Hydrobiologia 446/447: 63–69, 2001.L. Sanoamuang, H. Segers, R.J. Shiel & R.D. Gulati (eds), Rotifera IX.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Population growth of Lepadella patella (O. F. Müller, 1786) at differentalgal (Chlorella vulgaris) densities and in association with Philodinaroseola Ehrenberg, 1832

S. Nandini & S. S. S. SarmaDivision of Interdisciplinary Research, National Autonomous University of Mexico (UNAM – Campus Iztacala)AP 314, CP 54090, Los Reyes, Tlalnepantla, State of Mexico, MexicoE-mail: nandini@servidor.unam.mx

Key words: population growth, rotifers, littoral species, food density, Bdelloidea

Abstract

Population growth of Lepadella patella was studied using Chlorella as the sole food at five concentrations rangingfrom 0.25 × 106 to 4.0 × 106 cells ml−1 at 25 ◦C for 22 days. The population densities increased with increasingalgal concentration up to 1.0 × 106 cells ml−1. The population growth of L. patella was lower at algal concentrationof 2.0 × 106 cells ml−1 and above. In a separate experiment, we tested the influence of the bdelloid rotifer Philodinaroseola on the population growth of L. patella at different ratios of initial inoculation densities using 1.0 × 106 cellsml−1 of Chlorella at 28 ◦C. Despite lower initial inoculation densities compared with those in the controls, bothL. patella and P. roseola showed higher peak abundances when grown together. The maximum peak abundancevalues recorded for L. patella and P. roseola were 830 and 230 ind. ml−1, respectively, at an inoculation ratio of1:1.

Introduction

Population growth of planktonic rotifers is stronglycontrolled by several factors including food availab-ility, temperature, interactions with co-occurring spe-cies and initial inoculation density (Edmondson, 1965;DeMott, 1989; Sarma et al., 1996). A vast major-ity of the population growth studies on brachionidsand some littoral species have shown a positive re-lation between the growth rates of rotifers and theavailability of food (Stemberger & Gilbert, 1985a;Ooms-Wilms, 1998). Population growth studies on lit-toral and benthic species are less common than thoseon planktonic rotifers. Studies on bdelloids and othernon-planktonic species have so far shown food-densityrelated abundance until a certain food level, usuallylower than those for planktonic rotifers (Ricci, 1984;Pérez-Legaspi & Rico-Martinez, 1998).

The relation between rotifer body size and theoptimal concentration of food required to produce amaximal abundance have yielded inconclusive results.Based on some studies, larger species require morefood to reproduce than smaller ones and species may

be inhibited by high algal concentrations (Stember-ger & Gilbert, 1985b). Conversely, some small sizedrotifer species do well under very high food concen-trations (Dumont et al., 1995). Most of these studieshave been conducted using planktonic rotifer spe-cies. Littoral and benthic rotifers are equally importantin freshwater ecosystems as an intermediate step forthe transfer of energy from detrius and benthic algaeto invertebrate and vertebrate predators (Lampert &Sommer, 1997). Furthermore, compared to planktonicrotifers, littoral and benthic species are much more di-verse (Koste, 1978; Nogrady et al., 1993) and thus aremore intricately linked in the trophic web.

Since the planktonic system is relatively homo-genous, there is an intense competition as a resultof which only a few species can thrive. Competitionis a much slower process than predation. In naturalsystems, competitive exclusion in the planktonic com-munity can be detected in ancient lakes. This has beenelegantly documented with reference to Lake Baikaland Lake Tanganyika (Dumont, 1994). However, inthe benthic community, the situation is different. Dueto the high complexity of benthic systems leading to

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a variety of niches, inter- and intra-specific compet-ition among benthic organisms may be less intensethan among planktonic ones. At times, co-occurrenceof two benthic species may actually facilitate theirgrowth (Nandini et al., 1998). This has, however, notbeen experimentally well established under laboratoryconditions.

In this study, we present some quantitative inform-ation on the population growth of the benthic speciesLepadella patella under different food concentrationsand in association with Philodina roseola at varyinginoculation densities.

Materials and methods

We isolated the test zooplankton, Lepadella patellaand Philodina roseola, from local water bodies inMexico City. L. patella is a littoral monogonont rotifer(Koste, 1978), while P. roseola is a bdelloid, pre-dominantly found attached to littoral zone vegetation(Donner, 1965). Both species were cloned separatelyand maintained on the unicellular green alga Chlorellavulgaris at concentrations of 1–2 × 106 cells ml−1.

We tested the effect of five algal food densities(0.25 × 106, 0.5 × 106, 1.0 × 106, 2.0 × 106 and4.0 × 106 cells ml−1) on the population growth ofL. patella. For all experiments, we used EPA me-dium (prepared by dissolving 96 mg NaHCO3, 60 mgCaSO4, 60 mg MgSO4 and 4 mg KCl in 1 l of dis-tilled water) (Anon., 1985) and Chlorella vulgaris wasmass cultured using Bold Basal medium (Borowitzka& Borowitzka, 1988). The alga was harvested dur-ing log phase, centrifuged at 3000 rpm for 5 min,and resuspended in EPA medium; the density was de-termined using a haemocytometer. Into 25 ml capacitybeakers with 20 ml EPA medium, rotifers were intro-duced at a density of 1 ind. ml−1. Four replicates wereset up for each treatment. All beakers were kept indiffused and continuous fluorescent illumination (200lux) at 25±2 ◦C. Observations were taken every dayon the population density of the test organisms. Afterevery 24 h, rotifers were filtered using a small net of25 µm mesh and food suspension medium was re-newed 100%. When densities of rotifers were greaterthan 10 ml−1, counts from 3 to 4 aliquot samplesof 1 ml each were used to estimate population dens-ity. Population growth was followed for 22 days, bywhich time a declining trend in the growth curve wasobserved for all the food concentrations tested. Figure 1. Population growth of Lepadella patella at various con-

centrations of Chlorella vulgaris. Values are mean (± 1 standarderror).

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Figure 2. Rate of population increase of L. patella in relation tofood density. Values are mean (± 1 SE).

Based on the results of the above experiment, weselected a food concentration of 1.0 × 106 cells ml−1

of Chlorella vulgaris to study interactions betweenP. roseola and L. patella. The experiment was con-ducted at 28 ◦C, which was suitable for both thespecies. Into 25 ml beakers we introduced 20 mlof EPA medium with the appropriate algal concen-tration. We introduced the rotifers at varying intialdensities: Lepadella control (20 Lepadella individu-als), Lepadella:Philodina (15:5), Lepadella:Philodina(10:10), Lepadella:Philodina (5:15) and Philodinacontrol (20 Philodina individuals). Philodina had atendency to stick to the walls of the beaker; therefore,a delicate brush was used to detach them before count-ing and transferring them to fresh medium. Populationgrowth of rotifers in all treatments was followed for22 days, by which time a declining trend in the growthcurve was observed.

Population growth rate (r) was calculated from theexponential phase using the formula:

r = (lnNt − lnNo)/t,

where No = initial population density and Nt = popu-lation density after time t. The r was calculated foreach replicate separately, for different time intervals,and mean values were used for analysis. The maximalpopulation densities and time required to reach themwere derived from each replicate separately followingSarma et al. (1998).

The various population characteristics were com-pared using Analysis of Variance and multiple com-parison tests (Students–Newmans–Keuls test) (Sokal& Rohlf, 1981).

Results

Population growth of L. patella in relation to fooddensity

Population growth curves of L. patella offered fivedifferent concentrations of Chlorella vulgaris whichare presented in Figure 1. In general, the popula-tion abundance of L. patella increased with increasingfood concentration from 0.25 × 106 to 1.0 × 106

cells ml−1 after which a further increase in food con-centration resulted in a reduced population growth.The growth curves had a long lag phase (6–8 days)followed by an exponential phase for the next 8–12days and thereafter remained stationary or began todecline. The maximal population density reached byL. patella was 1000±96 (mean±se) ind. ml−1 un-der a food concentration of 1.0 × 106 cells ml−1.There was a significant difference in both, the max-imum population density and the day at which this wasreached (P<0.001, Table 1, ANOVA) with increasingfood concentration. Maximum population density wasreached earliest at the highest food concentration (4.0× 106 cells ml−1). The rate of population increaseper day (mean±standard error) varied from 0.09±0.02at 4.0 × 106 cells ml−1 to 0.41±0.01 at 1.0 × 106

cells ml−1 (Fig. 2). There was a significant differ-ence in the population growth rates at the various foodconcentrations tested (Table 1).

Growth of L. patella and P. roseola under conditionsof co-existence

Population growth curves of L. patella and P. roseolagrown together at a food concentration of 1.0 × 106

cells ml−1 using different inoculation densities arepresented in Figure. 3. Regardless of the inoculationratio, both L. patella and P. roseola showed a signi-ficantly higher population abundance, in the presenceof each other, when compared to controls (P< 0.05,SNK-test). The maximum population abundance ofL. patella (920±230 ind. ml−1) was obtained whengrown with P. roseola in an equal inoculation ratio.There was no negative influence of the presence of L.patella on P. roseola or vice versa; instead both speciesbenefitted from their association as reflected in theirgrowth rates. There was no significant difference inthe peak population density reached by Lepadella incontrol or in association with Philodina; on the otherhand, those reached by Philodina were significantlyhigher in association with Lepadella than in controls(P < 0.01; Table 1). The rate of population increase of

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Table 1. Analysis of variance (ANOVA) performed on population growth variables ofLepadella patella. DF = degrees of freedom, SS = sum of squares; MS = mean square,F = F-ratio

Source DF SS MS F

Population growth of L. patella

Peak population density

Among food levels 4 1888093.0 47202 3.013.98∗∗∗Error 15 506637.0 337775.0 –

Day at peak population density

Among food levels 4 246.00 61.5 12.47∗∗∗Error 15 74.00 4.93 –

Rate of population increase

Among food levels 4 0.252 0.063 23.62∗∗∗Error 15 0.04 0.002 –

Population growth of L. patella and P. roseola in single- and mixed-species culturesPeak population density (L. patella)

Among inoculation 1 141376.0 141376.0 1.76ns

densities

Error 14 1124760 80340 –

Peak population density (P. roseola)

Among inoculation 1 0.014 0.014 12.25∗∗densities

Error 14 0.016 0.0011 –

Rate of population increase of L. patella

Among inoculation 3 0.01 0.003 5.71∗densities

Error 12 0.007 0.0006 –

Rate of population increase of P. roseola

Among inoculation 3 0.014 0.0047 3.50∗densities

Error 12 0.016 0.0013 –

∗ = p< 0.05; ∗∗ = p< 0.01; ∗∗∗ = p<0.001; ns = non-significant (p>0.05).

both test rotifers was significantly higher in the mixed-species treatments than in their respective controls(P < 0.05; ANOVA; Table 1). However, on com-paring the three inoculation ratios tested, we foundthat they had no significant effect on the populationgrowth rate of either species (P > 0.05, SNK-test).There was no significant difference in the day at whichthe maximum population density was reached by bothspecies. Similar trends were observed with the popu-lation growth rate values of both rotifers which weresignificantly higher in mixed species culture than insingle-species cultures (p <0.05, Table 1). The rate of

population increase per day varied between 0.35±0.01(L. patella = 100%) and 0.42±0.02 (L. patella = 50%),while for P. roseola the range was 0.24±0.03 (P.roseola = 100%) and 0.32±0.01 (P. roseola = 25%)(Fig. 4).

Discussion

In this study, both Lepadella and Philodina showedtypical growth curves similar to those in other stud-ies on non-planktonic rotifers (Ricci, 1984; Nandini etal., 1998). These growth curves differ from those ob-

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Figure 3. Population growth of L. patella and P. roseola alone and when grown together at a Chlorella concentration of 1 × 106 cells ml−1.Shown in column A are 100% L. patella; 75% L. patella + 25% P. roseola; 50% L. patella + 50% P. roseola and 25% L. patella + 75% P.roseola. Values are mean (± 1 SE). Shown in column B are 100% P. roseola; 75% P. roseola + 25% L. patella; 50% P. roseola + 50% L. patellaand 25% P. roseola + 75% L. patella. Values are mean (± 1 SE).

served in planktonic species in the prolonged lag phaseof almost 8 days. Planktonic rotifers, on the otherhand, begin to show increases in population dens-ity within 3 days at comparable temperatures (Walz,1995). Among the littoral rotifers, data on popula-tion growth have concentrated on Euchlanis (Gulatiet al., 1987) and Lecane (Hummon & Bevelhymer,1980). Lepadella patella, a common rotifer speciesoften found in high abundance in many freshwaterbodies, has not previously been cultured for a pro-

longed period of time. In this study, this species wascultured exclusively on Chlorella for several monthsbefore the experiments were commenced. The factthat L. patella could grow well on Chlorella indicatesthe possible utilization of planktonic algae by benthicrotifers. In our experimental containers, we noticedthat L. patella individuals were often present in greatnumbers on the surface of the water column. Philod-ina roseola has been cultured earlier on green algae(Lebadeva & Gerasimova, 1985, 1987). The range of

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Figure 4. Rate of population increase of L. patella and P. ros-eola in single species cultures and in mixed-species cultures havingdifferent inoculation ratios. Values are mean (± 1 SE).

algal food density used in this study has been used inthe past for other rotifers including planktonic, littoraland benthic species (Sarma et al., 1996; Nandini et al.,1998).

The patterns of population growth of L. patellaindicate a preference for lower algal levels. In gen-eral, smaller species can survive and reproduce atlower algal levels compared to larger species (Stem-berger & Gilbert, 1985a). This has been documentedalso for Anuraeopsis fissa (70 µm body size) vs Bra-chionus calyciflorus (255 µm) (Sarma et al., 1996)and Brachionus patulus (97 µm) vs B. calyciflorus(175 µm) (Sarma et al., 1999). The maximal abund-ance reached by a species is also influenced by thebody size. Thus, for any given food concentration, thenumerical abundance of L. patella was significantlyhigher than that of P. roseola.

The range of population growth rates recordedhere for L. patella was within the range recorded formany other rotifers. In general, most rotifer specieshave a growth rate of 0.2–2.0 depending on food typeand density. When compared to monogonont rotifers,bdelloid rotifers have much lower rates of popula-tion increase (Ricci, 1984). Similarly, we found muchhigher rates of population increase in Lepadella com-pared to Philodina (Fig. 4), and the maximal abund-ance reached by L. patella was much higher than thatof Philodina (Fig. 3). Since the two species differstrongly in their body sizes, the capacity of L. pa-tella to reach higher mean population abundance maybe attributed to its smaller body size. The differencesin the abundance of L. patella in the controls of themixed-species experiment and in the food density ex-periment (especially under 1.0 × 106 cells ml−1) mayhave been due to the different temperatures of the twoexperiments.

Rotifer community structure in nature is controlledby various factors, food density and predation be-ing important among the biotic factors (Lampert &Sommer, 1997). Competition among planktonic ro-tifers is known to be intense and is also influencedby food availability (Rothhaupt, 1990). A higher di-versity of littoral and benthic rotifers (Koste, 1978)is probably due to less intense competitive interac-tions (Dumont, 1994). Among littoral and benthicrotifers, competition is less intense and, therefore, apossible co-existence can be expected. In the presentwork, both L. patella and P. roseola benefited byeach other’s presence, possibly by utilizing excretedand partially digested alga; it is established that boththese rotifer species are detritivorous (Donner, 1965;Koste, 1978). The best results in terms of growth wereobtained when both species were initially in equal pro-portions (Fig. 4). Nandini et al. (1998) have shown thatPhilodina roseola has an antagonistic influence on thepopulation growth of planktonic monogonont rotifers.It is thus possible that the influence of Philodina onother rotifers is species-specific.

The present study indicates that Lepadella patellais well adapted to low food levels and that P. roseolahad a positive effect on the population growth of L.patella. It thus appears that the ability of littoral andbenthic species to co-exist is related to their low foodrequirements as well as species-specific interactions.Further research on interactions among other littoralspecies may help clarify the reason for the high spe-cies diversity observed in littoral and benthic regionsof lakes and ponds.

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Acknowledgements

We thank The National System of Investigators (Mex-ico) (Ref. No. 20520 & 18723) for support.

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