Proc. Natl. Acad. Sci. USAVol. 77, No. 3, pp. 1716-1720, March 1980Population Biology

Population oscillations and energy reserves in planktonic cladoceraand their consequences to competition

(Daphnia/time lags/triacylglycerol/zooplankton)

CLYDE E. GOULDEN AND LINDA L. HORNIGDivision of Limnology and Ecology, Academy of Natural Sciences of Philadelphia, Philadelphia, Pennsylvania 19103

Communicated by Ruth Patrick, December 17,1979

ABSTRACT Time lags in an individual's response to de-creased food densities are responsible for oscillations in labo-ratory populations of Daphnia galeata mendotae. Visible energyreserves of triacyiglycerols accumulate in the body of animalsat low population ensities when food is abundant and are latermetabolized at high population densities when food is scarceto temporarily sustain activity and reproduction. After theseenergy reserves are metabolized many individuals, primarilyjuveniles, starve and die. The length of the time lags is a functionof the amount of energy reserve accumulated in the individual.Because this sustained activity and reproduction further de-creases food concentrations to very low levels, individuals ofa second smaller-body-sized species, Bosmina longirostris, thatdo not have sufficient energy reserves quickly starve and die.Thus the accumulation of energy reserves in individualsunderlies the time lags important in causing population oscil-lations and has consequences to interspecific competition.

The experiments described here were initiated to answer thequestion, why do large zooplankton species dominate in lakeswithout intense fish predation (1)? Because the dominance oflarge animals can at least partially be due to competitive ex-clusion, we believe the factors that bestow a competitive ad-vantage can be identified in controlled laboratory experimentsand their importance can then be tested in the animal's naturalhabitat. We began with competition experiments with indi-viduals of a large cladoceran species, Daphnia galeata men-dotae (0.6-2.4 mm long) and a small cladoceran species, Bos-mina longirostris (0.25-0.5 mm long). Both species primarilyreproduce parthenogenetically; males and sexual egg-bearingfemales were seldom encountered in our cultures. The char-acteristic oscillation pattern of Daphnia populations (2, 3)dominated these cultures; therefore, we concluded that we hadto understand this phenomenon before we could explain theresults of the two-species cultures.

Population oscillations have been studied in the laboratorywith Daphnia and other species. These oscillations are pre-sumably analogous to eruptive fluctuations of large mammals(4), fluctuations of microtine rodents (5), and fluctuations ofinsect species such as the desert locust (6), though the mecha-nism underlying each may differ.

Classical population dynamics theory assumes that anequilibrium density exists among the supply of available foodor other limiting resource, the number of individuals, and thebirth rate of the population. When food available exceeds thefood demand, birth rate will be high and the population willincrease in size. As the number of individuals increases and theamount of food per individual decreases, birth rate corre-

spondingly decreases and at the equilibrium density the birthrate should approximately equal the death rate. However, adensity equilibrium is not realized if there is a long time lagbetween the decrease of food concentrations and the corre-sponding decrease in birth rate. If fecundity of all or part of thefemales remains high, the density of individuals will eventuallyexceed the equilibrium density-i.e., the food demand of in-dividuals will exceed the food available. Individuals will starveand die. Hutchinson (7) suggested that such a time lag exists inspecies whose populations oscillate and Pratt's (2) and Slobod-kin's (3) laboratory studies of Daphnia support this idea.Our objectives in this paper are (i) to describe the mechanism

underlying the time lag in Daphnia-i.e., to explain how ani-mals can remain active and reproduce after the equilibriumdensity has been exceeded-and (ii) to discuss the consequencesof this behavior to interspecific competition.

METHODSIndividuals of the two species were collected from reservoirsnear Philadelphia and were maintained in filtered (Whatmanno. 4) and autoclaved lake water in a 20'C culture room witha 14-hr light/1-hr dark cycle. The food used in the experi-ments was a 50:50 mixture of Anktstrodesmus falcatus (un-known origin) cultured on modified ASM-1 medium* andChlamydamonas reinhardtii negative strain (wild-typeUTEX90) cultured on Woods Hole MBL medium (8) withoutbuffer. A vitamin mixture (9) was added to the algae as culturemedium was added. Algal cultures were maintained in a con-tinuous growth condition and were replaced after 1 month, orearlier if high bacteria counts were noted or if the culturechanged color. The algae were centrifuged to remove the cul-ture medium; the cells were resuspended in fresh water,counted on a hemocytometer, and diluted to the experimentalfood concentration.

Single cultures of Daphnia and Bosmina and cultures of bothspecies were maintained at 20'C in 220 ml of lake water in250-ml erlenmeyer flasks. Each culture was started with 10nonsister neonates, the offspring of females who had been iso-lated through two generations in the same food density as wouldbe used in the experiment. Five replicate flasks were used foreach experimental condition and each experiment was repeatedone or two times. One experiment consisted of single or twospecies cultured in 104 algae cells ml-l (referred to as the "lowfood" cultures); the animals were counted and transferred to

* One liter contains 170 mg of NaNO3, 24.07 mg of MgSO4, 40.67 mgof MgCl2-6H20, 22.19 mg of CaCl2, 17.42 mg of K2HPO4, 1.08 mgof FeCls-6H20, 2.47 mg of H3BO3, 1.39 mg of MnCI2-4H20, 0.44mg of ZnCl2, 19 ,ug of CoCl2-6H20, 0.1 ,ug of CuCl2'2H20, 7.45 mgof Na2EDTA, and 10 ug of NaMoO4-2H20.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

Proc. Natl. Acad. Sci. USA 77 (1980) 1717

N 100 -

50-

AAO

A* 0. A.c,

400

300

N 200

100

20 40 60 80Time, days

FIG. 1. Population oscillation in low (Upper) and high (Lower)food cultures. Ordinate scale is the number of individuals in 220 mlof water at 20'C. 0, Females with eggs; A, females without eggs plusfemales with eggs; 0, juveniles plus females without eggs and femaleswith eggs. Thus the number of individuals for each age or reproductiveclass is the difference between lines at any one time.

fresh water and food every two days. A second experimentconsisted of animals maintained in 105 algae cells ml-' (the"high food" cultures) and were counted and transferred everyfourth day. As the animals were transferred, each was countedand the daphnids were distinguished as neonates (instar 1),juveniles (instars 2-4), adults with eggs, or adults without eggs.However, it was not possible to distinguish juveniles fromneonates in crowded cultures because individual growth slowedconsiderably. Therefore, neonates are noted only when iden-tified and measured under the microscope. Bosmina individualsare too small to categorize without size measurements, so theywere not separated.Dead daphnids on the bottom of each vessel were counted,

but the probability of death (Pd) was calculated as the differ-ence in numbers between the peak number of a population andthe subsequent lowest number before a new oscillation began.This probability agreed well with counts of dead bodies.

Algal counts were made on water samples from vessels afterthe animals had been transferred. Bacteria were counted in

water samples from experimental vessels by the epifluorescencetechnique (10).

*The assimilation rates of Daphnia and Bosmina feeding on"4C-labeled A. falcatus were measured at 1 X 1(3, 2 X 103, 1X 104, 1 X 105, and 5 X 105 cells ml-l by Lampert's technique(11). Corrections for respiratory loss of 14CO2 could only bemade at the higher food densities. These were projected for useat the lower food level.

RESULTSThe results for one population of D. galeata mendotae main-tained at each of the food densities are illustrated in Fig. 1.Replicate populations exhibited patterns similar to those illus-trated; however, the period of oscillation differed slightly so thatcombining the data of replicates would have no meaning.The results show a typical oscillation pattern with a rapid

increase in the number of daphnids and a subsequent sharpdecline. Juveniles dominate in number as in populations witha high birth rate. Many of the original females survived the firstcycle and initiated the second. In the high food cultures addi-tional females matured early, but in the low food cultures newadults did not appear until the later stages of the culture. Almostall females carry young in the brood chamber when density islow, but as the density increases the frequency of femaleswithout eggs or embryos increases.

In both high and low food cultures, the extreme fluctuationof Daphnia numbers is largely due to the high birth rate andsubsequent death of juveniles, a result first noted by Slobodkin(3). Although the original food densities are restored at frequentintervals, counts of the algae indicate that the food is quicklyconsumed by the animals present. Malnourished adults andjuveniles starve and die but there is a lag between the time thefood is diminished and an increase in the death rates.To determine how much the amplitude of the population

exceeded the equilibrium density, we calculated the daily fooddemand of the population by measuring the assimilation rateof animals in a food concentration that would simulate equi-librium density conditions and compared this with the dailyfood ration as a food demand (FD) to food available (FA) ratio.tIn Fig. 2 the probability of deaths in the population during thedecline phase is plotted against this ratio. The food demand ofthe populations often exceeds the available food supply by 2-to 3-fold and can be as high as 7-fold. The probability of deathafter each peak is closely related to the amplitude of the oscil-lation. In most of the oscillations we have studied, when fooddemand exceeded available food by 2- to 3-fold, 40-70% of theindividuals of a population could be expected to die during the

tThe food demand (FD) was calculated by multiplying the amountof carbon as algae assimilated day'I daphnid-' of each age class ata food density of 50 gg of carbon liter-' or 2000 algae cells mlh' (theestimated reproductive threshold food concentration) by the numberof individuals of each age class. The food available (FA) was calcu-lated by dividing the amount of carbon as algae added (25 pg ofcarbon per algal cell) by the number of days between each census.The reproductive threshold is defined as the food concentration thatjust maintains egg production in an animal (12). This threshold isanalogous to the equilibrium density for the population, and bothshould occur at about the same food density. Lampert and Schober(12) have identified the food level for this threshold for Daphniapulex as 100 Iug of carbon per liter of food and for D. rosea, a speciesvery similar to D. galeata mendotae, as 50,gg of carbon per liter. Wehave found a high infant mortality and a low fecundity in individualsduring life table and birth schedule experiments when maintainedat 50 ,g of carbon per liter. We believe this food concentration is aclose approximation of the reproductive threshold and therefore ofwhat might be the food concentration at the equilibrium density ofthe population, if oscillations did not occur.

Population Biology: Goulden and Hornig

1718 Population Biology: Goulden and Hornig

5.0 6.0 7.0 8.0

FIG. 2. Probability of death (Pd) during the decline phase of anoscillation as a function of food demand (FD) to food available (FA)(see text for an explanation). X, Low food cultures; *, high food cul-tures.

ensuing decline. More than 90% of the individuals died invessels maintained at the low food density when the food de-mand ratio at peak densities was as high as seven.

Role of Energy Reserves. A time lag before birth rate de-creases is clearly involved in the oscillation of Daphnia popu-

lations, but there is also a time lag before death rate increasesas a result of starvation when food demand exceeds foodavailable. What is the mechanism that makes these two timelags possible?We suggest that both temporarily sustained activity and high

fecundity at such low food levels are possible only by the use

of energy reserves in the body. The reserves accumulate in thebody at high ambient food densities and are then used at lowambient food densities for metabolic maintenance and for re-

production.

0

There are visible reserves in the body in the form of oildroplets. Lipids of Daphnia have been extracted by RichardLarson with chloroform/ethanol, 1:1 (vol/vol). Thin-layerchromatography shows a large amount of triacylglycerol withthe same polarity as triolein. Triacylglycerols have been iden-tified in marine copepods and are metabolized as an energyreserve (13). Changes in these visible energy reserves inDaphnia during the course of an oscillation conform to theexpectation that these reserves are used to supplement the dietat low food densities. In addition, glycogen reserves may bepresent in cells (14) but are not visible unless stained. In ourstudies food reserves have been estimated based upon the visibleoil droplets.To test the conclusion about the use of energy reserves during

the oscillation, we developed a lipid index ranging from zeroto three to rank individuals in the cultures according to lipidcontent (Fig. 3). Individuals drawn from cultures at differentphases of an oscillation were given lipid index values; resultsfor one of the cultures are shown in Fig. 4.

As a population begins to increase, most females are gravidand have oil droplets in the body. Juveniles also contain oildroplets. As the population attains peak densities gravid femalesand juveniles lose most of their oil, whereas females withouteggs retain some droplets. As the death rate increases a fewadults still have oil droplets but juveniles have none visible. Aftera "crash" when population densities are low, there is a statisti-cally significant time delay before fecundity increases, ap-parently because adults must first accumulate new energy re-serves before they can produce large numbers of eggs. Each egghas a large triacylglycerol droplet that is not metabolized duringembryonic development but instead forms an energy reservefor neonates. Adult daphnids have more visible reserves thando juvenile daphnids and juveniles appear to use their reservesmore quickly than do adults. We believe this explains why a

I od M

2 3

FIG. 3. Lipid index for visible oil droplets in D. galeata mendotae. od, Oil droplets; M, mandible. animals with 04 oil droplets; 1, animals

with 5-20 oil droplets; 2, animals with many small droplets; 3, animals with dense aggregates.

1.0

0.75

Pd 0.50

x 9

X xX Xox

Sxx 0

0.25 [

01.0 2.0 3.0 4.0

FD/FA

Proc. Nati. Acad. Sci. USA 77 (1980)

x

I

Proc. Nati. Acad. Sci. USA 77 (1980) 1719

3f2

1o0L

Do

De 50

0

300,

2001

N

1ooFo-0 4;>'- ? ..

88 92 96 100 104 108Time, days

FIG. 4. Changes in oil content in Daphnia as percentage of animals(De, females with eggs; D. females without eggs; j, juveniles from in-stars 2-4) categorized by lipid index during an oscillation. N, numberof individuals in 220 ml of water at the high food density; symbols andscale are as in Fig. 1.

greater proportion of juveniles than adults die during the de-cline phase of the oscillation.Consequences of Oscillations to Interspecific Competi-

tion. The initial objective in these studies was to determinewhether a large plankton animal such as the cladoceranDaphnia had a competitive advantage over a smaller clado-ceran such as Bosmina. In single-species cultures, the increaseof Bosmina populations conforms to the classical pattern ofpopulation dynamics (Fig. 5). Population numbers attain adensity equilibrium when the amount of food is just adequateto support the number of animals present. We have estimated

1!

N

Q la

tr IW O.N O"y o

100

500

400A

300

N0

200 /

100 0 0,0

0

20 40 60 80 100Time, days

FIG. 6. Competition in replicate vessels ofD. galeata mendotae(0 ---0) and B. longirostris (0-0) cultured at high food den-sity.

the assimilation rate of Bosmina individuals at 2 X 103 algalcells ml-l and calculated a FD-to-FA ratio.t The mean valueof this ratio for five replicate samples at equilibrium densityis 1.15 with a range from 0.81 to 1.49.Under the conditions of our laboratory experiments, the re-

sults of the two-species cultures have consistently indicated thatDaphnia is the superior competitor (Fig. 6). These cultures weretreated similarly to the single-species cultures; the only dif-ference was the addition of 10 neonates of each species. Thenumbers of individuals of each species initially increased rap-idly. The basic pattern of the Daphnia population was adampened oscillation. The Bosmina population did not increaseto an -asymptotic level but instead, after attaining less thanone-half the density of a single species population (Figs. 5 and6), declined whereas Daphnia individuals continued to re-produce in the first phase of the oscillation.When a combined food demand ratio is calculated for the

two populations in the competition vessels, it is evident thatBosmina populations ceased growth at a ratio essentiallyidentical to the ratio determined for Bosmina when culturedalone. The mean ratio value is 1.18 with a range of 0.93 to 1.54in five replicate cultures.

DISCUSSIONThe role of energy reserves to support animals during periodsof low ambient food levels or as energy for production of eggshas been established in many phyletic groups including marinezooplankton (13, 15, 16). Abyssal species of marine copepodsaccumulate triacylglycerols and wax esters. The triacylglycerolsare metabolized first and supply an animal's energy require-ments during brief periods of starvation. Wax esters are me-

FIG. 5. Replicate growth curves of B. longirostris at high fooddensity in 220 ml of water.

t The FD-to-FA ratio for Bosmina was calculated as for Daphnia. Inthe absence of data we assumed that 2000 cells mlh' of algae sus-pension would approximate the threshold for reproduction as inDaphnia.

Population Biology: Goulden and Hornig

i

1720 Population Biology: Goulden and Hornig

tabolized when animals are exposed to several months of lowfood levels. Near-surface species of marine copepods accu-mulate only triacylglycerols that can be metabolized to supportactivity when food is temporarily scarce, as when swimmingin search of a new patch of food. The reserve energy of thefreshwater species of Daphnia is also triacylglycerol, and in thisrespect these freshwater zooplankton are similar to near-surfacedwelling marine zooplankton. However, in laboratory culturesof Daphnia the animals use energy reserves immediately tosustain activity, as in marine copepods, but Daphnia also con-tinues to reproduce and transfer part of its reserve to its off-spring. The feeding of the newborn individuals further sup-presses the remaining food density. The result is that most ofthe juveniles born after the food demand of all individualspresent exceeds food available will starve and die when theyhave metabolized both the small amount of food they can filterand the energy reserve supplied by their mother. Older indi-viduals'with inadequate reserves and those individuals thatinvest all reserve energy into reproduction will also starve anddie. The energy reserves make the time lags possible, and in turnthe time lags are the primary cause of the oscillation.

This energy reserve cycle and the resultant oscillations haveconsequences to individuals of other species that do not accu-mulate as much reserve. The decline of Bosmina populationsmay be the result of the death of Bosmina juveniles althoughwe have not yet tested this idea. Bosmina individuals are verysmall relative to Daphnia and have a more rapid growth rate.Whereas adult Bosmina have lipid reserves, juveniles seldomhave very much reserve. We suggest that juveniles are able tocollect sufficient food for activity and growth but little forstorage as do Bosmina adults and Daphnia adults and juveniles.We believe Bosmina juveniles survive for only a brief periodwhen available food is reduced below a concentration that willsupport their activity and growth. Bosmina populations werenot eliminated from the cultures within the time frame of ourexperiments but appeared to increase after Daphnia popula-tions decreased (Fig. 6). The survival of adult Bosmina is dueto the presence and metabolism of energy reserves much as inDaphnia. When food is again abundant, at low densities ofDaphnia, Bosmina reproduces and its populations increase fora short period of time until FD/FA again exceeds 1. It is possiblethat Bosmina could indefinitely persist in the vessel, or at leastuntil an external stress would lead to exclusion. This is not the

same as frequency-dependent competition, because resourcesare not limiting during the brief interval that Bosmina popu-lations increase.The greater ability of individuals of the larger species to

accumulate larger amounts of energy reserves at all ages thanhas been observed in the juveniles of the smaller species and theuse of these reserves by Daphnia to sustain activity and toproduce additional young that further suppress the availablefood results in an increase in death rates of the smaller speciesdue to starvation.

This paper is dedicated to Dr. John Langdon Brooks for his contri-butions in emphasizing the importance of predation to the biology oforganisms and to the structure of ecosystems and for his continuedenthusiastic encouragement of research into zooplankton biology. Weare grateful to Dr. Luigi Provasoli for many helpful suggestions madeduring the course of this study and to Mr. Alan Tessier for organiccarbon measurements on the algae and for several helpful discussionsof the data. This work was funded by National Science FoundationGrant DEB 76-20119 and by the Supporting Research Fund of theDivision of Limnology and Ecology of the Academy of Natural Sci-ences of Philadelphia.

1. Brooks, J. L. & Dodson, S. I. (1965) Science 150,28-35.2. Pratt, D. M. (1943) Biol. Bull. 85, 116-140.3. Slobodkin, L. B. (1954) Ecol. Monogr. 24, 69-88.4. Caughley, G. (1970) Ecology 51, 53-72.5. Krebs, C. J. & Myers, J. H. (1974) Adv. Ecol. Res. 8, 267-399.6. Uvarov, B. (1966) in Grasshoppers and Locusts (Cambridge

Univ. Press, London).7. Hutchinson, G. E. (1948) Ann. N.Y. Acad. Sci. 50,221-246.8. Nichols, H. W. (1973) in Handbook of Phycological Methods,

ed. Stein, J. R. (Cambridge Univ. Press, London), pp. 7-24.9. Shiraishi, K. & Provasoli, L. (1959) Tohoku J. Agri. Res. 10,

89-96.10. Daley, R. I. & Hobbie, J. E. (1975) Limnol. Oceanogr. 20,

875-882.11. Lampert, W. (1977) Arch. Hydrobiol. Suppl. 48,287-309.12. Lampert, W. & Schober, U. (1980) in The Evolution and Ecology

of Zooplankton Communities, ed. Kerfoot, W. C. (Univ. Pressof New England, Hanover, NH), in press.

13. Lee, R. F., Hirota, J. & Barnett, A. M. (1971) Deep-Sea Res. 18,1147-1165.

14. Smith, G. (1915) Proc. R. Soc. London Ser. B 88,418-435.15. Conover, R. J. & Corner, E. D. S. (1968) J. Mar. Biol. Assoc. UK

48,49-75.16. Fulton, J. (1973) J. Fish Res. Board Can. 30, 811-815.

Proc. Nati. Acad. Sci. USA 77 (1980)