the standard. All samples also contained a compound with a retention volume between those of myristic and niyristoleic acids. Another unknown compound overlapped the capric acid peak. These unidentifiable conipounds were interpreted as volatile components other than free fatty acids and were not included in the quantification of the latter.

:/
,
0

,
6

12 2025 2 8 22 MAY JUNE

20 3
JULY

17 31 AUGUST

Figure 3.

Total free fatty acid concentrations in C. demersum extracts during the 1982 season.

The highest total fatty acid concentrations were observed during the first part of the season (Fig. 3). However only minor variations were seen in the relative proportions of the freefatty acids during the season (Table l),as judged from the finding that the concentrations of all fatty acids were significantly positively correlated with each other (r=0.60-0.98, p=0.05-~0.001,n = 8 ) as well as with the total fatty acid concentrations (r =0.73-0.98, p =0.02- -=0.001, n = 8).

76

E. PIP and K. PHILIPP

Threc fatty acids roniprised the bulk of the total. The greatest proportion was consistently rwnposed of the (16 :0 + 16 : I ) coriiplex (madeup primarily of the former), varying froni 36 to 45 "loof the total free fatty acid content, followed by 18 : 2 and 18 :3, which were present in roughly equal amounts (Table 2). The remainder was coinposed of 7 :0, 8 : 0, 9 : 0 and 12 : 0, which were present in minor aniounts that were relatively coristant during the season. The seasonal concentrations of 10 :0 were' significantly positively correlated with total soluble carbohydrate content ( r = 0.80, p = 0.009, n = 8). Fatty acid concentrations did not appear t o be signifivantly related to any of the environiiiental paranieters examined. Table 2. Relative proportions (percent) of free fatty acids in G. demersum extracts during the 1982 season.
Sampling time June 2 June 8 June 22 7:o
-

x:o
4.3 1.3 1.1 2.0 1.4 0.9 1.2 1.2

Free fatty acids

9:o 2.8 1.5 2.1 1.9 1.4 0.9 1.2 1.0 10:o 8.0 3.6 5.0 9.2 6.8 2.5 3.3 9.0 12:o 1.7 1.2 1.1 2.7 1.4 0.8 2.6 1.2 16:0tIG:l 37.9 38.9 41.9 37.0 36.4 43.2 44.7 40.6 18:2 25.1 24.7 25.8 21.9 29.1 24.8 21.0 23.2

18:3
~

myli July 20 August 3 August 17 August 31

1.3 1.9 2.7 1.6 1.9 2.4 1.2

20.2 27.6 20.0 22.6 21.6 25.0 23.6 22.8

The silica gel chromatography yielded a single fluorescent, spot a t an E f of approximately 0.64, and an absorption niaxiriium in the region of 224 nm. This compound showed a series of peaks, with the largest at the end of June (Fig. 4),as judged by absorhance values a t 224 nrn. The absorbance values were not significantly correlated with carbohydrate, protein or any of the fatty acids, nor were they correlated with any of the environmental parameters.
ALKALOID CONCENTRATION

Figure 4. Relative alkaloid concentrations in G. demersum during the 1982 season. Alkaloid absorbances are based on extracts of 2 g dry tissue.

5. Discussion
Although C. demersum showed three seasonal peaks in total soluble carbohydrate concentration a t the present study site, the seasonal pattern for this species varies in different situations (e.g. PIP and SUTHERLAND-GUY, 1987) and thus a number of

Chemical Composition of Ceratophyllum

77

environmental as well as physiological variables may be important in influencing carbohydrate content (PIP and SUTHERLAND-GUY, 1989). Identification of stachyose, raffinose and melibiose in addition to the three major sugars supported similar findings of BEST and VAN DER WERF (1986) for this species. Total soluble protein concentrations also fluctuated during the season. While sonie correlations were observed between certain environmental variables and content of each of protein and carbohydrate, i t is not known t o what extent these relationships were fortuitous. The inverse correlation between total soluble protein and soluble carbohydrate levels suggested that protein and carbohydrate pools may be interrelated. Although some periphyton residue still remained on the plant material that was analyzed, it is not likely that this residue contributed significantly towards the plant tissue values; for example, if 10 O/, of the original periphyton still remained, the estimated error from this source for the plant values would be less than 1 Su and STABA(1972) examined fatty acids derived from triglycerides (but not free fatty acids) of C. demersum collected in Minnesota. These workers found 14 :0, 15 :0,16 :0,16 : 1,18 :0,18 :1, 18 : 2 , 1 8 : 3,20: 3 (Z), 24 : Oand 2 4 : 1, of which20: 3 (?), 24 :1, 18 : 2 and 16 : 0 were respectively the most abundant. The disparities with the findings of the present study may suggest that the free fatty acid pool contains a higher proportion of short chain acids than is found in triglycerides. However the lack of available literature prevents any speculations regarding the constancy of the fatty acid composition in different populations and under different ecologieal conditions. Fluorescent compounds have also been reported from C. demersum by Su and STABA (1972), who used a number of different solvent systems for alkaloid extraction and identifieda total of five fluorescent thin-layer spots for plants collected in late sunliner and early fall in Minnesota. However only one spot was isolated with the solvent system in the present study. It has been suggested that secondary metabolites such as alkaloids may function in C . demersum to deter grazers (LAWALREE, 1955) as this plant is known to be unattractive to gastropods (FROMMING, 1956; PIP, 1977 ; KOLODZIEJCZYK and MARTYNUSKA,1980; STERRY et al., 1983), particularly during the early part of the season. Additional investigation is needed to determine whether such conipounds are indeed active deterrents.

6 . References

BACON, J. S. D., and R. DICKINSON, 1957: The origin of mclezitose: a biochemical relationship
between the lime tree (Tilia spp.) and on aphis (hucnllipterus tiEiae I,.).-Bioehem. J . 66: 289-299. BEST, P. H., 1976: Nutriont contcnt of the aquatic macrophytes Elodea, canadensis and Ceratophyllurn in the course of the year.-Hydrobiol. Bull. 10: 15-16. BEST, E. P. H., 1979: Photosynthesis in Ceratophyllum demersum. Carbon fixation rates in relation to the plants physiological stage, and the contents of chlorophyll and non-structural cnrbohydrates. - Hydrobiol. Bull. 13: 112. BEST, E. P. H., and A. K. VAN DER WERF, 1986: Respiration in relation to reserve substances in the submerged macrophyte Ceratophyllum demersum L.-Aquat. Bot. 26: 235-246. BRADFORD, M. M., 1976: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.-Anal. Biochem. 72: 248-254. CROMWELL, B. T., 1955: The alkaloids. In: Modern Methods of Plant Analysis, K. PAECH and eds.-Springer-Verlag, Berlin. Vol. IV, pp. 367-516. M. V. TRACEY, FROMMINQ, E., 1956 : Riologie der MitteleuropLischen Siil3wasserschnecken.-Duncker 8: Humblot, Berlin. HARBORNE, J. B., 1973 : Phytochemical Methods.-Chapman and Hall, London.

78

E. PIP and

I ( .PmLIrr

KOLODZIEJCZYK, A., and A. MARTYNKJSKA, 1980: Lymnaen stognalis (L.)-feeding habits and production of faecrs.-Ekologia Polska 28 : 201-217. LAWALREE, A., 1955 : Spermatophytes. In: Flore generale de Belgique.-Ministere Agric. Jardin Botanique de IEtat. Brussels. 1-120. PIP,E., 1977: A study of aquatic plant-snail associations.-Ph. D. Thesis, University of Manitoba. PIP, E., 1979: Snrvcy of the ecology of submerged aquatic macrophytes in central Canada.Aquat. Bot,. 7 : 339-357. PIP, E., 1984 : Ecogeographical tolerance range variation in aquatic macrophytes.-Hydrobiol. 108: 37-48. PIP, E., and K. SIMMONS, 1986: Aquatic angiosperms a t unusual depths in Shoal Lake, ManitobnOntario.--Can. Field-Nat. 100: 354-358. PIP, E., and J. ill. STEWART, 1976: The dynamics of two plant-snail associations.-Can. J. Zool. 54: 1192-1206. Pip, E., and C. SUTHERLAND-GUY, 1987: Aquatic macrophytes in Shoal Lake (Manitoba-Ontario). I. Diversity, biomass and metabolic status in relation to water depth and light intensity.Arch. Hydrobiol. Suppl. 76: 197-222. PIP, E., and C. SUTHERLAND-GUY, 1989: Seasonal flux of nonfitructural carbohydrate in five species of submerged macrophytes in a Precambrian Shield lake. 1 1 . Fluctuation in relation t o chlorophyll content, temperature and water chemistry.-Acts Hydrochim. Hydrobiol. 1 7 : 533-536 ROE,J. H., 1955: Thr: determination of sugar in blood and spinal fluid with anthrone reagent.J. Biol. Chem. 212: 335-343. SMITH,l., 1960: Chromatographic and Electrophoretic Techniques. Vol. I.-Interscience Publ., New York. STERRY, P. R., J. n. THOMAS and R. T,. PATIENCE, 1983: Rohavionral responses of Riomphalaria gluhrata (SAY)to chemical factors from aquatic macrophytes including decaying Levma paucicostata (HEOELM. ex ENuhm).-heshwat. Biol. 15: 465-476. Su, K. L., and E. J. STA4BA, 1972: Aquatic plants from Minnesota. Part 1 - Chemical survey.Water Resources Research Center, University of Minnesota Graduate School, Bull. No. 46, sopp.

Dr. EVAPIP Department of Biology University of Winnipeg Winnipeg, Manitoba Canada R3B 2E9
Manuscript accepted: June ist, 1989