Organism Size, Life History,and N:P Stoichiometry

Toward a unified view of cellular and ecosystem processes

James J. Elser, Dean R. Dobberfuhl, Neil A. MacKay, and John H. Schampel

E cosystem science and evolution-ary biology have long been in-frequent and uncomfortablebedfellows (Hagen 1992, Holt 1995,Mclntosh 1985). However, the con-vergence of a global decline inbiodiversity and global alterationsin biogeochemical cycles providesmotivation to overcome past inhibi-tions. Currently, attempts are beingmade {Jones and Lawton 1995) tounderstand relationships between thefoci of evolutionary biology (the in-dividual in its species population)and ecosystem science (energy andmaterial flow and storage). Analysisof relationships between species andecosystems requires a framework ap-propriate for moving between levels

James J. Elser is ati associate professorin the Department of Zoology, ArizonaState University, Tempe, AZ 85287.His research interests include trophicinteractions and nutrient cycling inecosystems. Dean R. Dohberfuhl is adoctoral candidate whose research in-terests include phylogenetic and evolu-tionary detertninants of biochemical andelemental content of invertebrates. NeilA. MacKay is a doctoral candidatewhose research interests include behav-ioral ecology of zooplankton and zoop-lankton-cyanobacteria interactions.John H. Schampel is an assistant re-search scientist whose research inter-ests include zooplankton-phytoplanktoninteractions, zooplankton physiologi-cal ecology, and applied limnology. Hispresent address is Departmenr of Ecol-ogy, Evolution and Behavior, Univer-sity of Minnesota, St. Paul, MN 55108.© 1996 American Institute of Biologi-cal Sciences.

Elementalstoichiometry can

provide a new tool totrace the threads ofcausal mechanisms

linking cellular,ecosystem, and

evolutionary processes

in an imperfect hierarchy of bioticand abiotic components (O'Neill etal. 1986). Although various frame-works are possible, the history ofecology since Lindeman's 1942 pa-per on the trophic dynamic conceptmakes it clear that energy has beenthe currency of choice for ecologists(Hagen 1992).

Although the energetics perspec-tive has had wide application andsuccess, both in studies of individu-als and of ecosystems (Brown 1995,Pandian and Vernberg 1987, Wiegert1988, Wright et al. 1994), criticalexamination reveals inadequacies inthis paradigm. For example. White(1993) argues that, because of dis-parities between the nitrogen com-position of many foods and the ni-trogen demands of many consumers,the availability of energy is less im-portant than that of nitrogen in de-termining the reproductive successand population dynamics of animals.Mansson and McGlade (1993) have

also scrutinized energy-based ap-proaches to evolutionary biology andecosystem dynamics (in particularthose proposed by H. T. Odum) andconcluded that there are fundamen-tal problems in describing ecosys-tems using a framework that has asingle currency.

Reiners (1986) has presented amore balanced, multidimensionalview, proposing elemental stoichi-ometry as a complementary way tostudy questions about ecosystemsthat are unsuited for analysis withenergy-based models. Elemental stoi-chiometry considers relative propor-tions (ratios) of key elements inorganisms in analyzing how charac-teristics and activities of organismsinfluence, and are in turn influencedby, the ecosystem in which they arefound. In this article we introducethe main concepts and patterns ofecological stoichiometry and synthe-size literature from a variety of fieldsto forge connections, not only be-tween evolutionary and ecosystemsciences but also between the dispar-ate disciplines of cell biology andecology. Stoichiometry may have anatural advantage in making suchconnections because it offers an ex-plicit multiple-currency approachthat is potentially better suited thana one-currency approach to under-standing ecological and evolutionaryprocesses that more closely resembleoptimization rather than maximiza-tion (Krebs and Houston 1989).

Our approach in this article is asfollows. First, we describe recentdiscoveries that establish theimportance of consumer body

674 BioScience Vol. 46 No. 9

nitrogenrphosphorus (N:P) ratio inmodulating secondary productionand consumer-driven nutrient cyclingin ecosystems. Second, we reviewaspects of cellular biochemistry andultrastructure through the eyes of anecosystem scientist, focusing on therelative nitrogen and phosphoruscontents of important biomoleculesand cellular structures. Third, wepresent examples of how organismalcharacters such as growth rate andontogeny are linked with biochemi-cal and cellular investment and thuswith body N:P ratio. Finally, wepropose a general scenario for allo-metric variation in body N:P ratioamong consumers ranging from bac-teria to large vertebrates and use thescenario to predict patterns of con-sumer-driven nutrient cycling andfood quality constraints. In the spiritof Reiners (1986), we employ stoi-chiometric theory as a complemen-tary approach to the study of bio-logical processes, one that we hopewill both reinforce conclusions de-rived from energetic perspectives aswell as provide new insights intobiological phenomena that may bepuzzling when considered from moretraditional single-currency ap-proaches.

Ecological stoichiometry: basicconcepts and patternsEcological stoichiometry focuses onthe relative elemental compositionof participants in ecological interac-tions in ecosystems. Constraints ofmass balance must be met both insimple inorganic chemical reactions(Figure la) and more complex bio-chemical transformations (Figurelb); ecological interactions such ascompetition, predation, or herbivoryare also not exempt from thermody-namics (Figure lc). Thus, in the "eco-logical play," firm predictions canbe made about elemental ratios inthe "players" and their "stage"(sensu Lotka 1924) before and afterecological interactions.

One of the best-developed stoi-chiometric approaches in ecology isresource ratio competition theory(Tilman 1982). This theory, a modi-fication of the graphical approachesof MacArthur (1972), predicts out-comes of competition for inorganicnutrients among autotrophic taxa

a

b

c

stoichiometry

3C

Stoichiometry

Stoichiometry in

(M P )\ X y 'p r

n chemistry

/aoi- + ^i^a r^u ' ' ua ^r^u.L + DINCIL/I

in biology (respiration)

C,H,A + 6O, -^6CO,- .6Hp

ecology (predator-prey interaction with nutrient recycling)

Figure 1. The first law of thermodynamics dictates mass balance of mnltipleelements before and after: (a) inorganic chemical reactions, (b) simple biochemicaltransformations (e.g., respiration of glucose), and (c) complex ecological interac-tions (e.g., predation with nutrient recycling). In the stoichiometry of predator-preyinteractions, a prey item of a given elemental composition is consumed hy apredator of fixed elemental composition to increase predator biomass hy a factorQ, simultaneously producing waste of altered elemental composition. (That is,a':b' may be greater or less than aib, depending on the relative demands fornitrogen and phosphorus required for producing predator biomass; see Sterner1990.) The elemental ratio of recycled nutrients (a':b') contributes to the stoichi-ometry of another ecological interaction—nutrient competition among auto-trophs in the ecosystem.

differing in elemental requirements.In competitive situations, variationin nutrient supply ratios tips thecompetitive balance in favor of taxabest suited to the supply regime,altering the elemental compositionof autotroph community biomass andof the residual chemical environ-ment. Resource ratio theory has beenwidely supported by studies of com-petition among autotrophs (Sommer1989, Tilman 1982). However, stoi-chiometric approaches have rarelybeen applied to higher levels in foodwebs. In this article we highlightstudies of elemental ratios in con-sumers and how they may help inunderstanding the role of consumersin nutrient cycling and food webs.

Consumer-regulated nutrient cy-cling is increasingly attracting theattention of ecosystem scientists(DeAngelis 1992, and papers inNaiman 1988) who have tradition-ally focused on processes mediatedby autotrophs and microbes. In re-cent studies of consumer-driven nu-trient recycling in lakes, ecologicalstoichiometry explains unexpectedeffects of food web alterations onnitrogen and phosphorus availabil-ity (Sterner et al. 1992) and identi-fies qualitative differences in zoop-lankton-phytoplankton interactionsthat occur in marine and freshwaterhabitats (Elser and Hassett 1994).

These studies have focused on spe-cies-specific differences in body N:Pratio of zooplankton that dramati-cally affect the relative rates of recy-cling of nitrogen and phosphorus byelementally homeostatic consumers(Sterner 1990). For example, whenthe food web structure favors domi-nance by consumers with high bodyN:P (e.g., calanoid copepods, withbody N:P ratio greater than 30:1; allratios are given as atomic ratios),then the N:P ratio of nutrients re-cycled by those consumers is lowbecause food items tend to have lowerN:P ratios than consumers, whichwould therefore tend to retain nitro-gen and release phosphorus (see Fig-ure lc). Under such conditions,phytoplankton growth is limited pri-marily by nitrogen. By contrast, whenfish predation on zooplankton is low,permitting dominance by low N:Ptaxa (especially Daphnia, with N:Pratio approximately 12:1), recyclingN:P ratio is high and phytoplanktonare phosphorus limited (Sterner etal. 1992). Thus, body N:P ratio iscritical for understanding nutrientcycling in ecosystems because bodyN:P ratio directly determines therelative ratios of limiting nutrientsrecycled by consumers.

Variation in body N:P ratio is alsouseful in understanding a relativelynew aspect of consumer ecology: the

October 1996 675

Table 1. Major categories, exartiples, and biological functions of nitrogen- atidphosphorous-containing molecules. General information about structure, function,composition, and relative abundance of various molecules is from Lehninger et al.(1993).

Class of molecule Examples Eunctions Comments

Protein

Nucleic acids

Lipids

Collagen, actin Structure, regulation,communication

DNA, RNA

Phospholipids,glycolipids

Storage, transmission,and expression ofgenetic information

Ceil membranes

Phosphorylated ATP, phosphocreatine High-turnover energyenergy storage carrierscompounds

Average nitrogen con-rent of the 20 aminoacids in proteins is17.2%

DNA content (as a per-centage of cell mass)conservative. RNA:DNA greater than 5:1.

Carbon-rich, minorcomponent of cells (ap-proximately 5% of to-tal cell mass)

ATP oniy approxi-mately 0.05% of inver-tebrate body mass(DeZwann andThillartl985)

Structural Chitincarbohydrates

Structural support,protection

2 5 -

20 -

15 -

10-(

5 -

0 •

5 0 : 1 ^ 5 ' ,

; /' J'-^protein /;' / , /

I '' /'' / /'' cmtin/ / 'V phospholipibs' . - -" •

' pht3sphoarginine•

•phosphocrealine

%nucleic ATP •acids

_,..1:i"'

10 15

%P20

Figure 2. Stoichiometric diagram illus-trating the nitrogen and phosphoruscomposition of biomolecules contain-ing nitrogen and phosphorus. Valuesfor percentage nitrogen and percentagephosphorus are given in terms of weight.Dotted lines depict standard values ofatomic (molar) N:P ratio for the pur-pose of comparing various graphs.

role of mineral food quality in influ-encing consumer growth and repro-duction. Mineral nutrition has tra-ditionally been of interest primarilyto managers of livestock and gameanimals (McDowell 1992). How-ever, recent studies of mineral nutri-tion of freshwater zooplankton indi-cate that mineral limitation, inparticular phosphorus deficiency,may be commonplace in pelagic eco-systems (Elser and Hassett 1994,Sterner and Hessen 1994). In par-ticular, zooplankton taxa with high

phosphorus demands for growth(e.g., Dapbnia) experience reducedgrowth and reproductive outputwhen feeding on phosphorus-defi-cient food (Sterner and Hessen 1994).Knowledge of consumer N:P couldbe critical in identifying taxa mosthkely to suffer phosphorus limita-tion in nature and in assessing theextent to which the elemental stoi-chiometry of available food is likelyto affect production of higher trophiclevels.

The stoichiometric approachesjust described are largely phenom-enological, relying on direct mea-surements of body N:P ratio of domi-nant consumer taxa (Andersen andHessen 1991). Observations ofstrongly contrasting body N:P ratiosbetween taxa raise the question"What causes variation in bodyN:P?" In biology, answers to thatquestion are of two types (Mayr1961): proximate (biochemical andphysiological) and ultimate (evolu-tionary). Reiners (1986) addressedboth issues by distinguishing betweenbasic "protoplasmic life," which heargues has a standard chemical sto-ichiometry, and "mechanical struc-tures" (adaptations for specific func-tions, such as spines for defense orbones for support), which are highlyvariable in their stoichiometry. Thus,

selection for certain mechanical struc-tures in functionally dominant spe-cies will alter material cycling, in-cluding global ecosystem processes.

However, we believe that there isno characteristic elemental contentof "protoplasmic life" and that evenunicellular organisms exhibit con-siderable variation in elemental ra-tios as a function of their evolvedtraits. We therefore propose thatmajor changes in organism life his-tory (especially size and growth rate)require substantial changes in thecomplement of cellular components.Because different cellular compo-nents generally have contrasting bio-chemical constituents that differstrongly in elemental composition,major macroevolutionary patternsmust be accompanied by changes inorganism stoichiometry. In the fol-lowing, we focus on the limitingelements nitrogen and phosphorusand the role of heterotrophs (organo-heterotrophic bacteria, protozoa,and multicellular animals) in cyclingof those elements. We explore howrelationships between major life his-tory traits and cellular organizationare reflected in body N:P ratio andthus how evolved characters affectmaterial cycling by consumers at thelevel of the ecosystem.

How an ecosystem scientistsees cellsUnderstanding how selection onmajor life history traits alters el-emental content requires an under-standing of the biochemical func-tions of the various molecules usedby organisms and an appreciation oftheir elemental (especially nitrogenand phosphorus) composition.Wereview the function and structure ofimportant biomolecules, summarizetheir relative nitrogen and phospho-rus content, and then consider thebiochemical and elemental composi-tion of the cellnlar and subcellularstructures constructed from thesemolecules. As ecosystem scientists,we focus on biomolecules that con-tain relatively large amounts of ni-trogen and phosphorus and also con-tribute substantively to the cellularand extracellular make-up of organ-isms. This treatment is therefore asimplification of the actual varietyof biochemicals in organisms.

676 BioScience Vol. 46 No. 9

Biochemical stoichiometry. Majorfunctional classes of organic mol-ecules used in biological systems arelisted in Table 1, along with theirrelative nitrogen and phosphoruscomposition. For purposes of under-standing body N:P ratios, we canneglect most carbohydrates and stor-age lipids because most of these con-tain no nitrogen or phosphorus (butsee chitin, a structural carbohydrate;Figure 2). Nitrogen-rich moleculesinclude proteins, nucleic acids, andhigh-energy adenylates (ATP). Notethat these three classes of compoundsdiffer little in terms ofthe percentageof nitrogen (Figure 2); thus, cellularstructures or organisms with differ-ing protein:nucleic acid ratios willnot differ substantially in their per-centage of nitrogen. The main classesof phosphorus-rich molecules (Table1) are high-energy adenylates (par-ticularly ATP) and nucleic acids(DNA, RNA). Due to their high phos-phorus content, these molecules havelow N:P ratio (Figure 2). The ATPcontent of organisms is generallylow; thus the contribution of phos-phorus from ATP to whole-organ-ism phosphorus is also likely to below. For example, a study of theATP content of 22 invertebrate spe-cies (DeZwann and Thillart 1985)indicated that phosphorus in ATP asa percentage of dried weight variesfrom 0.02% to 0.2%, with a meanof 0.05%. Because whole-organismphosphorus content generally rangesfrom 0.2% to 2%, ATP contributeslittle more than a fifth of wholeorganism phosphorus.

The molecules likely to dominatecellular and organismal N:P stoichi-ometry are thus proteins, due to theirhigh nitrogen content and their sub-stantial contribution to biomass, andnucleic acids, due to their high nitro-gen and phosphorus content and theirrelatively high abundance. More spe-cifically, the strong contrast in N:Pstoichiometry between proteins andnucleic acids reflects the high phos-phorus content of nucleic acids andindicates that a critical determinantof N:P stoichiometry of cellular struc-tures and whole organisms will bethe relative abundance of proteinsversus nucleic acids.

Cellular stoichiometry. Now con-sider how major biomolecules are

20

1 5-

1 0-

5 -

200:1,50:1

bacteriai plasmalemma

eukaryoiic ,ribosome,-'

chromosome

nucleus ^ - ' '

,7:1

proloryoticribosome

. ' animal , - ''plasmalemma

mitochondrion; / _ , - 1:1 • - -

0 1 2 3 4 5 6

%PFigure 3. Stoichiometric diagram illus-trating the nitrogen and phosphoruscomposition of major organelles andother cellular structures. Values for per-centage nitrogen and percentage phos-phorus are given in terms of dry weightpercentage. Dotted lines depict standardvalues of atomic N:P ratio. As describedin the text, values for elemental compo-sition were calculated on the basis ofreported biochemical composition foreach structure, with the exception ofnuclei and mitochondria, for which dataare based on direct determinations ofpercent nitrogen and percent phospho-rus (Bowen 1979).

deployed in cells, and thus how struc-tures performing specific cellularprocesses themselves differ in bio-chemical and elemental composition,with potential consequences for eco-system processes affected by bodyN:P stoichiometry.

Cell membranes. In both prokary-otes and eukaryotes, membranesform selectively permeable physicalboundaries of compartments whosecomposition can be regulated to per-mit efficient biochemical processing(Fvans 1989). Membranes consistprimarily of a phospholipid bilayerand associated proteins, with a bio-chemical composition of 25%-56%lipids, 25%-62% protems, and 10%carbohydrates (Frausto da Silva andWilliams 1991). Thus, membranepercent nitrogen and percent phos-phorus are also variable (% N: 5.2-11.1; % P: 1.1-2.4), as is membraneN:P ratio (4.9-23:1; average pre-sented in Figure 3). Although mem-brane types differ in nitrogen andphosphorus content, increasing mem-brane contribution to overall cellu-lar biomass would generally drivethe N:P ratio down due to the phos-phorus-rich phospholipid compo-nent. However, the relatively smallinterspecies differences in contribu-tions of phospholipids to total bodymass that have been documented

(e.g., Reinhardt and Van VIeet 1986)appear unlikely to contribute greatlyto differences in organism N:P ratio.

Nucleus and chromosomes. Thenucleus is the largest organelle ineukaryotic cells and consists of chro-mosomes, nuclear membrane withexternally situated ribosomes, andproteinaceous nucleoplasm matrix.In eukaryotic chromosomes, DNA iscomplexed with organizing proteins(both histone and nonhistone) intochromatin, which has a protein:DNAratio of 1-2:1 by weight (Lehningeret al. 1993). Assuming that chroma-tin proteins are 17.2% N by weight(above) and are present in a 1.5:1ratio by weight with DNA, eukary-otic genetic material is thus 16.5%N by weight and 3.6% P by weight,with an atomic N:P ratio of approxi-mately 10:1 (Figure 3). The variablecomplex of materials other than chro-mosomes in nuclei complicates cal-culation of the elemental composi-tion of nuclei as a whole. However,cell fracture studies (Bowen 1979)have directly measured the nitrogenand phosphorus composition of nu-clei as approximately 12.6% N and2.5% P (N:P ratio of approximately11:1; Figure 3).

Ribosomes. Ribosomes are sitesof protein synthesis and thus arecentrally involved in growth. Ribo-somes are composed of ribosomalRNA (rRNA) and protein in ratiosof 1.22:1 for eukaryotes and 1.8:1for bacteria (Campana and Schwartz1981). Calculations of elementalcontent based on these data indicatethat ribosomes are a particularlyphosphorus-rich cellular constituent{eukaryotic ribosomes: 16.3% N,5.0% P, N:P approximately 7.2:1;prokaryotic ribosomes: 16.1% N,5.6% P, N:P approximately 6:1; Fig-ure 3). Because ribosomes appear tohave lower N:P ratio than other sub-cellular structures, increasing ribo-somal content will tend to lower theN:P ratio.

Mitochondria. Mitochondria aresites of oxidative ATP generation byrespiration in eukaryotic cells. Mito-chondria are composed of outer andinner membranes and a gel-like in-ner compartment known as the ma-trix. The matrix is approximately50% protein by weight and alsocontains DNA (approximately15,000 base pairs in human mito-

Octoberl996 611

chondria) and ribosomes (Becker1986). The outer membrane con-tains nearly equal proportions oflipids and proteins, whereas the in-ner membrane is nearly 80% proteinand only approximately 20% lipidby weight (data for liver mitochon-dria; Lehninger et al. 1993). Convo-luted foldings of the inner mem-brane increase its surface area,enhancing mitochondrial ATP gen-eration. Not surprisingly, the sur-face area of the inner membranecorrelates with the intensity of tis-sue respiration (Lehninger et al.1993). Cells that respire heavily havea larger percentage of inner mito-chondrial membrane, which by vir-tue of its high protein content has ahigh percent nitrogen and a high N:Pratio, than cells with smaller respi-ratory demands. Thus, mitochon-dria probably have an inherently highN:P ratio. Direct observation sup-ports this suggestion: data for per-cent nitrogen and percent phospho-rus from cell fracture studiessummarized by Bowen (1979) indi-cate a mitochondrial N:P ratio ofapproximately 80:1 in mammaliantissues (Figure 3). Consequently, in-creased mitochondrial contributionwould tend to increase cellular andorganismal N:P ratio.

Endoplasmic reticulum (ER) andthe Golgi complex. ER is a membra-nous network that constitutes ap-proximately 15% of total cell vol-ume (Mieyal and Blumer 1981). ERis the major component of the intra-cellular cytocavitary network, is theorganizing structure for some ribo-somes (rough ER), and is involved inhydroxlation reactions, detoxifica-tion, and other metabolic transfor-mations. ER membrane has a highprotein:!ipid ratio of 2.3 (Becker1986). Thus, we estimate that ERmembrane itself is 12.3% N and1.2% P by weight and has an N:Pratio of approximately 22:1 (Figure3). The Golgi complex mediatesflows of secretory proteins from theER to the exterior of the cell and iscomposed of proteins and lipids in-termediate in composition betweenER membrane (from which Golgimembrane is thought to arise) andplasma membrane (with whichGolgi-derived secretory vesicleseventually fuse in discharging theircontents to the outside of the cell;

0.0 2.0

Figure 4. Stoichiometric diagram illus-trating the characteristic difference innitrogen and phosphorus compositionfor two major herbivorous zooplanktongroups: calanoid copepods (circles; fourspecies plotted) and the cladoceranL)apbnia (triangles; five species plot-ted). Values for percentage nitrogenand percentage phosphorus are given interms of dry weight percentage. Dottedlines depict standard values of atomicN:P ratio. Data from Andersen andHessen (1991) and Hessen and Lyche(1991).

Becker 1986). Assigning the Golgi aprotein:hpid ratio of 1.7 (intermedi-ate between the protein:lipid ratiosof plasma membrane and ER), weestimate the elemental compositionof the Golgi apparatus as 11.2% Nand 1.5% P, with an N:P ratio of17:1 (Figure 3).

Cytoplasm. Cellular cytoplasm iscomposed of cytosol (the solubleportion of the cytoplasm) and cy-toskeleton (an internal frameworkthat gives eukaryotic cells their dis-tinctive shape and internal organiza-tion and governs the position andmovement of organelles). In typicalanimal cells, cytoplasm occupiesmore than half of cell volume. Pro-tein content of the cytosol exceeds20% (Lehninger et al. 1993), likelyreflecting localization of the major-ity of enzymes of intermediary me-tabolism. Proteinaceous microtu-bules dominate the structuralcomponents of cytoskeleton (Leh-ninger et al. 1993). Because cyto-skeleton probably contributes moreto total cell mass in larger, moredifferentiated cells than in small,undifferentiated cells, if all else wereequal we would expect such cytosk-eleton-rich cells to have a higher N:Pratio.

Extracellular materials. Unicel-lular organisms release materials tothe exterior ofthe plasma membraneto form cell walls, and in multi-cellular organisms, extracellular

materials are involved in cellularaggregation, tissue organization,maintenance of intercellular spaces,and construction of protective cov-erings and support structures. Thus,the biochemical and elemental com-position of these materials must beconsidered for a complete view oforganism composition. For example,invertebrates commonly use the ni-trogenous polysaccharide chitin (Fig-ure 2) for exoskeletons (insects) andcarapaces (crustaceans). In verte-brates the fibrous protein collagen isthe major extracellular structuralprotein in connective tissue and bone,making up one-third or more of totalbody protein in higher vertebrates(Lehninger etal. 1993). Also of criti-cal importance in determining wholeorganism elemental content in verte-brates is bone itself, which is depos-ited as apatite [Ca,(OH)(PO,)Jwithin a proteinaceous connectivetissue matrix (Frausto da Silva andWilliams 1991). The phosphorouscontent of bone is sufficiently highthat bone has a very low N:P ratio(0.8:1) despite the importance ofcollagen in the bone matrix.

Organismal N:P ratio and lifehistory charactersPrimary life history parameters oforganisms, especially specific growthrate, are linked to biochemical com-position and body N:P stoichiom-etry. We focus on crustacean zoo-plankton, the group with which weare most familiar, but data for othergroups illustrates how these linkagesare general across broad taxonomicand habitat categories.

Growth rate and N:P stoichiometry.Studies of nitrogen and phosphoruscomposition of zooplankton haverevealed patterns (both within andbetween taxa) that are particularlyinstructive in understanding linksbetween elemental composition, bio-chemical makeup, and life historystrategies. The two dominant groupsof crustacean herbivores in freshwa-ter plankton, calanoid copepods andcladocerans, have contrasting lifehistories. Cladocerans (e.g., Daph-nia) grow rapidly, reach sexual ma-turity (for parthenogenetic repro-duction) within days of birth undergood food conditions, and produce

678 BioScience VoL 46 No. 9

many generations within a growingseason. Calanoid copepods, by con-trast, grow slowly, reproduce sexu-ally, and generally complete onlyone or two generations during a year.In addition, copepods undergo com-plex metamorphosis, whereas cla-docerans do not.

Large differences in body N:P ra-tio accompany these life history con-trasts (Figure 4). Both marine andfreshwater calanoid copepods haveN:P ratios exceeding 30:1 (Andersenand Hessen 1991, Bamstedt 1986,Hessen and Lyche 1991). However,cladocerans, a predominantly fresh-water gronp, have an elemental com-position with slightly less nitrogenand considerably more phosphorus,resulting in lower N:P ratios (12-18:1; Andersen and Hessen 1991,Baudouin and Ravera 1972, Hessenand Lyche 1991). For example,Daphnia has an N:P ratio of 15:1(Figure 4). However, carnivorous cla-doceran species seem to have higherN:P ratios than herbivorous cladocer-ans of similar size (Hessen and Lyche1991).

Although data are limited, the bio-chemical basis of the differences inbody N;P ratio between cladoceraand copepods is becoming clear(Sterner 1995, Sterner and Hessen1994). First, nitrogen compositionvaries from only 8%-10% in bothcopepods and cladocerans {Andersenand Hessen 1991, Hessen and Lyche1991). Because nitrogen is a primaryconstituent of proteins, it is likelythat the protein pool varies littleacross taxa. In contrast, the specificphosphorus content of zooplanktonis apparently much more variable:the mean percentage phosphorusvalues for copepods and cladocerans(especially Daphnia) are less than0.6% and around 1.5%, respectively(Andersen and Hessen 1991, Bau-douin and Ravera 1972, Hessen andLyche 1991). As discussed above,phosphorus is a constituent of sev-eral prominent biochemicals in cells:phospholipids, ATP/ADP, andnucleic acids. Phospholipids are aminor constituent in cells, and high-energy adenylates likewise generallycontribute less than 1 % to dry weightin zooplankters (Bamstedt 1986).This leaves nucleic acids as the re-maining candidate to explain varia-tion in percent phosphorus among

Tuesday Lake Zooplankton Peter Lake Zooplankton

0.0 1.5

Figure 5. Stoichiometric diagram illustrating changes in estimated nitrogen andphosphorus composition of zooplankton cotnmunities during food web manipula-tions (introduction or removal of piscivorous bass) reported by Elser et al. (1988).Dotted lines depict standard values of atomic N:P ratio. Values for percentagenitrogen and percentage phosphorus are given in terms of dry weight percentage.Estimates of zooplankton community N:P stoichiometry were made using data forbiomass contribution of copepods and Daphnia and published values of percent-age nitrogen and percentage phosphorus for these groups (see Figure 4). Datapoints indicate weekly observations during periods of rapid zooplankton change.Shifts in the nature of phytoplankton nutrient limitation (nitrogen versus phos-phorus) as a result of changes in zooplankton community N:P stoichiometry areindicated. Tuesday and Peter Lakes are located at the University of Notre DameEnvironmental Research Center in Michigan's upper peninsula.

taxa. In fact, copepods are generallyaround 2% RNA by weight, whereasDaphnia can be as high as 10%RNA (Bamstedt 1986, Baudouin andScoppa 1975, Dagg and Littlepage1972, McKee and Knowles 1987).Assuming that RNA is 10% phos-phorous (Figure 2), the entire differ-ence in specific phosphorus contentof these zooplankton groups (0.8%phosphorous) can be explained bydifferences in RNA content (Sterner1995).

This example provides one of thebest illustrations of the fundamentallink between organism life history,biochemical investment, and bodystoichiometry. Cladocerans in gen-eral, and Daphnia in particular, haveevolved traits that favor rapid growthand high reproductive output,whereas copepods grow more slowlyand live longer. Daphnia's highgrowth rate requires a high riboso-mal complement for extensive pro-tein synthesis coupled to that highgrowth rate. Thus, its tissues havehigh rRNA and therefore high phos-phorus contents and low N:P ratio,making Daphnia a poor recycler ofphosphorus relative to nitrogen.

We can now see the study of Elseret ai. (1988), the first to documentqualitative effects of consumers onavailability of nitrogen and phos-phorus in ecosystems, in a new light.

In Tuesday Lake, predation pressureon zooplankton was reduced by in-troducing piscivorous bass that re-duced zooplankton-feeding minnowpopulations. Daphnia (a superiorcompetitor to copepods by virtue ofits high grazing and growth rates)rapidly came to dominate the zoo-plankton, replacing calanoid cope-pods (Figure 5). The replacement ofhigh N:P copepods with low N:PDaphnia likely produced a high re-cycling N:P ratio. Thus, the samefeature that enables Daphnia toachieve dominance under low pre-dation (its rapid growth rate) neces-sitates investment in biochemical andcellular machinery that lowers bodyN:P ratio (Fignre 3) and elevatesrecycling N:P ratio in the ecosystem(Figure .5). Conversely, when nearbyPeter Lake was manipulated to in-crease predation intensity on Daph-nia^ slower growing zooplanktonspecies (calanoid copepods and smallcladocerans) achieved dominanceand, by virtue of their reduced in-vestment in low N:P cellular con-stituents, generated a low recyclingN:P ratio (Figure 5). Such variationsin recycling N:P ratio as a functionof consumer N:P ratio are now be-coming more widely documented(Elser and Hassett 1994, Elser et al.1995, Urabel993,Urabe etal. 1995).

In addition to these nutrient cy-

Octoberl996 679

6-

4-

2 -

n -

200:1j

I;

Daphnia

, 6

50:1

/

magna5 "*

_ 15:1

1 3 2

7:1

. . . 1 : 1 • - -

Drosophila melanogaster

0.0 0.5 1.00 0.2 0.4 0.6 0.8 1.0

.0 0.2 0.4 0.6 0.8Growth rate (per d)

-1 0 1 2 3 4 5Growth rate (per d)

Figure 6. (a) Stoichiometric diagrams illustrating ontogenetic variation in percent-age nitrogen and percentage phosphorus in Daphnia magna and Drosophilamelanogaster. Dotted lines depict standard values of atomic N;P ratio. Values forpercentage nitrogen and percentage phosphorus are given in terms of dry weightpercentage. Numbers indicate the ontogenetic sequence of the observations, (b)Correlation between body N:P ratio and specific growth rate during ontogeny inD. magna and D. melanogaster. Data for D. magna are from McKee and Knowles(1987) and for D. melanogaster are from Church and Robertson (1966).

cling effects, researchers are begin-ning to explore how the nutritionalvalue of food (specifically food phos-phorous content) alters how con-sumers with different life historiesand N:P stoichiometries respond tochanges in food web structure. Forexample, we recently manipulatedthe food web of a severely phospho-rus deficient lake on the CanadianShield in Ontario by introducingpredatory pike to a minnow-domi-nated food web lacking Daphnia.Despite 100-fold reductions in min-now abundance, Daphnia increaseshave been modest and high N:Pcalanoid copepods remain domi-nant,' suggesting that the poor qual-ity of the lake's severely phosphoruslimited phytoplankton preventsdominance by low N:P Daphnia.Body stoichiometry thus provides aframework to establish direct mecha-nistic links between the molecularprocesses of growth at the level ofthe cell and the reciprocal interac-tions between organisms and the eco-systems in which they are found.

'J.J. Elser, T. H. Chrzanowski,R. W. Sterner,and K. H. Mills, 1996, manuscript in review.

Ontogeny and N:P stoichiometry.Many organisms undergo complexdevelopmental sequences (ontogeny)during which both body size andspecific growth rate vary consider-ably. Thus, we would also expectvariation in biochemical and ele-mental composition to accompanyontogeny. To the extent that N:Pstoichiometry changes during devel-opment we would also expect varia-tion in an organism's effects on ni-trogen and phosphorus cycling andits sensitivity to mineral f̂ ood qual-ity.

Data on biochemical compositionand specific growth rate during on-togeny in a variety of invertebratesshow that there are indeed strongontogenetic shifts in body stoichi-ometry. For example, daphnids ap-pear to maintain high concentrationsof RNA throughout ontogeny, re-sulting in modestly variable but gen-erally low N:P ratios in all life stages(Baudouin and Ravera 1972, McKeeand Knowles 1987; Figure 6a). Incontrast, complex metamorphosis incopepods appears to result in moredramatic changes in elemental com-position during ontogeny. Protein:RNA ratio in copepod nauplii (stages

immediately following hatching) islow (and thus body N:P ratio is low);as development progresses throughcopepodid (juvenile) stages, growthrate slows and investments in struc-tural proteins increase, causing in-creases in the protein:RNA ratio(Bamstedt 1986, Dagg and Littlepage1972) and thus in N:P ratio. Thus,effects of individual copepods onrelative nitrogen and phosphoruscycling in nature probably varystrongly during development.

Strong shifts in body biochemicaland elemental composition duringdevelopment are not confined tocopepods. Holometabolous terres-trial insects exhibit similar trends inbiochemical and elemental composi-tion (Church and Robertson 1966,Langet al. 1965). For example, bodyN:P ratio of Drosophila mela-nogaster varies from 9:1 in earlylarvae to 100:1 immediately beforepupation (Church and Robertson1966; Figure 6a). Variation in bodystoichiometry during ontogeny alsoprovides compelling evidence thatthe main determinant of body N:P ininvertebrates is specific growth rate:stage-specific body N:P ratio isstrongly (r̂ > 0.85) and linearly cor-related (P < 0.01) with stage-specificgrowth rate in both Daphnia andDrosophila (Figure 6b).

Organism size and N:P ratio:a predictionWe have discussed how evolved char-acteristics, such as growth rate anddevelopmental sequences, translateinto differences in biochemical andelemental composition in consum-ers. In this section we discuss howmacroevolutionary patterns in lifehistory traits also have implicationsfor organismal N:P stoichiometry,focusing on the central parameter oftraditional life history theory, or-ganism size. How does body N:Pratio in healthy, actively growingorganisms vary in consumer taxaranging from bacteria (less than 1picogram [pg]) to large vertebrates(7000 kg or more), a size range of 15orders of magnitude? Tbe study ofallometry in body stoichiometry hasconsiderable potential for new in-sights into the causes and conse-quences of evolutionary processes inconsumer taxa. These insights may

680 BioScience Vol. 46 No. 9

-16 -14 -12 -10 -B -S -4 -2 0 2 4 6 8

Log(Mass, g)

Figure 7. Predicted variation in organ-ism N:P ratio (hy atoms) as a function oforganism size (g dry weight). Dottedlines above and helow the main trend(black line) are meant to indicate thatthere is likely to be important ecologi-cally or evolutionarily derived variationin organism N:P ratio at any given bodysize. For large organisms, two trajecto-ries are possible. One trajectory (grayline) corresponds to a continuous in-crease in organism N:P ratio (which islikely for large invertebrates) and theother to a decline in organism N:P ratiodue to increasing bone investment invertebrates (the main group of animalsof large hody size).

complement those that have resultedfrom a widespread study of allo-metry in energetic relations (Schmidt-Nielsen 1984) in consumers.

The question of whether organis-mal N:P ratio varies significantly andsystematically with size distills towhether percent nitrogen and per-cent phosphorus vary differently withbody size. We have already shownthat, within certain taxa, percentnitrogen and percent phosphorus areclearly not isometric, because thehody N:P ratio varies ontogeneti-cally, and therefore with size, inDapbnia magna and, especially, D.melanogaster (Figure 6a). But whatabout allometric variation acrosstaxa?

Two factors probably influenceorganism N:P ratio as we proceedfrom bacteria to large vertebrates.First, the biochemical and elementalcomposition of protoplasm may varysignificantly with size. For example,organisms adapted for rapid growthmay contain a greater proportion ofRNA, and thus have a lower N:Pratio, than organisms adapted forslower growth. The second factorinfluencing the size-dependence ofbody N:P is that increases in sizemay bring concomitant increases instructural materials, as emphasized

by Reiners (1986). For example, thechitinous exoskeletons of arthropodscontain more nitrogen than verte-brate structural materials, includingbone, which has a very low N:Pratio.

One of the most widely docu-mented allometric patterns is thebroad relationship between organ-ism size and specific growth rate(Peters 1983). Allometric declines ofspecific growth rate (as indexed byproduction per unit biomass) withsize have similar slopes (approxi-mately -0.25) for unicells, poikilo-therms, homeotherms, tetrapods,mammals, and fish, although in somecases the intercepts are displacedsomewhat and the slope of the rela-tionship for invertebrates is steeper(-0.37). These patterns hold withinmore specific taxonomic categoriesas well. For example, in ciliates spe-cific growth rate declines signifi-cantly with cell volume (Fenchel1968).

If the link between organism N:Pratio and specific growth rate (Fig-ure 6) is general, it follows that asorganisms increase in size, theorganismal N:P ratio should increase(Figure 7). This view is supported byavailable but extremely limited data.For example, healthy bacterial cells(mass: 1 pg) have extremely rapidgrowth rates and can have N:P ratioas low as 5:1 (Bratbak 1985). Thesmall flagellate Parapbysomonasimperforata (mass: 5 nanogram [ng])has an N:P ratio of 10:1 (Caronet al.1985), whereas characteristic N:Pratios for crustacean zooplankton inthe 10-100 |i.g range are generally12-70:1 (Andersen and Hessen 1991)and those for late instar Drosophilalarvae (mass: 0.5 mg) are 100:1 (Fig-ure 6a). Variation around this gen-eral pattern is likely. For example,the different N:P ratios of the simi-larly sized crustacean zooplanktersDapbnia and calanoid copepods(12:1 versus greater than 30:1) prob-ably reflect differences in their spe-cific growth rates. Thus, just as allo-metric patterns of growth rate withbody size show considerable scatter,we would also expect to observesubstantial deviations from the over-all trend of increasing N:P ratio withorganism size over this size range.These deviations are likely to havesignificant evolutionary and ecologi-

1 4 -

1 2 -

1 0 -

8 :6 -

2 -0

skin

0 heartkidney

7^~-liver' brain'

muscle •

fl,blood , , . - • 1 : 1 *

6

% P10 12

Figure 8. Stoichiometric diagram illus-trating nitrogen and phosphorus com-position of mammalian organs and or-gan systems. Dotted lines depict standardvalues of atomic N:P ratio. Values forpercentage nitrogen and percentagephosphorus are given in terms of dryweight percentage. Data from Bowen(1966).

cal causes, as in the case oiDaphniaversus copepods.

However, the trend of increasingorganism N:P ratio with increasingbody size is unlikely to continuemonotonically throughout the com-plete range of organism size becauseother factors, in particular struc-tural investments, come into play atlarge organism size. In particular,vertebrates, which enter the size con-tinuum at around 100 mg, compli-cate the picture because contribu-tions of skeletal materials are likelyto strongly influence whole organ-ism N:P ratio. Thus, for large organ-isms, it is necessary to consider howprotoplasmic and structural compo-nents combine to affect organismalN:P ratio. For both invertebratesand vertebrates of large body size,specific growth rate declines withbody size (Peters 1983). Indeed, spe-cific RNA content is known to de-cline with body size in certain mam-malian tissues (Peters. 1983). So, N:Pratio of large invertebrate biomassand of "soft-tissue" biomass of ver-tebrates likely continues to increasewith size. However, the structuralsupport investments of vertebratesbegin to dominate body N:P ratioand necessitate a different approachto predicting size dependence of N:Pratio within the vertebrates, thedominant group of organisms largerthan 10 g.

Evaluating N:P ratio at large bodysize is facilitated by information onthe biochemical and elemental com-position of major tissues and organsof vertebrates (Bowen 1966). In ad-

Octoberl996 681

dition, the allometry of contribu-tions of these tissues and organs tototal body mass is also known (Calder1984). From these data we can esti-mate N:P stoichiometry of "theo-retical vertebrates" of various bodysizes. Various body components varystrongly in elemental content (Fig-ure 8). Tissues such as skin and bloodhave high protein contents and thushave high N:P ratio (greater than100:1); in contrast, skeletal materialis phosphorus rich due to the depo-sition of apatite within collagen-based connective tissue (Frausto daSilva and Williams 1991) and haslow N:P ratio (0.8:1). The relativecontributions of various tissues varystrongly with body size; data formammals were analyzed by Calder(1984). Of particular interest in thiscontext is an increase in percent skel-etal mass with body size from 3.8%of body mass in shrews to 13.6% inelephants (Prange et al. 1979).

To evaluate N:P variation withbody size in vertebrates, we usedpercent nitrogen and percent phos-phorus data for various organ sys-tems compiled by Bowen (1966) asfixed values (although they likelyvary with body size as well; seediscussion above) and calculatedbody percent nitrogen and percentphosphorus based strictly on Calder's(1984) equations regarding contri-butions of tissue and organ systemsas a function of body size. Thesecalculations predict a strong size de-pendence for N:P stoichiometrywithin vertebrates: we estimate thata 10-g vertebrate is 10.8% N and0.98% P (N:P ratio: 24:1), whereasa 1000-kg vertebrate is 7.0% N and1.6% P (N:P ratio: 9.6:1).

The substantial shifts in organismN:P ratio with size (Figure 7) haveimplications for impacts of consum-ers on ecosystem nntrient cycling.The increase in organism N:P ratiofrom bacteria through metazoansimplies that larger organisms withinthis size range will generally be moreefficient recyclers of phosphorus thanof nitrogen. Relative recycling effi-ciencies for nitrogen and phospho-rus by a consumer depend on the N:Pratios of both prey and consumer(Sterner 1990); thus, the degree towhich the consumer differentiallyrecycles nitrogen and phosphorus willbe determined by the size dichotomy

between predator and prey. Becausepredators are generally larger thantheir prey, the increasing N:P ratiowith organism size implies that el-emental imbalance (food N:P ratio -consumer N:P ratio; Elser andHassett 1994) will generally be nega-tive for consumers eating other con-sumers and thus will result in a lowrecycling N:P ratio. This is in strongcontrast to the interaction betweenherbivorous consumers and primaryproducers (which commonly haveextremely high N:P ratios); elemen-tal imbalance for the herbivore-pro-ducer interaction is frequentlystrongly positive in lakes, resultingin high recycling N:P ratio (Elser andHassett 1994).

Decreases in N:P ratio with largebody size also probably influencedirect effects of large animals onnutrient cycling, an impact that hasbeen increasingly emphasized (Nai-man 1988). For example, many stud-ies have shown that migration ofanadromous fishes from oceans tolakes, where adnlts spawn and die,represents a significant source ofphosphorus to the lake (Northcote1988). The low N:P ratio of largevertebrates (Figure 7), including fish(e.g., body N:P ratio of northernpike [Esox lucius] is around 12:1[George 1994]), implies that suchfluxes differentially introduce phos-phorus (relative to nitrogen) to lakes.Moreover, Carpenter et al. (1992)have shown that fish transport phos-phorus from the littoral zone (wherethey capture prey) to the pelagic zone(where they excrete wastes). How-ever, most studies of fish nutrientcycling have focused exclusively onphosphorus and have not considerednitrogen (but see Vanni 1995).

Our predictions of N:P ratio (Fig-ure 7) indicate that fish have bodyN:P ratios lower than those of mostof their prey (size range: 50 |ig-l g).Thus, stoichiometry predicts that fishshould differentially recycle nitro-gen relative to phosphorus (Sterner1990); if fish alter the phosphorusbudgets of pelagic ecosystems, thenthey are also likely to alter the nitro-gen budgets even more strongly. Thus,an indirect effect offish on phytoplank-ton communities not yet emphasizedby aquatic ecologists is an alterationof the N:P supply ratio and thereby ashift in competitive relations among

phytoplankton species.The size dependency of organism

N:P ratios also has implications forthe role of phosphorus-based foodquality in consumer ecology. Organ-isms with low body N:P ratio, eitheras adults or at sensitive points intheir life history, will have high phos-phorus demands for growth andmaintenance; these organisms willthus require food that meets not onlytheir energetic demands but also theirsomatic elemental demand. The re-cent discovery of effects of phospho-rus-deficient food quality on low N:Pzooplankton {e.g., Daphnia; Sternerand Hessen 1994) provides an in-triguing complement to examples ofphosphorus-based food quality con-straints on vertebrate herbivores(livestock and game populations;McDowell 1992), which also havelow N:P ratio. Thus, we suggest thatorganism N:P ratio provides a toolfor identifying taxa, or key life his-tory stages within taxa, that are mostlikely to be affected by variation inphosphorus-based food quality inecosystems. This stoichiometricknowledge may lead to a better un-derstanding of consumer foragingbehavior and population regulationthat may not be obtained from stud-ies that view production and forag-ing solely in terms of energetic pa-rameters.

ConclusionsWe have shown how the mechanis-tic bases of a phenomenon occurringat the level of the ecosystem (differ-ential recycling of nitrogen and phos-phorus as affected by food web struc-ture) can be traced to the level of thecell and molecule by focusing on N:Pstoichiometry. This application ofstoichiometric thinking was possiblebecause species that are dominantunder contrasting ecological condi-tions have contrasting life historiesrequiring different cellular and bio-chemical investments that necessar-ily result in differences in body N:Pratio. Elemental stoichiometry canthns provide a new tool to trace thethreads of causal mechanisms link-ing cellular, ecosystem, and evolu-tionary processes. It offers a meansof integrating not only the tradition-ally disparate disciplines of evolu-tionary biology and ecosystem sci-

682 BioScience Vol. 46 No. 9

ence but also ecology and cell biol-ogy, two fields that have developednot only independently but oftenantagonistically. Stoichiometry thuscomplements energetic perspectivesby addressing situations in whichenergy acquisition and use may notbe primary factors dictating fitnessor ecological dynamics.

Exploration of the role of stoichi-ometry in regulation of biologicalprocesses is just beginning, evenwithin ecology. Nevertheless, con-sideration of biochemical and el-emental consequences of macroevo-lutionary trends sets the stage for aunified evolutionary view of the cel-lular mechanisms that drive ecosys-tem processes. The power of stoi-chiometric perspectives arises fromthe unavoidable demands for chemi-cal elements and the first law ofthermodynamics, which applies toall processes by which organisms areborn, grow, develop, and die. Theessence of this idea was also capturedby the Greek philosopher-scientist,Fmpedocles (quoted by Lotka 1924):

There is no coming into being ofaught that perishes, nor any endfor it...but only mingling, andseparation of what has beenmingled.

AcknowledgmentsThis work was made possible byNational Science Foundation grantDFB-9119269 to J. J. Elser. We aregrateful to the members ofthe Plank-ton Ecology discussion group, wherethis project was born. T. H.Chrzanowski, J. F. Harrison, R. P.Hassett, J. Alcock, and three anony-mous reviewers provided severalimportant and stimulating commentson the manuscript, and D. E. Chan-dler helped the authors learn moreabout cell biology with a gift ofseveral useful texts. We also ac-knowledge our creative interactionswith R. W. Sterner, for whom sto-ichiometric thinking is now secondnature. Flser is especially grateful toJ. P. Collins for providing both en-couraging comments and sufficienttime for creative thinking while thiswork was in progress.

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684 BioScience VoL 46 No. 9