Sexallocationa ndpopulationst ructure inapicomplexan(protozoa)parasites Stuart A.West *,Todd G.Smithand AndrewF .Read Institute of Cell,Animal and Population Biology,University of Edinburgh, Edinburgh EH9 3JT,UK Establishingthe sel¢ng rate ofparasites isimportantfor studies inclinical and epidemiological medicine aswell as evolutionary biology .Sexallocation theory o¡ ers arelativelycheap and easy way to estimate sel¢ng rates innatural parasite populations. Local mate competition(LMC) theorypredicts thatthe optimalsex ratio ( r*;de¢ned as proportion males) is relatedto the sel¢ng rate ( s)bythe equation r* (17s)/2.In this paper,we generalize the applicationof sex allocation theory across parasitic protozoaˆ in the phylumApicomplexa. This cosmopolitan phylum consists entirelyof parasites, and includesa number ofspecies ofmedical and veterinary importance. W esuggestthat LMC theoryshould applyto eimeriorin intestinal parasites. Aspredicted, datafrom 1 3eimeriorin species showeda female- biasedsex ratio, with the sexratios suggesting high levels of sel¢ng (0.8^1 .0).Importantly,ourestimate of the sel¢ng rate inone of these species, , isinagreement with previous genetic analyses. In contrast, wepredict that LMC theorywill not apply to the groupsin which syzygy occurs (adeleorins, gregarinesand piroplasms) .Syzygyoccurs whena singlemale gametocyte and a singlefemale gameto - cyte pairtogether physically or in close proximity ,just priorto fertilization. As predicted, datafrom four adeleorinspecies showedsex ratios not signi¢ cantly di¡ erent from0.5. Keywords: gametocytes; ;sel¢ng ;syzygy; Toxoplasma; virulence

complete outcrossing( s 0)to 0 forcomplete sel¢ng 1.INTRODUCTION ˆ (s 1),the latter interpreted asmeaning that afemale ˆ Aspects ofparasite population structure, such assel¢ ng shouldproduce the minimum number ofsons required to rate andthe number ofgenotypes infecting each host, fertilize allof her daughters.Thisis oneof the most quanti- haveconsequences for the evolutionof drug resistance tativelyveri¢ ed aspects ofevolutionary biology (Charnov (Mackinnon& Hastings 1998),parasitevirulence (Herre 1982;Godfray 1 994;Herre et al. 2000).Severalrecent 1993;F rank1 996),disease diagnosis(Tibayrenc et al. studies haveapplied LM Ctheorysuccessfully tomalaria 1990;Hastings &Wedgwood-Oppenheim1 997),andthe andother closelyrelated blood parasites (Read et al. 1992, developmentand assessment ofvaccines and curative 1995;Pickering et al.2000).However,the evidencelinking drugs(Dye 1992;Gupta et al.1997).Despite considerable the sel¢ng rate andsex ratio is notperfect (Schall1 989; recent attention,the populationstructure ofparasitic Paperna& Landau1 991;Shutler et al. 1995)and so it isstill protozoaspecies, andin particular estimates ofsel¢ ng notclear how widely sex-ratio theory can be used toesti- rates, haveremained highly controversial (Tibayrenc mate populationstructure inparasites. 1995).Thiscontroversy has arisen in part because much Inthis paper,wegeneralize the applicationof sex alloca- previousevidence has come from indirect genetic tiontheory across parasitic protozoa in the phylumApicom- measures such aslinkage disequilibrium, which are open plexa.This cosmopolitan phylum consists entirelyof tomultiple explanations(Paul & Day1 998).However, parasites,and includes a number ofspecies ofmedicaland direct geneticmeasures areextremely laboriousand veterinaryimportance such asmalariaparasites ( Plasmodium expensiveto obtain (Paul et al. 1995),andso there is a spp.),piroplasms( spp.cause babesiosis in humans needfor more accessible indirect methods. andred waterfever in cattle, and spp.cause East Sexallocation theory o¡ ers atoolfor inferring sel¢ ng Coastfever in cattle) ,adeleorins( spp.cause an rates innatural populations of parasites (Read et al. 1992). oftenfatal hepatozoonosis in dogs) and (various Ifmating takes place between the o¡spring of one or a species of , , , , fewmothers (asubdivided population) ,then afemale- andToxoplasma arepathogenic to immunocompro- biasedsex ratio (where sex ratio is de¢ned as the propor- mised humansor are the causativeagents of veterinary tionof males) is favouredby a process termed localmate coccidiosis).Our speci¢c aims areto: (i) maketheoretical competition(LM C;Hamilton1 967).Thisfemale bias predictionsfor when sel¢ ng should (and should not) lead to arises becauseit reduces competitionamong brothers for biasedsex ratios in apicomplexan species; (ii) test these mates, andbecause it increases the number ofmates for theoreticalpredictions; and (iii) use the sex-ratiodata to eachof the mother’s sons (Taylor1 981).Theoptimal sex estimate sel¢ng levels in apicomplexan species. ratio (r*)canbe shown to depend upon the sel¢ng rate (s;de¢ned as the proportionof a mother’s daughtersthat 2.BACKGROUND BIOLOGY arefertilized byher sons) bythe equation r* (17s)/2 ˆ (Hamilton1 967).Theoptimal sex ratio favoured by Thephylum , class Sporozoasida,can be naturalselection shouldthus declinefrom 0.5 for dividedinto ¢ vetaxonomic groups following R oberts & Janovy( 1996).Thegregarines (subclass ) *Author forcorrespondence ([email protected]) . aregenerally one-host parasites ofinvertebrates. The

Proc. R.Soc.Lond. B (2000) 267, 257^263 257 © 2000The RoyalSociety Received 14September 1999 Accepted 4November1 999 258S. A.West andothers Sexratios of protozoan parasites adeleorins(subclass Coccidiasina,suborder ) Theproduction of gametocytesand the process offerti- areone-host parasites ofinvertebrates orvertebrates, or lizationoccur by di¡ erent means ina varietyof host two-hostparasites that alternatelyinfect haematopha- tissues dependingon which of the ¢vemajor groups of gous(blood-feeding) invertebrates andthe bloodof Apicomplexaa particularspecies belongsto. F orthe vertebrates. Allspecies ofpiroplasms (subclass Piroplas- purposes ofthis paper,there is animportant distinction masina)are two -host parasites infectingticks and betweenthe groupsin which syzygy occurs (gregarines, vertebrates. Haemospororins(subclass Coccidiasina,sub- adeleorinsand piroplasms) and those inwhichit doesnot orderHaemospororina) ,oftenknown as the (haemospororinsand eimeriorins) .Syzygyis de¢ned as parasites, aretwo-host Apicomplexa that parasitize the process wherebya singlemale gametocyte and a blood-feedingdipteran £ ies andthe bloodof various singlefemale gametocyte pair together physically or in tetrapodvertebrates. Finally,the eimeriorins (subclass closeproximity ,either inhost cells orinthe lumenof host Coccidiasina,suborder ) ,frequentlycalled organs,just priorto gametogenesis (Barta 1 999).The the coccidia(although this term is oftenused toinclude crucialconsequence of syzygy is that gametes froma the adeleorins),area diversegroup that includesone- singlemale gametocyte are only able to fertilize the host species ofinvertebrates, two-host species ofinverte- gametefrom a singlefemale gametocyte. The excess of brates, one-hostspecies ofvertebrates andtwo -host malegametes die.In contrast, inspecies wheresyzygy species ofvertebrates. doesnot occur, sexual development involves gametes Apicomplexanlife histories involvethe alternationof pairing.In this case,di¡ erent gametes arisingfrom a sexualand asexual reproduction, and the features rele- singlemale gametocyte are able to fertilize gametes vantto understanding their sexallocation can be arisingfrom a number offemalegametocytes. summarized asfollows(Roberts &Janovy1996) .Haploid infectivestages calledsporozoites infect host tissues to 3.SEX ALLOCATION INTHE APICOMPLEXA: formfeeding stages calledtrophozoites. These stages THEORETICAL PREDICTIONS undergoa periodof asexual proliferation and become multicelled stages calledmeronts (orschizonts) .These (a) Species withoutsyzygy rupture toproduce merozoites, some orall of whichtrans- Sex-ratiomodels havebeen developed previously for forminto sexual stages, whichare termed gametocytesfor malariaand related haemospororin parasites where the haemospororinsand eimeriorins, andgamonts for the fertilizationoccurs inthe bloodmeal of the invertebrate gregarines,adeleorins and piroplasms. F orthe purposes host (Read et al. 1992,1 995;Dye &Godfray1993; ofconsistency among groups of Apicomplexa and also Pickering et al. 2000).Inthese species, gametes competing withprevious malaria parasite sex-ratio papers, we refer formatings will generally be those foundin a singleblood togamonts as gametocytes. In the gregarinesand piro- meal,and so mating will occur between the di¡erent para- plasms, gametocytesare thought to be isogamous, and so site genotypes(clones) that arein asinglehost (andprodu- wecannot present sexallocation data. However ,inmost cinggametocytes) rather thanthose foundin many hosts. species ofthe otherthree groups,the gametocytesare Thisleads to the potentialfor LMC andsel¢ ng ifthere are sexuallydimorphic. lownumbers ofparasitegenotypes infecting each host. Inthese three groups(adeleorins, eimeriorins and Wesuggestthat the naturalhistory of eimeriorin haemospororins),`male’microgametocytes rupture to species wherefertilization occurs inthe intestines ofthe release anumber ofmale gametes, while`female’ macro- host shouldalso lead to LMC andappreciable levels of gametocytesgive rise toa singlefemale gamete. W ewill sel¢ng. As with the species wherefertilization occurs in refer tomicro- ormacrogametocytes and micro -or the blood,this shouldoccur when low numbers ofpara- macrogametesas male or female gametocytes and male site genotypesinfect eachhost (Shirley& Harvey1 996). orfemale gametes, respectively.Themale gametes fertil- However,the extent ofLM Candsel¢ ng may be ize the largerfemale gametes toform diploid zygotes. In increasedfurther insome eimeriorins because(i) sexual terms ofsex allocation, male gametocytes are function- developmentand fertilization occur on a verylocalized allyequivalent to males, andfemale gametocytes are scalein a small portionof the intestinal cells ofinfected equivalentto females. Similarly,the di¡erent male hosts (Marquardt1 973;Long 1993) ,and(ii) malegametes gametes arisingfrom a singlemale gametocyte are onlydisperse ashort distanceto fertilize femalegametes equivalentto multiple matingsby a malegametocyte, (Hammond1973; Dubey 1 993).Consequently,ifdi¡ erent witheach being able to fertilize asinglefemale gamete. genotypesinfect di¡erent areasof the intestine, then high When wediscuss the sexratio we mean the gametocyte levelsof LM Candsel¢ ng may occur even if the host is sexratio, de¢ ned as the proportionof gametocytes that infected bya largenumber ofgenotypes ( Johnson1 997). aremale. In all cases weassume that maleand female Aswith blood parasites, enoughmale gametes must be gametocytesare equally costly to produce, as observed producedto fertilize the femalegametes, andso a lower in Plasmodium falciparum (T.G. Smith, P.Laurenco,R. boundis placedon the sexratio of 1 /(1+ c), where c is the Carter,D .Wallikerand L. C.Ranford-Cartwright, averagenumber ofviable gametes released froma male unpublisheddata) .Thediploid zygotes undergo , gametocyteafter ex£agellation (¢ gure 1 ).Thislower includinggenetic recombination involving random segre- boundmay be increased by mortality or if gametes have gationof chromosomes andcrossing over of homologous troublelocating mates. regionsof DNA, whichrestores the haploidstate. Zygotesthen dividemitotically to form spores containing (b) Species inwhich syzygy occurs the infectivestages that initiatethe periodof asexual Syzygyoccurs inthe gregarines,adeleorins and proliferationonce again. piroplasms.These groups consist ofspecies inwhich

Proc. R.Soc.Lond. B (2000) Sexratios of protozoan parasites S.A. West andothers 259

0.5 byother workers, and (iii) we madecounts from samples, in the syzygy formof slides that we hadprepared ourselves. Blood ¢ lmswere stainedwith Giemsaor Di¡ -Quik 1 (DadeDiagnostics, 0.4 Aguada,Puerto Rico) ,whereastissue sections were stained variouslywith ironhaematoxylin, haematoxylin and eosin, or

o no syzygy i periodicacid ^Schi¡reaction, depending on the source of the t a

r 0.3

material(see references in table1 ).Sampleswere examined x

e undera lightmicroscope, with gametocytessexed according to s

l

a Gardiner et al. (1988).The sourcesfor all data and type of infec- m

i 0.2 c = 4 t tion(natural or experimental) are given in table1 .Wehavenot p

o includeddata from haemospororin species, because these have c = 8 alreadybeen analysed elsewhere from an LMC perspective 0.1 (Read et al. 1992,1995; Shutler et al. 1995;Pickering et al. 2000). c = 20 We¢rsttested for biased sex ratios, de¢ ned as a signi¢cant c = 50 deviationfrom a sexratio of 0.5,in eachspecies. Ideally ,we would 0 testwithin eachspecies by using samples from di¡ erent hosts as 0 0.25 0.5 0.75 1 independentdata points. However, biological (rarity) and ethical selfing rate considerationsmean that generally only a singleor small number Figure1. The optimalsex ratio ( r*)plottedagainst the sel¢ ng ofhostshas been sampled for each parasite species. W etherefore rate (s)forapicomplexan species in which syzygyoccurs used a G-testto look for biased sex ratios within eachindividual (gregarines,adeleorins and piroplasms), and syzygy does not host,using the total counts made from a numberof samples on occur(eimeriorins and haemospororin s).The relationshipfor slides.W ethenexamined the average sex ratios of the species in speciesin which syzygydoes not occur is plottedfor various which syzygydid and did not occur, testing for a signi¢cant devia- values of c, theaverage number of viablegametes released tionfrom 0.5. Proportion data such as sexratio usually have non- froma malegametocyte after ex£ agellation. normallydistributed error variance and unequal sample sizes. T o avoidthese problems, we initiallyanalysed the data with a fertilizationtakes place in the tissues ofblood-feeding generallinear model analysis of deviance, assuming binomial invertebratehosts (two-host adeleorinsand piroplasms) errors,and a logitlink function in theGLIM statistical package andin the intestines ofinvertebratehosts (most gregarines (Crawley1993) .However,after ¢ ttingthe explanatory variables, andone-host adeleorins) ,andso appreciable levels of theratio of the residual deviance to the residual degrees of sel¢ng may occur for the same reasonsas in the species freedomwas greater than 10, showing considerable over- wheresyzygy does not occur . dispersion,suggesting that the data did not ¢ ttheassumption of However,weargue that female-biasedsex ratios will binomialerrors (McCullagh & Nelder1983) .Consequently,the notbe selected forspecies wheresyzygy occurs. Thecrucial sexratios were arcsine square-root transformed and used as the consequencesof syzygy for sex allocation are that (i) an responsevariable assuming normal errors. increase inthe proportionof female gametocytes would notdecrease competitionbetween male gametes fromthe 5. RESULTS same malegametocyte, and (ii) anincrease inthe propor- tionof female gametocytes would not allow gametes from Weobtainedsex-ratio data (proportion of gametocytes the same malegametocyte to fertilize additionalfemale that weremale) on1 3eimeriorin species, representing gametes. Thismeans that syzygyremoves the twofactors sevengenera in which syzygy does not occur (table 1 ).In that favourfemale-biased sex ratios under conditions of allof these cases, afemalebias was recorded. In ¢ ve LMC (Taylor1 981).Consequently,evenwhen sel¢ ng species the sexratio was not quanti¢ ed and merely occurs, LMCofa formthat favoursfemale-biased sex recordedas female biased, whereas for eight species we ratiosdoes not occur .Instead,the reproductivesuccess ofa havequantitative estimates ofthe sexratio. In all of these parasiteis alwaysoptimized by ensuring that there are cases, the sexratio was signi¢ cantly female biased enoughmale gametocytes to form pairs with the female (table1 ).Theaverage sex ratio of these species was0. 11 gametocytes.A sexratio of 0.5,equal numbers ofmale and andsigni¢ cantly di¡ erent from0.5 ( t 9.20, p50.01). 7 ˆ femalegametocytes, is therefore alwaysfavoured (¢ gure 1 ). Weobtainedsex-ratio data on four adeleorin species, Weshowthis more formallyin Appendix A. representing fourgenera where syzygy occurs (table1 ).In Toconclude,we make two predictions. First, inspecies onespecies the sexratio was not quanti¢ ed, and merely wheresyzygy does not occur (haemospororins and eimer- recordedas equal numbers ofmale and female gameto- iorins),the sexratio should be equal (0.5) or female cytes. Inthe otherthree species the sexratio was quanti- biased (50.5),andwill provide information on sel¢ ng ¢edandnot signi¢ cantly di¡ erent from0.5 (table 1 ).Data rates (¢gure 1 ).Second,in species wheresyzygy occurs arelacking on species wheresyzygy occurs becausesexual (adeleorins,gregarines and piroplasms) ,the sexratio dimorphism ofthe gametocytestage at the lightmicro- shouldbe equal(0.5) and not provide any information on scopelevel is rare amongadeleorins, and absent alto- sel¢ng rates (¢gure 1 ). gether inthe piroplasms.The average sex ratio of the species wheresyzygy occurs wasnot signi¢ cantly di¡ erent from 0.5 (t 0.51, p40.2). 4. METHODS 2 ˆ Our sex-ratiodata estimated sel¢ng rates of0.44 to 1.0 Weobtaineddata in threeways: (i) from the literature ; (mean 0.79)in the eimeriorin species (table1 ).Theesti- ˆ (ii)we madecounts from samples, in theform of slides provided mates ofsel¢ ng rates intable 1 havebeen given ranges to

Proc. R.Soc.Lond. B (2000) 260S.A.West andothers Sexratios of protozoan parasites

Table 1. Characteristics of19eimeriorin species, inwhich syzygy does not occur, and 15 adeleorin species, inwhich syzygy does occur

(The datawere obtained from ( ng,numberof gametocytescounted which, forliterature data, is only given when reportedin the originalsource): (Bonnin et al.1995,personal communication ) 1; (Hammond et al. 1961)2; (Pakandl et al. 1996)3;(Haberkorn1971) 4; 5 6 7 8 9 6^9 (Chauve et al. 1994) ;(thisstudy, slides provided by S. Desser; ng 72 ; ng 96 ; ng 102 ; ng 110 ) ;(thisstudy, slides 10 23 10,23,24 ˆ 11 ˆ ˆ 12ˆ 13 providedby M. Cameron; ng 225 ; ng 109 ) ;(Dubey1993) ;(Dubey& Fayer1976) ;(Dubey1978; ng 2256 ; 14 13,14 ˆ 15 ˆ 16 17 18 ˆ ng527 ) ;(Dubey1979; ng 21) ; (Baker et al. 1996) ;(Dubey1982) ; (Dubey et al. 1982; ng 300) ;(Bristovetsky& 19 ˆ 20 21 ˆ 22 Paperna1990; ng 143) ;(Paperna& Finkelman1996 a,b) ;(Dubey& Frenkel1972) ; (Omata et al. 1997) ;(Lainson1981, 25 ˆ 26 27 28 1992; ng 300) ;(Minchin& Woodcock1910) ;(Siddall& Desser1992) ;(Siddall& Desser1990) ;(thisstudy, slide ˆ 29 30 31 providedby M.Siddall; ng 235) ;(referencesin Smith& Desser1997) ;(Smith1996; ng 228) .The secondcolumn shows infectiontype, i.e. whether ˆthe data were taken from experimental or natural infections. ˆThe majorityof the experimental infectionswere initiated with sporesfrom natural infections. The followingcolumns give the average number of gametesobserved permale gametocyte, the average sex ratio (proportion of male gametocytes) ,a G-teston the hypothesis that there are equal numbersof maleand female gametocytes (a sexratio of 0.5),and estimated sel¢ ng rate given the observed sex ratio.)

gametesper male estimated species infectiontype gametocyte sex ratio G-test sel¢ng-rate

Eimeriorin species(no syzygy) Cryptosporidiumparvum 1 experimental 16 50.50 ö 40 Eimeriaauburnensis 2 experimental 41000 ö ö ö Eimeriacoecicola 3 experimental 41 50.50 ö 40 Eimeriacontorta 4 natural 40^60 ö ö ö Eimeriamulardi 5 natural ö 50.50 ö 40 Eimeriaperforans 6 natural 133 0.13 133.40b 0.75 Eimeriastiedae 7 natural 317 0.04 317.33b 0.92^1.0 Eimeriastiedae 8 natural 271 0.14 271.00b 0.73 Eimeriatenella 9 experimental 97 0.05 97.33b 0.89^1.0 Eimeriatenella 10 natural 97 0.11 159.10b 0.78^1.0 Frankeliabuteonis 11 natural 12 ö ö ö Isosporabigemina 12 experimental 6^12 ö ö ö Isosporaohioensis 13 experimental 20^50 0.07 1977.00b 0.86^1.0 Isosporaohioensis 14 experimental ö 0.05 535.40b 0.91^1.0 Isosporarivolta 15 experimental 470 0.19 8.66a 0.62 Lankesterella sp.16 natural 416 50.50 ö 40 Sarcocystiscruzi 17 experimental 3^11 50.05 ö 0.90^1.0 Sarcocystistenella 18 experimental 8^11 0.01 382.30b 0.99^1.0 Schellackiaagame 19 experimental ö 0.28 28.73b 0.44 Schellackiaptyodactyli 20 natural ö 50.50 ö 40 Schellackiamuriviperae 20 natural 5^12 ö ö ö Toxoplasmagondii 21 experimental 12 (6^21) 0.02^0.04 ö 0.92^1.0 Toxoplasmagondii 22 experimental ö 50.50 ö 40 Toxoplasmagondii 23 natural 30 0.06 75.52b 0.89^1.0 Toxoplasmagondii 24 natural 41 ö ö ö Adeleorin species(syzygy) Cyrilialignieresi 25 natural ö 0.50 0 ö Desseriarovigensis 26 natural ca. 0.5 ö ö Desseriamyxocephali 27 natural 4 ö ö ö Haemogregarinaballi 28 natural 4 ö ö ö Haemogregarinaballi 29 natural ö 0.49 0.21 ö Hepatozoonaegypti 30 natural 4 ö ö ö Hepatozoonbreinli 30 natural 4 ö ö ö Hepatozooncatesbianae 30 natural 2 ö ö ö Hepatozoonerhardovae 30 natural 2 ö ö ö Hepatozoongracilis 30 natural 4 ö ö ö Hepatozoonmauritanicum 30 natural 4 ö ö ö Hepatozoonmocassini 30 natural 4 ö ö ö Hepatozoonrarefaciens 30 natural 4 ö ö ö Hepatozoonsipedon 30 natural 2 ö ö ö Hepatozoonsp.31 natural ö 0.53 0.90 ö a p50.005. b p50.001. include1 .0in cases wherethe sexratios are su¤ ciently Weobtainedcounts of the number ofgametes per male biasedthat complete sib matingoccurs, andthat the gametocytein 1 6eimeriorin species (table1 ).Across species is merely producingenough male gametocytes to species, the number ofgametes per malegametocyte, fertilize the femalegametocytes (see } 3(a)). rangedfrom 1 2in Frankelia buteonis togreater than 100 0in

Proc. R.Soc.Lond. B (2000) Sexratios of protozoan parasites S.A. West andothers 261

Eimeria auburnensis. Inaddition, we obtained counts of the sex-ratiodata predict extremely highsel¢ ng rates in number ofgametes per malegametocyte in 12adeleorin eimeriorin species. Importantly,ourestimates ofsel¢ ng species (table1 ).Across species, the number ofgametes rates fromsex-ratio data include a species forwhich per malegametocyte, ranged from two to four . geneticanalyses of population structure havebeen carried out, Toxoplasma gondii .Theestimates fromboth the genetic analyses(Tibayrenc et al. 1991;Sibley& Boothroyd1 992) 6.DISCUSSION andthe sex-ratiodata (table 1 )arein agreement, with Ithaspreviously been argued that LMCtheoryshould e¡ective clonalitypredicted. Suchsel¢ ng is probablya applyto species wherefertilization occurs inthe blood,as consequenceof matings occurring on a verylocal scale with Plasmodium,andother haemospororins. W ehave withinintestinal tissue (see } 3).Incontrast, the life suggestedthat LMC theoryshould apply to other histories ofother apicomplexan species (two-host adele- apicomplexanparasites, the eimeriorins, wherefertiliza- orins,piroplasms and haemospororins) provide a means tionoccurs inthe intestine ofthe host.As predicted, a forgametocytes and gametocytes from di¡ erent geno- female-biasedsex ratio was observed in 1 3eimeorin types (clones) tomix before mating occurs. Thiswill lead species (table1 ).Incontrast, wepredicted that,indepen- tosel¢ ng levels that varyenormously ,dependingupon dent ofthe sel¢ng rate, LMC theorywould not apply to otherbiological variables, such asinfection and trans- species inwhich syzygy occurs, the adeleorins,gregarines mission rates andthe feedinghabits of the invertebrate andpiroplasms. As predicted, unbiasedsex ratios were hosts (Read et al. 1995;Shutler &Read1 998).Thesex- observedin fouradeleorin species (table1 ). ratiodata from haemospororin species areconsistent with Thephylogeny of the Apicomplexais currently a this prediction,showing a rangeof sex ratios from extre- matter ofmuch debate(Barta 1989; Barta et al. 1991; melyfemale biased to 50% males (Schall1989; Read et Allsopp et al. 1994;Escalante & Ayala1995 ;Lang- al. 1992,1995;Shutler et al. 1995;Pickering et al. 2000). Unnasch et al. 1998).Inall resolutions, syzygy is the Toconclude,our results illustrate twogeneral points ancestralcondition for the phylum.However ,the number aboutthe prosand cons of applying optimality models oftimes that syzygyhas been lost is unclear,withsome andan evolutionary biology approach to infectious studies suggestingonly once (e.g. Barta 1 989),andothers disease research (Stearns 1999).First, ourdata show that, more thanonce (e.g. Lang-U nnasch et al. 1998). By despite assumingequilibrium states whichare not an allowingfor LMC ofa formthat favoursfemale-biased obviousfeature of microparasite populations ( Anderson sexratios we suggest that the loss ofsyzygy has allowed &May1991 ),the optimalityapproach is ableto explain biasedsex ratios to evolve in species wherehigh levels of variationin a life-historytrait (sex ratio)across ataxono- sel¢ng occur. The evolution of female-biased sex ratios micallydiverse range of microparasites. Inparticular, couldthen havefavoured the productionof more gametes sex-ratiodata provide the clearest demonstrationof the per malegametocyte, which are observed for haemospor- importanceof population structure asa determinant of orinsand eimeriorins, especiallyif there arehigh the directionof natural selection. Theorysuggests that mortalityrates inthe intestine. Givenhigh enough sel¢ ng populationstructure is oneof the keydeterminants of rates, this couldhave allowed more female-biasedsex parasitevirulence (F rank1 996).Second,the conse- ratiosto evolve. This potential for coevolution between quences ofsyzygy provides an example of when a basic sexratio and gamete numbers couldhave resulted inthe assumptionof models developedfor does not largenumbers ofgametes per malegametocyte that are apply,emphasizingthe importanceof checking basic observedin the eimeriorin species whichhave female- assumptions whenapplying such models tomicropara- biasedsex ratios (table 1 ).However,it shouldalso be sites. stressed that becausesyzygy may have only been lost once ortwice, our ability to test such scenarioswith formal WethankD. Baker, A. Buckling,C. Chauvre,M. Cameron, comparativemethods willbe limited bya lackof degrees A.Davies,S. Desser,J. Dubey ,M. A.Fernando,I. Hastings, offreedom (Harvey & Pagel1991 ;Read& Nee 1991, R.Lainson,M. Mackinnon,Y .Omata,I. Paperna, P .Preston, L.Ranford-Cartwright,A. Riveroand S. Uptonfor supplying 1993,1995) .Notethat inother contexts, some authors slides,reprints and discussion. W earesupported by the UK Bio- havefelt comfortableapplying formal tests toanalogous technologyand Biological Sciences Research Council (S. W.and situations(e.g. Brook¢ eld 1993; Orr 1998).Inthis case, A.R.)andN aturalSciences and Engineering Research Council such tests woulddemonstrate highlysigni¢ cant associa- ofCanada (T .S.)fellowships,and an MR Cgrantto D .Walliker tions. (T.S.).Wearegrateful to three anonymous referees for help Thesex ratios of the eimeriorin species suggesthigh clarifyingour arguments. levelsof sel¢ng, averaging 0.8^1 .0(table 1 ).Theextremely female-biasedsex ratios observed in these parasitespecies APPENDIX A aresimilar tothose observedin species whentotal sel¢ng (s 1)occurs (Green et al. 1982;Hardy & Cook Inthis section weshow that syzygyleads to equality ˆ 1995;W est &Herre 1998).Thenumber ofgametes (0.5)being the optimalsex ratio independent of sel¢ ng producedper malegametocyte ( c)is much greaterin the rates. Assume that matingoccurs between n parasites ina eimerionspecies ( c 12^1000;table 1 )thanin haemo- host andthat allparasites areequally abundant in the host. ˆ spororins (c 4^8; Read et al. 1995),allowingmore Assume syzygyoccurs. Inorder to determine the optimal ˆ female-biasedsex ratios and a greateraccuracy when strategywe consider the ¢tness ofamutant producinga sex estimating highsel¢ ng rates (¢gure 1 ). ratio r 0 ina populationof individuals who produce a sex Howdo our data contribute to the debateon the ratio r.Theequation for the ¢tness ofthe mutant ( W 0) is populationstructure ofparasitic protozoa species ?Our given by W 0 r 0 z (1 r 0)z , where z and z are the ˆ m ‡ ¡ f m f Proc. R.Soc.Lond. B (2000) 262S. A.West andothers Sexratios of protozoan parasites probabilityof male and female gametocytes being mated, Dubey,J.P.&Frenkel,J .K.1972Cyst-induced in respectively.Theequations for zm and zf depend upon cats. J.Protozool. 19, 155^177. whetherthe sexratio in the patchis maleor femalebiased. Dubey,J.P .,Speer,C. A., Callis,G. & Blixit,J. A. 1982 If there is afemale-biasedsex ratio then z 1, and Developmentof the sheep ^canidcycle of Sarcocystistenella . m ˆ Can.J .Zool. 60,2464^2477. z r 0 (n 1)r/((n 1)(1 r) r 0).If there is amale- f ˆ ‡ ¡ ¡ ¡ ¡ Dye,C. 1992Models for investigating genetic exchange in proto- biasedsex ratio then z (1 r 0 (n 1)(1 r)/(r 0 m ˆ ¡ ‡ ¡ ¡ zoanpopulations. In Modellingvector- borneand other parasitic (n 1)r), and z 1.The optimal sex ratio, r*, is that ‡ ¡ f ˆ diseases (ed.B. D.Perry & J.W.Hansen),pp.165^1 75. whichcannot be beatenby any other strategy.Thiscan be Nairobi,Kenya: ILRAD. determined throughiterative simulations (see West & Dye,C. &Godfray,H.C. F.1993On sex ratio and inbreeding Godfray1 997)or graphically.Inall cases it canbe shown in malariaparasite populations. J.Theor.Biol. 161, 131^134. that r* 0.5,independent of the valueof n (¢gure 1 ).Note Escalante,A. A. &Ayala,F .J.1995 Evolutionary origin of ˆ that this theoreticalargument could apply to other life Plasmodium andother Apicomplexa based on rRNA genes. histories, such aswhenmales andfemales formlong-term Proc.N atlAcad. Sci. USA 92,5793^5797. pairbonds and in whichextra pair copulations do not take Frank,S. A.1996Models of parasite virulence. Q.Rev.Biol. 71, place. 37^78. Gardiner,C. H.,F ayer,R. &Dubey,J.P.1988 Anatlasof proto- zoanparasites in animal tissues ,AgricultureHandbook no. 651 . REFERENCES USDepartment of Agriculture. Allsopp,M. T.E.P.,Cavalier-Smith,T .,DeW aal,D .T.& Godfray,H.C. J.1 994 Parasitoids.Behavioural and evolutionary Allsopp,B. A.1994Phylogeny and evolution of the piro- ecology.PrincetonU niversityPress. plasms. Parasitology 108, 147^152. Green,R. E., Gordh, G. & Hawkins,B. 1982Precise sex ratios Anderson,R. M. &May,R.M. 1991 Infectiousdiseases of humans. in highlyinbred parasitic wasps. Am. Nat. 120, 653^665. Dynamicsand control .OxfordUniversity Press. Gupta,S., Ferguson,N. M. &Anderson,R. M. 1997 Baker,D. G., Speer, C. A.,Yamaguchi,A., Gri¡ey ,S. M. & Vaccinationand the population structure of antigenically Dubey,J.P .1996An unusualcoccidian parasite causing pneu- diversepathogens that exchange genetic material. Proc.R. Soc. moniain anortherncardinal ( Cardinaliscardinalis ). J. Wildl. Lond. B 264,1435^1443. Dis. 32, 130^132. Haberkorn,A. Z.1971ZurWirtsspezi¢ tat von Eimeriacontorta Barta,J .R.1989Phylogenetic analysis of the class Sporozoea n.sp.(Sporozoa: ) . Parasitenkd 37, 303^334. (PhylumApicomplexa Levine, 1 970):evidence for the inde- Hamilton,W .D.1 967Extraordinary sex ratios. Science 156, pendentevolution of heteroxenous life cycles. J.Parasitol. 75, 477^488. 195^206. Hammond,D. M. 1973Life cycles and development of coccidia. Barta,J. R. 1999Suborder Adeleorina Leger, 1 911.In Illustrated In TheCoccidia (ed.D. M. Hammond& P.L.Long) ,pp.45^ guideto the protozoa (ed.J .J.Lee,G. F .Leedale,D .J.Patterson 79.Baltimore, MD: UniversityPark Press. &P.C. Bradbury),pp.70^107 .Lawrence,KS: Societyof Hammond,D .M., Clark,W .N.& Miner, M. L.1961 Protozoologists. Endogenousphase of the life cycle of Eimeriaauburnensis in Barta,J. R., Jenkins, M. C. &Danforth,H. D.1 991 calves. J.Parasitol. 47, 591^596. Evolutionaryrelationships of avian Eimeria speciesamong Hardy,I.C. W.&Cook,J. M. 1995Brood sex ratio variance, otherapicomplexan protozoa: monophyly of theApicomplexa developmentalmortality and virginity in agregariousparasi- issupported. Mol. Biol.Evol. 8, 345^355. toid wasp. Oecologia 103, 162^169. Bonnin,A., Gut,J., Dubremetz, J. F .,Nelson,R. G.& Harvey,P.H.&Pagel,M. D.1 991 Thecomparative method in evolu- Camberlynck,P .1995Monoclonal-antibodies identify a subset tionarybiology .OxfordUniversity Press. ofdense granules in Cryptosporidiumparvum zoites and Hastings,I. M. &Wedgwood-Oppenheim,B. 1997Sex, strains gamonts. J.Eukar.Microbiol. 42, 395^401. andvirulence. Parasitol.Today 13, 375^383. Bristovetsky,M. &Paperna,I. 1 990Life cycle and transmission Herre,E. A. 1993Population structure and the evolution of of Schellackiacf .agamae ,aparasiteof thestarred lizard Agama virulencein nematodeparasites of ¢ gwasps. Science 259, stellio. Int.J .Parasitol. 20, 883^892. 1442^1445. Brook¢eld, J. 1993. Haldane’ sruleis signi¢ cant. Evolution 47, Herre,E. A., Machado,C. A.&West,S. A.2000Selective 1886^1888. regimeand ¢ gwaspsex ratios: towards sorting rigor from Charnov,E.L.1982 Thetheory of sex allocation . Princeton pseudo-rigorin testsof adaptation. In Adaptionismand UniversityPress. optimality (ed.S. Orzack& E.Sober).CambridgeUniversity Chauve,C. M., Reynaud,M. C.&Gounel,J .M. 1994 Press.(In the press. ) Descriptiond’ Eimeriamulardi N.sp. chez le canard mulard. Johnson,A. M. 1997Speculation on possible life cycles of the E¨ tudede la phase endoge © nedeson cycle e ¨ volutifavec mise en clonallineages in thegenus Toxoplasma. Parasitol.T oday 13, e¨videncedu de ¨veloppementintranucle ¨aire. Parasite 1, 15^22. 393^397. Crawley,M. 1993 GLIMfor ecologists .Oxford,UK: Blackwell. Lainson,R. 1981On Cyriliagomesi (Neiva& Pinto,1926) gen. Dubey,J.P .1978Life-cycle of Isosporaohioensis in dogs. nov.(Haemogregarinidae)and Trypanosomabourouli Neiva & Parasitology 77, 1^11. Pinto,in the¢ sh Synbranchusmarmoratus :simultaneoustrans- Dubey,J.P .1979Life cycle of Isosporarivolta (Grassi,1 879)in missionby the leech Haementerialutzi . In Parasitologicaltopics catsand mice. J.Protozool. 26, 433^443. (ed.E. U.Canning) ,pp.1 50^158.Journal of Protozoologists Dubey,J.P .1982Development of ox ^coyotecycle of Sarcocystis SpecialPublication 1. Lawrence, KS: Societyof cruzi. J.Protozool. 29, 591^601. Protozoologists. Dubey,J.P .1993 Toxoplasma,, , Sarcosytstis , Lainson,R. 1992A protozoologistin Amazonia:neglected para- andother cyst-forming coccidia of man and animals. In sites,with particularreference to members of the Coccidia Parasiticprotozoa, vol.6 (ed.J. P .Kreier),pp.1^1 58.New Y ork: (Protozoa:Apicomplexa) . Cienc.Cult. 44, 81^93. AcademicPress. Lang-Unnasch,N., Reith, M. E.,Munholland, J .&Barta,J. R. Dubey,J.P .&Fayer,R. 1976Development of Isosperabigemina in 1998Plastids are widespread and ancient in parasitesof the dogsand other mammals. Parasitology 73, 371^380. phylumApicomplexa. Int.J .Parasitol. 28,1739^1742.

Proc. R.Soc.Lond. B (2000) Sexratios of protozoan parasites S.A. West andothers 263

Long,P .L.1993A viancoccidiosis. In Parasiticprotozoa , Roberts,L. S. &Janovy,J.1996 Foundationsof parasitology , 5th vol.4 (ed.J. P .Kreier),pp.1^88. New Y ork:Academic Press. edn.IA: WilliamBrown. McCullagh,P .&Nelder,J. A. 1983 Generalizedlinear models . Schall,J. J. 1989 The sexratio of Plasmodium gametocytes. London:Chapman & Hall. Parasitology 98, 343^350. Mackinnon,M. J.& Hastings,I. M. 1998The evolutionof Shirley,M. W.&Harvey,D.A.1996 Eimeriatenella : infection multipledrug resistance in malariaparasites. Trans.R. Soc. with asinglesporocyst gives a clonalpopulation. Parasitology Trop.M ed.Hyg . 92, 188^195. 112, 523^528. Marquardt,W .C. 1973Host and site speci¢ city in thecoccidia. Shutler,D. & Read,A. F.1998Extraordinary and ordinary In TheCoccidia (ed.D. M. Hammond& P.L.Long),pp.23^ bloodparasite sex ratios. Oikos 82, 417^424. 44.Baltimore, MD: UniversityPark Press. Shutler,D .,Bennett,G. F .&Mullie, A.1995Sex proportions of Minchin, E.A.&Woodcock,H. M. 1910Observations on bloodparasites and local mate competition. Proc. certainblood-parasites of ¢ shesoccurring at Rovigno. Q. J. NatlAcad. Sci. USA 92,6748^6752. Micro. Sci. 55, 113^154. Sibley,L.D.&Boothroyd,J .C.1992Virulent strains of Omata,Y .,Taka,A., Terada,K., Koyama,T .,Kanda,M., Saito, Toxoplasmagondii comprisea singleclonal lineage. Nature 359, A.&Dubey,J.P.1997Isolation of coccidian enteroepithelial 82^85. stages of Toxoplasmagondii fromthe intestinal mucosa of cats Siddall,M. E.&Desser,S. S. 1990Gametogenesis and sporo- byPercoll density-gradient centrifugation. Parisitol.Res. 83, gonicdevelopment of Haemogregarinaballi (Apicomplexa: 574^577. Adeleina:Haemogregarinidae) in theleech Placobdellaornata . Orr,H. A.1998Haldane’s rule. A.Rev. Ecol. Syst. 28, 195^218. J.Parasitol. 77, 426^436. Pakandl,M., Gaca,K., Drouet-Viard, F .&Coudert,P .1996 Siddall,M. E.&Desser,S. S.1992Ultrastructure of gameto- Eimeriacoecicola Cheissin1 947:endogenous development in genesisand sporogony of (sensu lato) myxocephali gut-associatedlymphoid tissue. Parasitol.Res. 82, 347^351. (Apicomplexa:Adeleina) in themarine leech Malmianascorpii . Paperna,I. & Finkelman,S. 1996 aSchellackiaptyodactyli sp. n. of J.Protozool. 39, 545^554. thefan-footedgecko Ptyodactylushasselquistii fromthe rift escarp- Smith,T .G.1996 The genus Hepatozoon (Apicomplexa: mentof thelower JordanValley. FoliaP arasitol. 43,161^172. Adeleina). J.Parasitol. 82, 565^585. Paperna,I. & Finkelman,S. 1996 b Ultrastructuralstudy of Smith,T .G.& Desser,S. S. 1997Phylogenetic analysis of the Sarcocystismuriviperae developmentin theintestine of its snake genus Hepatozoon Miller, 1908(Apicomplexa: Adeleorina) . host. FoliaP arasitol. 43, 13^19. System.Parasitol. 36, 213^221. Paperna,I. & Landau,I. 1 991 Haemoproteus (Haemosporidia)of Stearns,S. C. 1999 Evolutionin health and disease . Oxford lizards. Bull.Mus. N atlHist. N at. 13, 309^349. UniversityPress. Paul,R. E.L. &Day,K.P.1998Mating patterns of Plasmodium Taylor,P .D.1981 Intra-sex and inter-sex sibling interactions as falciparum. Parasitol.Today 14, 197^202. sexdeterminants. Nature 291, 64^66. Paul,R. E. L.,Packer, M. J.,W almsley,M., Lagog,M., Tibayrenc,M. 1995Population genetics of parasitic protozoa Ranford-Cartwright,L. C., Paru,R. &Day,K.P.1995 andother microorganisms .Adv.P arasitol . 36, 47^115. Matingpatterns in malariaparasite populations of Papua Tibayrenc,M., Kjellberg,F .&Ayala,F .J.1990A clonaltheory New Guinea. Science 269,1709^1711. ofparasitic protozoa: the population structures of Entamoeba, Pickering,J., Read, A. F.,Guerrero,S. &West,S. A. 2000Sex Giardia,Leishmania, Plasmodium, T richomonas , and Trypanosoma ratioand virulence in twospecies of lizard malaria parasites. andtheir medical and taxonomic consequences. Proc. Natl Evol.Ecol. Res . 2, 171^184. Acad.Sci. USA 87,2414^2418. Read,A. F.&Nee,S. 1991Is Haldane’s rule signi¢ cant ? Tibayrenc,M., Kjellberg,F .,Arnaud,J .,Oury,B., Breniere, Evolution 45,1707^1709. S. F.,Darde,M. L.&Ayala,F .J.1 991Are eukaryoticmicro- Read,A. F.&Nee,S. 1993Haldane’s coincidence: a replyto organismsclonal or sexual ?Apopulationgenetics vantage. Brook¢eld. Evolution 47,1888^1889. Proc.N atlAcad. Sci. USA 88,5129^5133. Read,A. F.&Nee,S. 1995Inference from binary comparative West,S. A.&Godfray,H.C.J.1997 Sex ratio strategies after data. J.Theor.Biol . 173, 99^108. perturbationof thestable age distribution. J.Theor.Biol. 186, Read,A. F.,Narara,A., Nee,S., Keymer,A. E.&Day,K.P. 213^221. 1992Gametocyte sex ratios as indirect measures of West,S. A.&Herre,E. A. 1998Stabilizing selection and outcrossingrates in malaria. Parasitology 104, 387^395. variancein ¢gwaspsex ratios. Evolution 52, 475^485. Read,A. F.,Anwar, M., Shutler,D .&Nee,S. 1995Sex alloca- tionand population structure in malariaand related parasitic Asthispaper exceeds the maximum length normally permitted, protozoa. Proc.R. Soc.Lond. B 260, 359^363. theauthors have agreed to contribute to production costs.

Proc. R.Soc.Lond. B (2000)