Received 18April 2003 Accepted 8 July 2003 Publishedonline 10September 2003

Costlytraumatic inseminatio nanda female counter-adaptationin bed bugs Edward H.Morrow * and Go¨ranArnqvist UppsalaUniversity, EvolutionaryBiology Centre, Department of Animal Ecology, Norbyva ¨gen18D, SE-752 36 Uppsala, Sweden Male bedbugs pierce femalesthrough thebody wall andinseminate directly intothe body cavity. It has previously beenshown that suchtraumatic insemination carries costsfor females,and sexual conflict regarding themode of insemination shouldthus propel male–female coevolution.Since males accumulate sexually antagonistic adaptations,females should evolve counter-adaptationsthat efficiently abate thecosts tofemales of sexual interactions.Yet, unambiguous experimental evidencefor female counter-adaptations islacking. In bedbugs, the spermalege (a highly modifiedregion ofthe abdomen where the male usually piercesthe female) may representa female counter-adaptation.We assess the female costsof traumatic insemination by varying therate ofinsemination onthe one hand, and the rate andmode of piercing trauma tofemales on the other. Our resultsshow that female mating costsare notextreme— elevated mating rate shortenedfemale lifespan buthad nosignificant effecton lifetime egg production.More importantly, additional abdominal piercing in thespermalege had noeffect on females whereas even a very lowrate ofsuch piercing outsidethe spermalege reducedfemale lifetime egg productionby 50%. Thus,females are well counter-adaptedto theintrusive modeof insemination exhibited bymale bedbugs andthe costs of elevated mating are comparable with thosein other insects,as predicted by theory. We thereforedemonstrate that thespermalege efficiently reducesthe direct costs of piercing trauma tofemales, andhence provide experimental evidencefor afemale counter-adaptationto a sexually antagonistic male trait. Keywords: sexual selection;multiple mating; Cimex lectularius ;costsof mating; sperm competition; coevolutionary arms race

1. INTRODUCTION thuseffectively hiding thecosts and conflict from the observer (Chapman &Partridge 1996). Thus,counter to Differencesin theevolutionary interestsof the sexes are naive expectations,we do not expect sexual interactions rife in sexually reproducing organisms (Parker 1979; tobecome more costlyas antagonistic adaptations become Chapman &Partridge 1996; Rice1996, 2000). Suchsex- more escalatedin agiven sex,because matching counter- ual conflictis predictedto lead tocoevolutionary ‘arms adaptations will occurin theother sex(Arnqvist &Rowe races’between the sexes (Parker 1979; Rice& Holland 2002a).Nevertheless,convincing empirical (Rice1996) 1997). Sucharms racesproceed over evolutionary time andcomparative (Arnqvist &Rowe2002 a)evidencenow whereadaptations in onesex select for counter- existsto demonstrate that sucharms racesdo occur. adaptations in theother sex,where the latter are aimed at Male bedbugs (Heteroptera; Cimicidae) donot copu- ameliorating thecosts imposed by theformer. Thus,as late via thegenital opening ofthe female. Instead they Dawkins& Krebs(1979, p.57) phrasedit, ‘asswords get pierce thefemale abdominal wall with their sharp intro- sharper, soshields get thicker, soswordsget sharper still’. mittent organ (paramere) andinject sperm andaccessory Sexually antagonistic coevolutionplays asignificant role gland fluidsdirectly intothe blood, leaving visible melan- in theevolution of sexual dimorphism in general (Rice izedscars (Carayon 1966). This unusualmode of copu- 1984), aswell asin theevolution of reproductive mor- lation, termedtraumatic extragenital insemination,occurs phology (Arnqvist &Rowe2002 b),physiology (e.g.semi- in only asmall numberof taxa butit isfound in all mem- nal fluid proteins;Rice 1996) andbehaviour (e.g. bersof the bed bug family (Usinger1966). In many bed courtship;Arnqvist &Rowe2002 a).It has also been bugs,the area at whichthe male piercesthe integument implicated asan important engineof speciation (Parker & ofthefemale showsseveral unilateral modifications called Partridge 1998; Rice1998; Arnqvist et al. 2000; thespermalege. For example, in thehuman bedbug Gavrilets 2000). Cimex lectularius aspecialized notchand a cuticular thick- Despitethe fact that sexually antagonistic coevolution ening ofthe right sideof the fifth sternite(the generatesadaptations, in both sexes,which are costlyfor ectospermalege) lies directly over adistinctpocket filled theother sex,the process is notoriously difficult todetect with haemocytoid cellson the inner surface of theabdomi- empirically. This is becausewe expect adaptation in one nal wall (themesospermalege) (Carayon 1966). sexto be balanced by counter-adaptationin theother, Mating at very high ratesis costly to female insectsin general (seeArnqvist &Nilsson2000 for areview) and bedbugs are noexception (Stutt & Siva-Jothy 2001). *Authorand address for correspondence: Department of Ecology, Evolution& Marine Biology,University of California SantaBarbara, CA Despitethe apparently damaging natureof traumatic 93106,USA ([email protected]). insemination,we do not expect male– female interactions

Proc.R. Soc.Lond. B (2003) 270, 2377–2381 2377 Ó 2003 TheRoyal Society DOI10.1098/ rspb.2003.2514 2378E. H.Morrowand G. Arnqvist Costsof traumatic insemination in bedbugs to carry extreme costsfor tworeasons. First, (b) Experimental treatments becauseof continual sexually antagonistic coevolution, Weindependentlyvaried the followingfactors: (i)the rate of femalesshould be well counter-adaptedto copewith any insemination;(ii) the rate of piercingtrauma; (iii)the siteof damaging effectsof mating. The spermalege is anobvious piercingtrauma; and (iv)the probability of infectionduring candidatefor sucha female counter-adaptation.The evol- piercingtrauma. The first variablewas based upon mating utionof the spermalege within theCimicidae wasfirst regime,using two treatments.Females were either housed with consideredby Carayon (1966) whoexplicitly suggested four virginmales (high mating rate)or were housed with four that thespermalege evolved toreduce direct costs to virginmales, three of which had had theirparamere glued into femalesassociated with thetrauma ofinsemination (see the parameregroove (lowmating rate),using asmallamount of also Stutt& Siva-Jothy 2001). Suchcosts may include:(i) quick-dryingglue (Super Attak, Loctite).This gluingtreatment repair ofthewounded cuticle; (ii) leakage ofhaemolymph; had no observableeffect on malebehaviour. The lattertreat- (iii) increasedrisk ofinfection through thepuncture mentcontrols for any confoundingeffects of exposure to males wound;and (iv) immune defenceagainst sperm oraccess- ormaleharassment, thus isolatingthe effectof copulation,since ory gland fluidsthat are introduceddirectly intothe blood threeout of the four maleswere unable actually to copulate. ofthe female. The protectionafforded by thespermalege Any malesthat diedduring the periodof the experimentwere tothe female during andafter copulation may operate in replacedwith amaleof the sametype (gluedor normal). Each anumberof ways:(i) by localizing damage toone area on replicateof four malesand asinglefemale was housed inawell theabdomen; (ii) by restricting thediffusion of theejacu- (volumeof 6ml)of amodifiedcell-culture plate fittedwith a late insidethe female; (iii) by reducingleakage ofblood gauze panelin the lid.A smallslip of filterpaper was provided through thewound site; and (iv) by restricting entry of as an ovipositionsubstrate. Femaleswere included in the experi- pathogensinto thebloodstream. It shouldbe noted, how- mentuntil their death. ever,that thedegree of elaboration ofthe spermalege var- Inaddition to the trauma experiencedduring mating, all iestremendously across different heteropterans exhibiting femaleswere subjected to additionalexperimentally imposed traumatic insemination (Carayon 1966). abdominalpiercing trauma. Femaleswere either: (i) simply Second,all elsebeing equal,males will sufferfrom handledfor the sameamount of timethat the treatments below harming their mates(Parker 1979). There shouldthere- took to complete( ca.1min);(ii) pierced through the ectosperm- forebe selection in both sexesto reduce the direct costs alegewith asterilizedpin (rinsed briefly in 98% ethanol and oftraumatic insemination tofemales, favouring males that allowedto dry priorto piercing);(iii) pierced through the ectos- inseminatefemales in alesscostly manner andleaving permalegewith anon-sterilizedpin that, priorto piercing,had only thosecosts that are more or less ‘unavoidable ’ to per- beenwiped over the surfaceof acontaminatedfilter paper taken sistover evolutionary time (seeHosken et al. 2003; Mor- froma jar containinga bedbug colony;(iv) pierced outside of row et al. 2003). For thesereasons, the costs of mating in the spermalegewith asterilizedpin, through the integument bedbugs should not be extraordinary. betweenthe fifth and sixth segments of the ventralsurface of The purposeof this reportedwork is twofold.By inde- the abdomenon the leftside of theirbody. This positionmirrors pendentlyvarying therate ofinsemination andthe type of exactly the positionof the ectospermalegeon the ventralsurface puncturetrauma in abedbug species,we first quantify ofthe right sideof the femaleabdomen. These treatments were female mating costsand second we dissectthe proximate carriedout oncea week,and allowedus to evaluatestatistically causesof such costs. We predict that thecosts of mating the effectson females of rate of piercingtrauma, siteof piercing shouldnot be dramatic. Further, thecost to females of trauma and probability of infectionduring piercing trauma. mating trauma in thespermalege shouldbe minimal, in Allpiercings were carried out undera stereobinocular micro- contrastto extra-spermalege trauma, whereasthe costs of scope (´ 20magnification) at roomtemperature and humidity insemination may besignificant (cf.Arnqvist &Nilsson duringdaylight hours. The femalewas heldventral side upper- 2000). most ina spring-loadedclamp, the jaws of which were cushionedwith piecesof closed-cellfoam. The pins usedfor puncturing the femaleswere stainless steel entomological pins 2. MATERIALAND METHODS (sizeA1; Watkins and Doncaster, UK)that had beenglued into (a) Rearingconditions apipette tip for easy handling.This toolwas then mountedon Weused the human bedbug ( C.lectularius )for the experi- amicromanipulator(Narishige MM-3), which enabledthe fine mentsdescribed below. All animals were taken froma stock and precisemovements of the pintip necessaryfor the piercing population (CBstrain)that has beenreared in constant environ- procedure.The dimensionsof the tip ofthis grade ofpincorre- mentchambers (Sanyo) at 27 ± 1 °Cand 70%relative humidity spond veryclosely to that of the maleparamere (maximum incontinualdarkness for the past two years.Both the stock and diameterof 0.05mm at 0.2mm from the tip of the needle). experimentalanimals were fed weekly. Experimental animals Using the micromanipulator,the pintip couldbe inserted wereisolated from the stock population as eitherfourth- orfifth- through the abdominalwall of afemaleat an obliqueangle and instar nymphs and kept individuallyin glass test tubes with a to adepth that closelysimulated the length of the maleparamere slipof filterpaper, plugged with apieceof foam.All nymphs that isnormallyinserted ( ca.0.2mm; personal observation). werefed communally but werereturned to theirindividual tubes after feeding,ensuring the virginityof any eclosedadults. The (c) Female fecundity andlifespan tubes werechecked weekly for any eclosedadults, which were The numberof eggs laidby eachfemale on the substrate was then sexedand placedinto either a maleor a femaleholding countedevery week, and the substrate was replaced.The date tube priorto usein the experiment.Any femalesnot usedwithin that eachfemale died was recorded,and femalelifespan as an aweekof eclosioninto adulthood wereexcluded from the adult was calculatedas beginningfrom the date fromwhich the experiment. femalewas includedin the experiment.

Proc.R. Soc.Lond. B (2003) Costsof traumatic insemination E.H.Morrowand G. Arnqvist 2379

Table1. Analysis of deviance of theeffects of mating rate and trauma per se wassupported by ouranalyses: when com- experimental piercing on adult femalelifespan ( n = 76) (test of paring femalesexperimentally piercedoutside of the sper- 2 model: x = 11.71, d.f. = 4, p = 0.019). malege with thosepierced in thespermalege with a (Addition of thetwo-way interactions between mating rate and focusedtest, the latter suffereda 50% reductionin lifetime theother variables to thismodel did not further improve model 2 fecunditycompared with theformer (9.0 ± 3.2 versus fit (x = 3.32, d.f. = 3, p = 0.345).) 2 18.8 ± 3.2; x = 3.72, d.f. = 1, P a/2 = 0.027). factor d.f. x2 p 4. DISCUSSION mating rate 1 5.99 0.014 pierced 1 2.33 0.127 Despitethe apparently dramatic natureof the mode of piercing site 1 4.20 0.040 mating in bedbugs, our results show that traumatic contaminated piercing 1 0.14 0.712 insemination doesnot carry extreme coststo females.This may seemsurprising, butit is actually what wewould expectbased on sexually antagonistic coevolutionary Table2. Analysis of deviance of theeffects of mating rate and theory (see § 1). Despitethe fact that ourmating-rate experimental piercing on femalelifetime fecundity ( n = 76) treatment inflated thepotential mating rate by afactor of (test of model: x2 = 4.51, d.f. = 4, p = 0.341). four,no detectable effects on female lifetime egg pro- (Addition of thetwo-way interactions between mating rate and ductionwere detected although female lifespan was theother variablesto thismodel did not improve model fit reducedby 30%. Theseresults are similar tothose of (x2 = 0.40, d.f. = 3, p = 0.940).) other experiments involving insectswithout traumatic insemination,which showan average effectsize of 2 factor d.f. x p 0.84 ± 0.08 95% confidencelimits (seeArnqvist &Nilsson 2000 for areview),and suggest that anincreased rate of mating rate 1 0.27 0.603 insemination in bedbugs elevates female reproductiverate pierced 1 1.00 0.317 withoutsignificantly reducingfemale fitness.In theonly piercing site 1 3.70 0.054 earlier experimental studyof thecosts of mating in atrau- contaminated piercing 1 0.13 0.718 matically inseminating speciesof which we are aware, Stutt& Siva-Jothy (2001) performedan experiment where they varied mating rate for female bedbugs. They docu- 3. RESULTS menteda lifespan reductiondue to increased mating rate (a) Adult female longevity that wasquantitatively very similar tothat reportedhere, The effectsof ourexperimental variables onfemale lon- butdid not find any effectson reproductive rate, and gevity andlifetime fecunditywere analysed in agen- consequentlyfound that lifetime fecunditywas reduced by eralized linear model,with Poissonerrors anda logit-link frequentmating. The differencesbetween our results and function,using GLIM (version 3.77; Payne 1986). Since thoseof Stutt & Siva-Jothy (2001) canbe attributed to both ofthese response variables representcount data, this thefacts that theabsolute mating rateswere lower in their modelis preferable toconventional linear modelsthat experiment andtheir experimental designdid not allow a assumea continuousresponse variable (McCullagh & separation ofthe effects of copulation per se from thoseof Nelder 1989). Statistical inferenceswere based on an thetrauma ofinsemination. analysis ofdeviance(ANDEVA), a procedurethat is anal- It is clear from ourstudy that female bedbugs are well ogousto an analysis ofvariance that followstraditional adaptedto both thephysical trauma ofpiercing ofthe linear models(Crawley 1993). To compensatefor overd- abdominal wall that occursduring copulation andto any ispersion,an empirically derivedscale factor wasused in associatedcomplications that may arise from pathogens theanalysis (Aitkin et al. 1989). This analysis (seetable 1 gaining entry through thewound site. Instead, the costs andfigure 1) revealed asignificant effectof mating rate ofa high mating rate documentedhere (see also Stutt& uponfemale longevity. Females includedin thehigh mat- Siva-Jothy 2001) apparently derive primarily from the ing rate treatment suffereda ca.30% reductionin adult receipt oftheejaculate into the haemolymph, whichcould lifespan ((average in days ± s.e.):low 36.53 ± 3.24, n = 38; beproblematic tofemales for tworeasons. First, sperm high 25.84 ± 2.65, n = 38). Neither thesterility ofthe pin and/or other ejaculatecomponents are likely toinitiate a usednor whether females were experimentally piercedhad female immune responsesince they will appear asforeign any significant effects,whereas females pierced outside of matter. Second,male bedbugs transfer accessory gland thespermalege suffereda significantly reducedlifespan productsto females at mating (Davis 1956, 1966). Such (24.06 ± 4.09) compared with thosepierced through the seminal substancesmay bemanipulative, andnegative spermalege (36.05 ± 3.33). effectsto females of receiving large amountshave been documentedin several insectswithout traumatic insemi- (b) Lifetime fecundity nation(e.g. Das et al. 1980; Arnqvist &Nilsson2000; The effectsof our treatment variables onfemale lifetime Chapman 2001). Interestingly, in bedbugs, aggregations fecunditywere analysed in ageneralized linear model, ofphagocytic cellspresent within thefemale become identical tothe one described above. The overall model active directly after insemination,and apparently take up wasnot significant (table 2), suggesting that ourtreat- anddigest seminal fluid material (Davis 1956; Carayon mentsdid not collectively affectfemale lifetime fecundity 1966). However,the fact that themagnitude ofthe nega- significantly. However,the prediction that thespermalege tive effectsto female bedbugs of elevatedmating ratesare functionsto reduce the direct fitness cost of the piercing comparable with thosein insectswithout traumatic

Proc.R. Soc.Lond. B (2003) 2380E. H.Morrowand G. Arnqvist Costsof traumatic insemination

40 40

30 30

20 20 ) s

y 10 10 a d (

n a

p 0 0 s

e high low

f no yes i l mating rate e pierced l a 40 40 m e f

t l u

d 30 30 a

20 20

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Figure 1. Theeffects of various experimental treatments on adult femalelifespan. Figure showsleast-squares means ( ± s.e.) from ageneral linear model analogue of themodel presented in table1. insemination (cf.Arnqvist &Nilsson2000) suggeststhat costsof physical piercing trauma andthat themesosperm- femalesare well adaptedto the special challenges that may alege functionsto minimize thecosts of receiving ejacu- beassociated with receiving theejaculate in thebody cav- lates by restricting diffusionof sperm and/orseminal fluid ity. Moreover,the costs to female bedbugs of elevated within thefemale body (Davis 1956; Carayon 1966; mating rates are primarily dueto the effects of theejacu- Stutt& Siva-Jothy 2001). Here,we have at least provided late, aphenomenonnot unique to traumatically insemin- experimental supportfor theformer suggestion.The fact ating species. that femaleshave investedin theevolution of counter- Our resultsreveal that experimental traumatic piercing adaptations toan antagonistic behaviour in males raises in thespermalege, closely mimicking that occurring during tworelevant issues.First, given ourresults, it is easyto natural copulation,had nosignificant effectson female seewhy males shouldevolve topierce andinseminate performance.By contrast,when females were subjected to femalesin thespermalege rather than in any other part suchpiercing outsidethe spermalege, it causeda dramatic ofthe body. All elsebeing equal,males will benefitfrom decreasein both lifespan andlifetime egg production harming their matesas little aspossible (Morrow et al. despitea very lowrate ofpiercing (oncea week).This 2003) andthe spermalege clearly offersan opportunity for resultunambiguously showsthat thespermalege effec- lesstraumatic piercing. Second,it isinteresting tonote tively reducesthe direct costs of trauma tofemalesduring that thedegree of elaboration ofthe spermalege varies insemination in bedbugs, and hence provides supportfor tremendouslywithin theCimicidae (seeCarayon 1966). thesuggestion that thespermalege representsa female At oneextreme, we find the genus Primicimex where counter-adaptationto anantagonistic adaptation in males femaleslack any tracesof a spermalege altogether. Atthe (traumatic insemination) (Davis 1956; Carayon 1966; other,we findthe genus Stricticimex in whichthe ectosper- Thornhill &Alcock1983; Stutt& Siva-Jothy 2001). malege is well developedand the mesospermalege essen- Although suchcounter-adaptations are nodoubt very tially formsa completeand complex copulatory tube widespreadand appear in many differentforms (e.g. Hol- leading tothe oviducts. In thelatter case,this derivedand land &Rice1998; Chapman et al. 2003) ourstudy is, to uniquereproductive tract ensuresthat theejaculate does ourknowledge, the first toprovide experimental evidence notcome into direct contact with thehaemolymph. If this showingthat afemale morphological trait reducesthe fit- variation is indeedthe result of sexually antagonistic nesscost of exposureto an antagonistic male trait. Earlier coevolution,we expect the degree of elaboration ofthe experimental studiesof putative counter-adaptationshave spermalege tocovary with antagonistic traits in males (cf. either notmeasured net fitness costs to females or have Arnqvist &Rowe2002 a).Unfortunately,we currently beenunable to identify thetrait in femalesthat mediates lack both awell-resolvedphylogeny for this group anda resistanceto males (Arnqvist &Rowe1995; Holland & clear idea ofwhich male traits might antagonistically coev- Rice 1999). olve with thefemale spermalege. Futurecomparative stud- It haspreviously beensuggested that thebed bug ectos- iespromise toshed light onthe dynamics of intersexual permalege is afemale counter-adaptationto cope with the coevolutionin this group.

Proc.R. Soc.Lond. B (2003) Costsof traumatic insemination E.H.Morrowand G. Arnqvist 2381

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