Functional Ecology 2010 doi: 10.1111/j.1365-2435.2010.01696.x A hitchhiker’s guide to a crowded : how do fig find the right ride?

Anusha Krishnan, Subhashini Muralidharan, Likhesh Sharma and Renee M. Borges*

Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560 012, India

Summary 1. Organisms with low mobility, living within ephemeral environments, need to find vehicles that can disperse them reliably to new environments. The requirement for specificity in this pas- senger–vehicle relationship is enhanced within a tritrophic interaction when the environment of passenger and vehicle is provided by a third organism. Such relationships pose many interesting questions about specificity within a tritrophic framework. 2. Central to understanding how these tritrophic systems have evolved, is knowing how they function now. Determining the proximal cues and sensory modalities used by passengers to find vehicles and to discriminate between reliable and non-reliable vehicles is, therefore, essential to this investigation. 3. The ancient, co-evolved and highly species-specific nursery between figs and fig is host to species-specific plant-parasitic nematodes which use fig wasps to tra- vel between figs. Since individual globular fig inflorescences, i.e. syconia, serve as incubators for hundreds of developing pollinating and parasitic wasps, a dispersal-stage within such a chemically complex and physically crowded environment is faced with the dilemma of choos- ing the right vehicle for dispersal into a new fig. Such a system therefore affords excellent oppor- tunities to investigate mechanisms that contribute to the evolution of specificity between the passenger and the vehicle. 4. In this study of fig––nematode tritrophic interactions in racemosa within which seven wasp species can breed, we demonstrate using two-choice as well as cafeteria assays that plant- parasitic nematodes ( racemosa) do not hitch rides randomly on available eclosing wasps within the fig syconium, but are specifically attracted, at close range, i.e. 3 mm distance, to only that vehicle which can quickly, within a few hours, reliably transfer it to another fig. This vehi- cle is the female pollinating wasp. Male wasps and female parasitic wasps are inappropriate vehicles since the former are wingless and die within the fig, while the latter never enter another fig. Nema- todes distinguished between female pollinating wasps and other female parasitic wasps using volatiles and cuticular hydrocarbons. Nematodes could not distinguish between cuticular hydro- carbons of male and female pollinators but used other cues, such as volatiles, at close range, to find female pollinating wasps with which they have probably had a long history of chemical adaptation. 5. This study opens up new questions and hypotheses about the evolution and maintenance of specificity in fig–wasp–nematode tritrophic interactions. Key-words: cuticular hydrocarbons, host specificity, plant– interactions, phoresy, plant– interactions, tritrophic interactions, volatiles

of the passenger or its progeny either due to crowding, Introduction habitat deterioration, sibling rivalry or for mate finding Phoresy is a phenomenon in which the phoretic organism (Farish & Axtell 1971; Binns 1982; Colwell 1986; Kruitbos, (the passenger) actively seeks out its vehicle for dispersal or Heritage & Wilson 2009). Therefore, ephemeral, patchy or migration out of areas unsuitable for further development unpredictable habitats coupled with low vagility of the passenger select for the evolution of a passenger–vehicle *Correspondence author. E-mail: [email protected] relationship (Houck & OConnor 1991; Zeh & Zeh 1992)

2010 The Authors. Journal compilation 2010 British Ecological Society 2 A. Krishnan et al. wherein passengers must be able to efficiently locate their ado 2008). The fig system is also host to two genera of vehicles for transmission. This relationship is under further parasitic nematodes [Schistonchus (: evolutionary constraints when the vehicle and the passenger Aphelenchida) and Parasitodiplogaster (Diplogasteridae: are in close association with a third organism as occurs in Rhabditida)] found so far exclusively in fig inflorescences tritrophic interactions. Thus, considerable specificity has called syconia (Giblin-Davis et al. 2006; Gulcu et al. 2008; been observed, for example, in phoretic tritrophic interac- Powers et al. 2009). The short-lived ephemeral fig syconium tions involving flower mites carried by hummingbirds or (see Section on Natural History in Materials and methods) bats between patchily distributed flowers (Colwell 1979, within which the nematodes and wasps develop provides an 1985; Tschapka & Cunningham 2004; but see Garcı´ a- ideal situation for the development of a passenger–vehicle Franco, Martı´ nez & Pe´ rez 2001) or those involving annelids relationship since the passenger must necessarily disperse to and ostracods carried by lizards and frogs between find another suitable syconium. Nematodes have also been epiphytes (Lopez et al. 2005). Indeed, tritrophic interactions found in fossil associations with fig wasps (15–45 mya), in general are often characterised by their specificity indicating a long evolutionary association (Poinar 2003; (Mumm et al. 2005; Singer & Stireman 2005; Blu¨ thgen, Pen˜alver, Engel & Grimaldi 2006). Parasitodiplogaster and Mezger & Linsenmair 2006; Heil 2008; Rasmann & Turlings Schistonchus are phoretic on the pollinating fig wasp; how- 2008). However, high species specificity, whatever its origin, ever, Parasitodiplogaster is parasitic on its wasp host (Poinar necessitates suitable proximal mechanisms to ensure its & Herre 1991; Herre 1993, 1996), while Schistonchus is a plant maintenance. Questions about the mechanisms contributing parasite feeding on male or female floral tissue within which it to specificity between partners engaged in multitrophic can also induce tissue hypertrophy (Vovlas, Inserra & Greco interactions, such as those that occur in phoresy, are conse- 1992; Giblin-Davis et al. 1995; Center et al. 1999). Parasito- quently fundamental to understanding evolutionary and diplogaster occurs in New World, African and Australian co-evolutionary processes (Thompson 2009). figs (Bartholomaeus et al. 2009) while Schistonchus also In phoretic interactions which are often considered a occurs in European and Australasian figs and its association prelude to the development of parasitism (Anderson 1984), with figs is presumed to have a more ancient origin (Giblin- passengers may use chemical (Soroker et al. 2003), visual Davis et al. 1995; Giblin-Davis et al. 2006). The tritrophic (Harbison, Jacobsen & Clayton 2009), heat and vibration interaction between fig, wasp, and nematode is thus a highly (Owen & Mullens 2004) cues from their vehicles, which aid in specific one, and therefore lends itself to an examination of their localization. The use of a particular sensory modality in questions regarding the processes that select for and maintain passenger–vehicle localization would depend on the nature of such specificity. Furthermore, although plant-parasitic nema- the tritrophic system, since certain cues, e.g. organic chemical todes and phytophagous are extremely speciose, the compounds of low volatility and therefore short range of tritrophic interactions between nematodes, plants and insects action, would be inappropriate if the localization distance is have scarcely been studied (Kaplan, Sardanelli & Denno necessarily large. This ability to successfully find targets is 2009). also linked to discrimination ability; studies of tritrophic Female pollinating wasps are appropriate and reliable interactions indicate that discrimination ability depends phoretic vehicles for the nematodes since the males of polli- considerably on the degree of specialisation of the interaction nating and non-pollinating wasps are usually wingless and die (Vet & Dicke 1992). In some phoretic interactions, passengers within their natal figs, and female non-pollinating fig wasps lure their vehicles to themselves and thus use the discrimina- do not enter a fig once they have exited their natal syconium tion ability of their vehicles instead (Saul-Gershenz & Millar (see Section on Natural History in Materials and methods for 2006). While varied biochemical and sensory processes may details). The generalisation that non-pollinating wasps do not mediate tritrophic interactions (Dicke & Hilker 2003; Heil enter the syconium is true for most fig species; however, there 2008), their specificity crucially depends on ecological are some exceptions (see Section on Natural History in Mate- context, e.g. selection for increased discrimination of hosts in rials and methods for details). Nematodes also do not usually sympatric communities compared to those in allopatry. The enter fig syconia via ovipositors of the externally-ovipositing success of a mechanism that guarantees specificity would thus wasps since nematodes have been found only in one species of ensure the uniqueness of a response given the ecological non-pollinator parasitising a dioecious fig species (Vovlas & context and attendant evolutionary constraints (Thrall et al. Larizza 1996). Therefore, a fig nematode must typically seek 2007; Poullain et al. 2008). out only a female pollinator for a ride into the next fig The fig–fig wasp–nematode system is ideal to investigate syconium. Conditions within the fig syconium are extremely the evolution and maintenance of host specificity especially crowded; hundreds of galls containing male and female polli- the phenomenon of phoresy. This is because this 70–90 nating and non-pollinating wasps are very closely packed million year-old seed-destroying (wasp) and seed-producing together, and nematodes must therefore make decisions at (fig) mutualism (Machado et al. 2001; Rønsted et al. 2008) is short range about the appropriate phoretic vehicle. Given highly species-specific with usually one pollinator species that there is also high species specificity between figs and their (: ) and several species of non-polli- nematodes (Poinar & Herre 1991; Gulcu et al. 2008), such nating parasitic fig wasps for each of the 700+ species of figs that each of the 700+ fig species is predicted to have a unique (Moraceae) (Cook & Rasplus 2003; Herre, Jande´ r & Mach- species of Schistonchus nematode (Giblin-Davis et al. 1995;

2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology Nematode dispersal between fig syconia 3

Gulcu et al. 2008; Bartholomaeus et al. 2009), we hypothes- STUDY SITE AND SPECIES ised that this specificity has led to each Schistonchus species Fig syconia of the monoecious Ficus racemosa L. in wasp-dispersal or developing specific attraction to the only vehicle that can D-phase were collected in and around the Indian Institute of Science reliably allow it to disperse between fig syconia, i.e. the female campus in Bangalore, India (1258¢N7735¢E). The syconia were cut pollinating wasp. open to collect pollinators (Ceratosolen fusciceps Mayr) and non-pol- Since nematodes show chemotaxis to specific host chemi- linating fig wasps (Apocryptophagus testacea Mayr, Apocryptophagus cals (Hong & Sommer 2006; O’Halloran, Fitzpatrick & fusca Girault, Apocryptophagus agraensis Joseph, Apocrypta west- Burnell 2006; Zhao et al. 2007; Rasmann & Turlings 2008), woodi Grandi and Apocrypta sp. 2) which mostly constitute the fig we investigated the role of whole insects, cuticular hydrocar- wasp community available in Ficus racemosa at this site (Proffit et al. bons, and volatiles of potential vehicles in attracting plant- 2007; M. Ghara, Y. Ranganathan and R.M. Borges, personal obser- vations). Wasps were dissected in distilled water and examined under parasitic Schistonchus nematodes of Ficus racemosa.Inthis a microscope for the presence of nematodes. Apocryptophagus first-ever study of the chemical ecology of fig nematodes, we stratheni Joseph which also occurs in this community was too rare to specifically (i) determined whether the nematodes used short- be thus examined. Nematodes were found only in female C. fusciceps range or long-range cues in locating potential vehicles, (ii) and were identified as Schistonchus racemosa Reddy & Rao. Nema- determined whether they preferred pollinating over parasitic todes were only observed in the lumen of the fig syconia, and were wasps and if they preferred one sex over the other, and (iii) often observed performing nictation behaviour; i.e. the dispersing identified which chemical classes, volatile vs. non-volatile stages stand up on their ‘tails’ and engage in waving their bodies. This cues, were useful in determining preference differences among is a typical behaviour exhibited by nematodes for transmission to a the species and between the sexes. new niche, and in this case occurs to increase encounters with mobile insects (Croll & Mathews 1977). The frequency of nematodes varied within syconia from complete absence to about a thousand individu- Materials and methods als. While S. racemosa is a plant-parasitic nematode specialising on floral tissue, we have been unable to quantify the damage it causes. Individual syconia varied greatly in the presence of wasps of the NATURAL HISTORY OF THE FIG–WASP–NEMATODE different species with pollinator and non-pollinator numbers in a TRITROPHIC SYSTEM syconium ranging from zero to several hundred in a highly stochastic In the fig brood-site pollination mutualism, the nursery is the fig manner (M. Ghara, A. Krishnan and R.M. Borges, unpublished syconium (globular enclosed inflorescence) in which seeds are pro- data). Nematodes were present only in those figs into which female duced and pollinators breed. In typical monoecious figs, pollinating pollinating wasps had entered. foundress female wasps enter the syconium at the -receptive or B-phase, pollinate some female flowers, and also oviposit into CHOICE ASSAYS other flowers resulting in galls (Galil & Eisikowitch 1968). The foundresses die shortly after pollination and oviposition, and their Nematodes were collected from the lumen of opened D-phase fig offspring develop within the galled flowers during the interfloral or syconia. Assays were designed to score the behaviour of individual C-phase. The non-pollinating wasps [Hymenoptera: Agaonidae nematodes, rather than sets of nematodes, since the numbers of nema- (paraphyletic)] do not enter the fig syconium but oviposit into the todes within syconia were highly variable. Some syconia were devoid syconium from the outside, using long ovipositors (Proffit et al. of nematodes. Choice assays with individual nematodes were carried 2007; however, there are some exceptions; see Cook & Rasplus out in six-well plates (12Æ5cm· 7Æ5 cm, each well = 3Æ6 cm diame- 2003; Herre, Jande´ r & Machado 2008). The parasites could be ter). The number of nematodes used for each choice assay was flower gallers, parasitoids, cleptoparasites or inquilines (Cook & variable (24–80) based on availability; the actual numbers used are Rasplus 2003; Ghara & Borges 2010) and have variable impacts on given in the Results section. Each well was half-filled with 1Æ6% the fig and fig wasp mutualism (Herre, Jande´ r & Machado 2008) agarose in a buffer containing 1 mM CaCl2, 1 mM MgSO4,and since they breed within the syconium at the expense of seeds and 50 mM potassium phosphate (pH 6Æ0) (after Brenner 1974). The pollinator progeny. Male wasps are usually wingless, emerge first, solidified agarose was layered with 2% BaSO4 suspension in the same and mate with freshly eclosed females. Female pollinators collect buffer. The BaSO4 layer formed a background on which nematodes pollen from freshly dehisced male flowers and leave the syconium left a clear trail as they moved on the plate. Since fig wasp nematodes through an exit hole prepared by the cooperative efforts of pollina- are much smaller (400 lmlength,13 lm width; Vovlas & Larizza tor males who die within their natal syconium (D-phase or wasp 1996) than the model nematode Caenorhabditis elegans (1400 lm emergence ⁄ dispersal phase). The winged parasitic females usually length, 80 lm width; Mo¨ rck & Pilon 2006), their tracks could not exit the syconia at the same time as the female pollinators through be directly visualised on agarose alone. Therefore BaSO4 was used, as the exit hole prepared by male pollinators. The juvenile or dispersal it is known to be an inert, non-toxic compound, was easily available, stage plant-parasitic nematodes of figs enter the dispersing female and convenient to handle. All choice assays were conducted with pollinating wasps (in the D-phase syconium) and are carried in the competing wasps or competing extracts rather than against solvent abdomen (‘abdominal folds’) or in the hemocoel into a receptive controls, since the objective of this study was to determine nematode stage fig syconium (B-phase) where the nematodes disembark from choices in chemically crowded environments and thus to replicate the body of the foundress female wasp and feed on tissues of plant natural conditions as much as possible. All choice assays were origin particularly floral ones (Reddy & Rao 1984; Vovlas, Inserra conducted in the dark to mimic natural conditions and to prevent & Greco 1992; Giblin-Davis et al. 1995; Vovlas & Larizza 1996; variation in ambient light from interfering with the experiments. The Vovlas et al. 1998). Thus nematodes need to disperse between translation of laboratory nematode assays to natural conditions is ephemeral syconia in order to continue their life cycles. vital (Spence, Lewis & Perry 2008).

2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology 4 A. Krishnan et al.

DISTANCE RESPONSE ASSAY WASP VOLATILE CHOICE ASSAYS

Male and female pollinator wasps were collected and freeze killed Wasps (male and female pollinators, and female non-pollinators at )20 C overnight. Two wasps (one male and one female) were of A. testacea and Apocrypta sp. 2) collected from D-phase figs placed at different distances (10 mm, 5 mm or 3 mm) on oppo- were placed in )20 C and freeze-killed 3 h before the assay, site sides of a central point in each well (Fig. 1a). In this and in and were stored at )20 C until the assay was performed. Two all subsequent two-choice assays, the positions of the wasps were slits were made in the agarose at a distance of about 2Æ5mmon interchanged between trials to counter direction biases in nema- either side of the central point (Fig. 1b) to prevent diffusion of todes. Single nematodes were picked up from figs using a single non-volatile cues from the wasps towards the nematodes. The bristle from a brush mounted on an insect pin, placed between wasps were placed outside the slits, and the assay was carried the wasps, and left in darkness for 2 h. At the end of this time, out as described earlier. the assay plates were inverted and either stored at 4 Ctoimmo- bilise the nematodes and scored later, or scored under a light microscope immediately for nematode choices. The nematode was WASP CUTICULAR HYDROCARBON CHOICE ASSAYS deemed to have made a choice if trails led from the central point Cuticular hydrocarbon extracts of wasps (male and female pollina- to a particular wasp. Wells which contained no trails or in which tors, and female non-pollinators of A. testacea and Apocrypta sp. 2) trails led to both wasps were classified as ‘no choice’. A distance collected from D-phase figs were made by adding 400 lLofpentane response curve was plotted and the appropriate distance for to twenty live wasps of each species in a glass vial. The vials were further experiments was determined to be 3 mm (see Results). vortexed gently for 1 min and incubated for ten minutes at room temperature. The wasps were removed and the pentane was allowed to evaporate completely. The extracts were stored at )20 C till they WHOLE WASP CHOICE ASSAYS were used. Each vial containing the cuticular hydrocarbon extract Whole wasp choice assays were conducted by giving the nema- from twenty wasps was re-suspended in 50 lL of pentane, and used todes choices between males and females of pollinator wasps for five assays. Into each agar-filled well, 10 lL each of two different and females of pollinating (C. fusciceps) and non-pollinating extracts were added such that the extracts were separated by approxi- wasps (A. testacea and Apocrypta sp. 2). Apocrytophagus agraen- mately 3 mm. The pentane was allowed to evaporate, and the spread sis and A. fusca were not available at the time of these experi- of the extracts were traced out with pin pricks on the surface of the ments and Apocrypta westwoodi was too rare to be used. Only BaSO4 (Fig. 1c). A single nematode was placed at a central point freshly eclosed wasps were collected by opening D-phase figs, as between the two extracts and left in darkness for 2 h, after which the they would be most suitable as phoretic vehicles at that time. In choice of the nematode was scored as above. In all cases, nematodes all experiments, wasps were placed 3 mm from the central point were noted to have sampled both extracts before making their final where the nematode was placed (Figs. 1a,b). The assay was choice; the assay would have been unacceptable had this not held carried out in the same manner as described above. true.

(a) (b)

Fig. 1. Experimental set up for nematode assays. Single wells of the six-well plate (c) (d) showing the assay set up for (a) whole wasp choice assays between male pollinator (mP) and female pollinator (fP); (b) volatile choice assays between male pollinator (mP) and female pollinator (fP); (c) cuticular hydrocar- bon choice assays between male pollina- tor ⁄ female non-pollinators (X) and female pollinators (fP); (d) cafeteria choice assays with male pollinators (mP), female pollina- tors (fP), male non-pollinators (mNP), females of non-pollinator Apocrypta sp. 2 (fNP1) and females of non-pollinators A. testacea or A. fusca (fNP2). Scale bar indicates 3 mm distance.

2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology Nematode dispersal between fig syconia 5

CAFETERIA CHOICE ASSAYS placed at a distance of 5 mm (n = 12) or 10 mm (n =12), In order to mimic nematode choice of a vehicle in the crowded the nematodes were unable to make a choice and remained syconial environment, cafeteria assays were conducted. Each cafeteria stationary. At a distance of 3 mm from the wasps, 75% choice assay was carried out with eight freeze-killed wasps (one polli- (n=20) of the nematodes made a choice and moved towards nator female, one pollinator male, two non-pollinator males and four one of the wasps. All assays described in this study therefore non-pollinator females) arranged in a ring around a central point in used this distance. the assay well (see next section on data analysis for the rationale for this wasp ratio). The non-pollinator wasps used in each assay consisted of two females of Apocrypta sp. 2 (fNP1), and one female WHOLE WASP CHOICE ASSAYS: SPECIES AND SEX each of A. testacea and A. fusca (both labelled fNP2). The male non- PREFERENCE pollinating wasps were picked randomly and belonged to Apocrypta On being given choices of whole wasps, a significantly larger sp. 2, A. testacea or A. fusca. The random selection of non-pollinator percentage of nematodes chose female pollinators over male males was due to the fact that fig syconia can contain unpredictable 2 numbers of non-pollinator males with some syconia being devoid of pollinators (v =4Æ54, P =0Æ033) or female non-pollinators 2 males; thus availability of fresh males was the limiting factor which (fNP1 Apocrypta sp. 2: v =8Æ05, P =0Æ004; fNP2 A. testa- 2 led to the adoption of this experimental design. The positions of the cea: v =6Æ76, P =0Æ009; Fig. 2). wasps were varied between trials to counter direction biases. Each wasp was placed 3 mm from the central point (Fig. 1d). The nema- WASP VOLATILE CHOICE ASSAYS: SPECIES AND SEX tode was placed in the centre of the ring of wasps, and left in darkness for 2 h, after which the choices made by the nematode were scored. PREFERENCE When nematodes were presented with only volatile cues, a

DATA ANALYSIS significantly larger percentage of nematodes chose female pollinators over male pollinators (v2 =4Æ41, P =0Æ035), The data from all two-choice assays were analysed using chi-square female non-pollinators of Apocrypta sp. 2 (fNP1: v2 =6Æ37, tests conducted with the software package STATISTICA (Tulsa, Okla- P =0Æ012) and A. testacea (fNP2: v2 =6Æ53, P =0Æ011) homa, USA). All assays labelled ‘no choice’ were excluded from the (Fig. 2). analyses. In most of the cafeteria choice assays, the nematode was seen to have made more than one choice; therefore a weighted scoring system was used to analyse the data. For convenience, based on the WASP CUTICULAR HYDROCARBON CHOICE ASSAYS: maximum number of choices made by a nematode within each assay SPECIES AND SEX PREFERENCE (recorded post hoc) and the least common multiple (LCM) of this maximum number, each assay was given a total of twelve points, In cuticular hydrocarbon choice assays, a significantly larger which was divided equally among the different choices made by the number of nematodes chose female pollinator extracts over nematodes. For example, if in a well, nematode trails led to three female non-pollinator extracts (fNP1 Apocrypta sp. 2: different wasps, then the twelve points were divided by three and a v2 =9Æ85, P =0Æ002; fNP2 A. testacea: v2 =10Æ70, score of four points was awarded to each wasp. The total number of P =0Æ001; Fig. 2). In choice tests between male pollinator points scored by the female pollinators, male pollinators, male non- and female pollinator cuticular hydrocarbons, there was no pollinators and female non-pollinators was recorded and compared difference between the number of times male pollinator (using a chi-square test) against an expected set of values calculated under the assumption that the nematode’s choices were random. The expected set of scores was calculated based on the numbers of each wasp in the assay. The ratios of the different wasps in each assay were fP : mP : mNP : fNP = 1 : 1 : 2 : 4 (fP = female pollinator; mP, male pollinator; mNP, male non-pollinator; fNP, female non-pollina- tor). This ratio was chosen to skew the assay towards the non-pollina- tors, since any preference for female pollinators under such an experimental condition would be considered evidence of a strong biological effect. A total of 40 trials were carried out, with each trial carrying 12 points. Therefore, the total number of points = 40 · 12 = 480, which when divided between each of the wasps according to their ratios in the trials results in an expected score set of fP : mP : mNP : fNP = 60 : 60 : 120 : 240.

Results Fig. 2. Responses of nematodes to whole wasp, volatile and cuticular hydrocarbon cues when given choices between female pollinators DISTANCE RESPONSE ASSAY (fP), male pollinators (mP), females of non-pollinator Apocrypta sp. 2 (fNP1) and females of non-pollinator A. testacea (fNP2). The choices The distance response assays carried out using male and (excluding ‘no choice’ responses) were analysed using chi-square tests. female pollinators indicated that the nematodes were able to n.s. = non-significant difference (P >0Æ05); *P <0Æ05; **P < sense the presence of the wasps at a distance of 3 mm. When 0Æ01; n includes nematodes exhibiting no choice.

2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology 6 A. Krishnan et al.

probably had a long evolutionary history of chemical adapta- tion. Our studies have shown that nematodes use cues from whole wasps, and they can also use cues from the cuticular hydrocarbon and volatile signatures of the wasp vehicles. Nematodes are known to respond to specific host hydrocar- bons (Stamps & Linit 2001; O’Halloran, Fitzpatrick & Burnell 2006), host pheromones (Hong & Sommer 2006), and host volatiles (O’Halloran & Burnell 2003; Hong & Sommer

2006; Zhao et al. 2007) including CO2 (Pline & Dusenbery 1987; Bretscher, Busch & de Bono 2008). In tritrophic interac- tions, entomopathogenic soil nematodes also respond to belowground volatiles such as b-caryophyllene emitted by infested roots (van Tol et al. 2001; Rasmann & Turlings 2008; Fig. 3. Observed and expected scores for female pollinators (fP), Degenhardt et al. 2009). Plant-parasitic nematodes also ori- male pollinators (mP), male non-pollinators (mNP) and female non- ent to specific phytohormones such as auxins (Curtis 2007). pollinators (fNP) in the cafeteria choice assay. The observed and Free-living nematodes, on the other hand, are more sensitive expected distribution of scores were significantly different (v2 =826Æ3, P <0Æ00001, n = 40, d.f. = 3). to cues of their bacterial prey (O’Halloran & Burnell 2003; O’Halloran, Fitzpatrick & Burnell 2006), or to CO2 (Bretscher, Busch & de Bono 2008). This chemosensitivity of extracts were chosen over female pollinator extracts nematodes enables them to distinguish between host plant (v2 =0Æ0, P =1Æ0, Fig. 3). However, a significantly larger species (Zuckerman & Jansson 1984; Zhao, Schmitt & Hawes number of nematodes chose the male pollinator extracts over 2000) and host insects (Hong & Sommer 2006). Similarly, we female non-pollinator (Apocrypta sp. 2) extracts (v2 =25Æ30, found that Schistonchus nematodes of F. racemosa were able P =0Æ0001, Fig. 2). to distinguish between female pollinators and female non-pollinators using cuticular hydrocarbons and volatiles. Furthermore, they were able to distinguish between male and CAFETERIA CHOICE ASSAYS: SPECIES AND SEX female pollinators when whole wasps and volatile cues were PREFERENCE provided to them, but were unable to distinguish the pollina- Most of the nematodes in the cafeteria choice assays made tor sexes based on cuticular hydrocarbons. This may be more than one choice, and trails leading to several wasps were because (1) cuticular hydrocarbons of male and female fig observed in these assays. The observed scores were very wasps are similar since they serve as species recognition sig- different from the expected scores (Fig. 3), with the highest nals as occurs in some insects (Howard & Blomquist 2005; attraction score being obtained by the female pollinator, Smadja & Butlin 2009), or (2) the cuticular hydrocarbons of followed by the male pollinator, male non-pollinator and the sexes are different as also occurs in insects (Howard & female non-pollinator. The observed scores were significantly Blomquist 2005; Peterson et al. 2007; Van Homrigh et al. different from the expected scores (v2 =826Æ3, P <0Æ0001, 2007), but the nematodes may lack sensitivity to these differ- d.f. = 3) and the nematodes preferred whole female pollina- ences. The fact that nematodes chose cuticular hydrocarbons tors over other wasps. of male pollinating wasps over those of female non-pollinat- ing wasps is further evidence of the similarities between male and female cuticular hydrocarbons of the pollinating wasps. Discussion It is noteworthy that the percentage of non-responding nema- In the chemically complex and physically crowded environ- todes was the least when whole wasps were offered compared ment of the syconium of F. racemosa, where there are a maxi- to choices made when volatiles or cuticular hydrocarbons mum of fourteen possible choices (seven females and seven alone were used (Fig. 2). This might mean that whole wasps males belonging to pollinator and non-pollinator wasp taxa), provide a complete set of cues (volatiles and cuticular the nematode S. racemosa was particularly attracted to hydrocarbons) including those that were not tested in these female pollinating wasps over other tested taxa, via cues from experiments, or that nematodes need a hierarchy of cues for intact female bodies, or their volatiles and cuticular hydrocar- complete response (Lewis, Grewal & Gaugler 1995). Only bons. Nematodes could not distinguish, however, between future experiments will be able to distinguish between these the cuticular hydrocarbons of male and female pollinators. possibilities. The distance response assay demonstrated that Yet, they were more attracted to the cuticular hydrocarbons nematodes necessarily need close-range cues (at distances of of the male pollinator compared to those of female non-poll- at least 3 mm) in order to respond within such a chemically inators. Nematodes were only responsive to chemical cues of crowded environment. In the case of volatiles, these could be potential vehicles at short distances. Thus, passenger nema- long-chain carbon compounds (up to C29) which have low todes are able to use short-range chemical cues to find their volatility but can still be recovered in the volatile headspace reliable vehicle, i.e. female pollinators, with which they have around insects (Schmitt et al. 2007).

2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology Nematode dispersal between fig syconia 7

There are many reasons why nematodes specialise on considerable inter- and intraspecific variation in non-pollinat- pollinating fig wasps as vehicles. Within the pollinators, ing wasps with regard to the timing of their oviposition into nematodes should also use only females as vehicles since the syconium during its developmental cycle (Ghara & Borges pollinator males are wingless and usually die within the fig 2010). This variation may prevent the evolution of precise (Weiblen 2002). While winged males occur in some species of matching of development time between fig-specific nematodes Old World non-pollinating wasps, whose females enter fig and non-pollinating wasps. While pollinator females live for a syconia for oviposition (Herre, Jande´ r & Machado 2008), the short time (24–72 h) in several fig species examined (Kjell- males themselves do not enter syconia, and hence would be berg, Doumesche & Bronstein 1988; Dunn et al. 2008; [24 h considered dead-ends as vehicles into new fig syconia. There- recorded in Ghara & Borges (2010)]), the non-pollinator fore, males of all species of fig wasps are inappropriate species have extended and variable life spans of up to 27 days vehicles and should be avoided by nematodes. Indeed, nema- in some cases (Ghara & Borges 2010). The precise and short todes have not been reported in male fig wasps in any study life span of the pollinator female can also drive nematodes to (Vovlas, Inserra & Greco 1992; Vovlas et al. 1998; Poinar & preferentially match their developmental time with that of the Herre 1991; DeCrappeo & Giblin-Davis 2001). pollinating female compared to other wasp species within the There are many reasons why nematodes do not use non- syconia. Furthermore, the evolution of specific innate mecha- pollinating fig wasps as vehicles. Firstly, from an evolutionary nisms to recognise vehicles would preclude the need for learn- perspective, since the mutualism between figs and pollinating ing (Vet et al. 1993; Huigens et al. 2009) which is certainly fig wasps is very old (70–90 million years), and nematodes are selected against when the lifespans of vehicle and ⁄ or passen- also an ancient taxon (Poinar 2003), there has probably been ger are short, as in this case. sufficient time for a specific tritrophic relationship between Thirdly, nematodes should choose pollinator wasps over figs, pollinators and nematodes to have evolved, as also non-pollinator wasps because in many fig species, as in our revealed by fossil nematodes in this system (Poinar 2003; study system, only the pollinating wasps enter the syconium Pen˜alver, Engel & Grimaldi 2006). Indeed, nematodes seem through the ostiole while the non-pollinating wasps oviposit to be highly co-evolved with and species-specific to figs and from the outside (Cook & Rasplus 2003; Herre, Jande´ r& fig pollinators (Poinar & Herre 1993; Giblin-Davis et al. Machado 2008). This means that nematodes that use female 2003; Gulcu et al. 2008; Bartholomaeus et al. 2009). Of the pollinators as vehicles can disembark into the next syco- numerous nematode species identified within the fig system, nium through the entire body of the pollinator. Nematodes whether of the entomopathogenic Parasitodiplogaster or of that enter a female non-pollinator could only enter the fig the plant-parasitic Schistonchus, almost all have been through the narrow ovipositor. Moreover, in such non- recorded to use only the fig pollinator as vehicle or host pollinating species, oviposition is often interrupted, being (Kumari & Reddy 1984; Reddy & Rao 1984; Vovlas, Inserra disturbed by predatory ants and other external parasites & Greco 1992; Vovlas et al. 1998; Herre 1993, 1996; Poinar & (Schatz et al. 2006; Ranganathan & Borges 2009). In the Herre 1993; Lloyd & Davies 1997; DeCrappeo & Giblin- few fig systems where non-pollinating wasps also enter the Davis 2001; Zeng, Giblin-Davis & Ye 2007). There is to date syconium (Cook & Rasplus 2003; Herre, Jande´ r & Mach- only one record, in the domesticated dioecious fig Ficus ado 2008), nematodes may be expected to also develop carica, of the nematode Schistonchus caprifici also found in a passenger–vehicle relationships with them. While nematodes non-pollinating fig wasp Philotrypesis caricae (Vovlas & have been found in the ovipositor of one parasitic wasp Larizza 1996). The age of the relationship between figs and within a dioecious fig species (Vovlas & Larizza 1996), they parasitic fig wasps is, however, unknown. Since the species- have not been found in the parasitic wasps of our monoe- specificity of pollinators to the figs is higher than that of the cious fig system or elsewhere. Whether there is a difference non-pollinators (Weiblen & Bush 2002; Marussich & Mach- in nematode loads and nematode strategies between dioe- ado 2007; but see Jousselin et al. 2006, 2008), nematodes cious and monoecious fig species is worth investigating, should be selected to evolve or co-evolve with pollinators because in dioecious figs, pollinators can breed only in male rather than non-pollinator wasps, and to specifically adapt to fig trees and not in female fig trees (Cook & Rasplus 2003). pollinators as phoretic vehicles. This should be especially true While pollinating fig wasps that enter female figs will die for plant-parasitic nematodes such as Schistonchus since they without progeny (Cook & Rasplus 2003), nematodes that parasitise specific syconial tissues. are transported into such figs by female pollinators are Secondly, an important constraint that selects for species- doomed to reproduce without any chance of dispersal. specificity in this tritrophic interaction is that the timing of Thus, it is intriguing to hypothesise that a nematode within the development of wasps (vehicles) and nematodes (passen- a male fig syconium should hedge its bets and attempt to gers) must coincide since the eclosed wasps and their phoretic even enter non-pollinating fig wasps if by this strategy the nematodes must exit the fig syconium at the same time. There- chances of getting dispersed to another male fig are fore, development synchrony between vehicle and passenger enhanced. If future studies reveal that plant-parasitic nema- must also evolve, and this constitutes an important evolution- todes are found in parasitic non-pollinating wasps in other ary necessity before phoresy can arise in a nematode system fig systems, including monoecious species, we also predict (Giblin-Davis et al. 2003; Baldwin, Nadler & Adams 2004). that they will only be found in those systems where there is In the F. racemosa system presented in this paper, there is high species-specificity between parasitic wasps and figs.

2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology 8 A. Krishnan et al.

Thus this study, which is the first on the chemical ecology Dunn, D.W., Yu, D.W., Ridley, J. & Cook, J.M. (2008) Longevity, early of nematodes in the mutualism between figs and fig wasps, emergence and body size in a pollinating fig wasp – implications for stability in a fig–pollinator mutualism. Journal of Animal Ecology, 77, illustrates how nematodes choose their appropriate ride to 927–935. another fig syconium within their chemically complex and Farish, D.J. & Axtell, R.C. (1971) Phoresy redefined and examined in Macroch- physically crowded environment. The study also provides eles muscaedomesticae (Acarina: Macrochelidae). Acarologia, 13, 16–29. Galil, J. & Eisikowitch, D. (1968) Flowering cycles and fruit types of Ficus new testable hypotheses in this very exciting area of species sycomorus in Israel. New Phytologist, 67, 745–758. co-evolution and species-specific interactions within a Garcı´ a-Franco, J.G., Martı´nez, B.D. & Pe´ rez, T.M. (2001) Hummingbird tritrophic framework. flower mites and Tillandsia spp. (Bromeliaceae): polyphagy in a cloud forest of Veracruz, Mexico. Biotropica, 33, 538–542. Ghara, M. & Borges, R.M. (2010) Comparative life-history traits in a fig wasp community: implications for community structure. Ecological Entomology Acknowledgements DOI: 10.1111/j.1365-2311.2010.01176.x. Giblin-Davis, R.M., Ye, W., Kanzaki, N., Williams, D., Morris, K. & Thomas, This research was funded by the Ministry of Environment and Forests, Govern- W.K. (2006) Stomatal ultrastructure, molecular phylogeny, and description ment of India. We thank R. Yettiraj for fig collection, C.M. Brijesh for the of Parasitodiplogaster laevigata n. sp. (Nematoda: Diplogastridae), a para- BaSO4 idea, D. Dey for the six-well plates, V.V. Ramamurthy and S. Ganguly site of fig wasps. 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