A hitchhiker’s guide to a crowded syconium: how do fig

nematodes 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 pollination mutualism between

figs and fig wasps 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 nematode 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–wasp–nematode tritrophic interactions inFicus racemosawithinwhich seven

wasp species can breed, we demonstrate using two-choice as well as cafeteria assays that plant-

parasitic nematodes (Schistonchus 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 pollinatingwasp.Malewasps and female parasiticwasps 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 pollinatingwaspswithwhich 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–animal interactions, phoresy,

plant–insect interactions, tritrophic interactions, volatiles

Introduction

Phoresy is a phenomenon in which the phoretic organism

(the passenger) actively seeks out its vehicle for dispersal or

migration out of areas unsuitable for further development

of the passenger or its progeny either due to crowding,

habitat deterioration, sibling rivalry or for mate finding

(Farish & Axtell 1971; Binns 1982; Colwell 1986; Kruitbos,

Heritage & Wilson 2009). Therefore, ephemeral, patchy or

unpredictable habitats coupled with low vagility of the

passenger select for the evolution of a passenger–vehicle

relationship (Houck & OConnor 1991; Zeh & Zeh 1992)*Correspondence author. E-mail: renee@ces.iisc.ernet.in

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Functional Ecology 2010, 24, 741–749 doi: 10.1111/j.1365-2435.2010.01696.x

106

wherein passengers must be able to efficiently locate their

vehicles for transmission. This relationship is under further

evolutionary constraints when the vehicle and the passenger

are in close association with a third organism as occurs in

tritrophic interactions. Thus, considerable specificity has

been observed, for example, in phoretic tritrophic interac-

tions involving flower mites carried by hummingbirds or

bats between patchily distributed flowers (Colwell 1979,

1985; Tschapka & Cunningham 2004; but see Garcı́a-

Franco, Martı́nez & Pérez 2001) or those involving annelids

and ostracods carried by lizards and frogs between

epiphytes (Lopez et al. 2005). Indeed, tritrophic interactions

in general are often characterised by their specificity

(Mumm et al. 2005; Singer & Stireman 2005; Blüthgen,

Mezger & Linsenmair 2006; Heil 2008; Rasmann & Turlings

2008). However, high species specificity, whatever its origin,

necessitates suitable proximal mechanisms to ensure its

maintenance. Questions about the mechanisms contributing

to specificity between partners engaged in multitrophic

interactions, such as those that occur in phoresy, are conse-

quently fundamental to understanding evolutionary and

co-evolutionary processes (Thompson 2009).

In phoretic interactions which are often considered a

prelude to the development of parasitism (Anderson 1984),

passengers may use chemical (Soroker et al. 2003), visual

(Harbison, Jacobsen & Clayton 2009), heat and vibration

(Owen &Mullens 2004) cues from their vehicles, which aid in

their localization. The use of a particular sensory modality in

passenger–vehicle localization would depend on the nature of

the tritrophic system, since certain cues, e.g. organic chemical

compounds of low volatility and therefore short range of

action, would be inappropriate if the localization distance is

necessarily large. This ability to successfully find targets is

also linked to discrimination ability; studies of tritrophic

interactions indicate that discrimination ability depends

considerably on the degree of specialisation of the interaction

(Vet &Dicke 1992). In some phoretic interactions, passengers

lure their vehicles to themselves and thus use the discrimina-

tion ability of their vehicles instead (Saul-Gershenz & Millar

2006). While varied biochemical and sensory processes may

mediate tritrophic interactions (Dicke & Hilker 2003; Heil

2008), their specificity crucially depends on ecological

context, e.g. selection for increased discrimination of hosts in

sympatric communities compared to those in allopatry. The

success of a mechanism that guarantees specificity would thus

ensure the uniqueness of a response given the ecological

context and attendant evolutionary constraints (Thrall et al.

2007; Poullain et al. 2008).

The fig–fig wasp–nematode system is ideal to investigate

the evolution and maintenance of host specificity especially

the phenomenon of phoresy. This is because this 70–90

million year-old seed-destroying (wasp) and seed-producing

(fig) mutualism (Machado et al. 2001; Rønsted et al. 2008) is

highly species-specific with usually one pollinator species

(Hymenoptera: Agaonidae) and several species of non-polli-

nating parasitic fig wasps for each of the 700+ species of figs

(Moraceae) (Cook & Rasplus 2003; Herre, Jandér & Mach-

ado 2008). The fig system is also host to two genera of

parasitic nematodes [Schistonchus (Aphelenchoididae:

Aphelenchida) and Parasitodiplogaster (Diplogasteridae:

Rhabditida)] found so far exclusively in fig inflorescences

called syconia (Giblin-Davis et al. 2006; Gulcu et al. 2008;

Powers et al. 2009). The short-lived ephemeral fig syconium

(see Section on Natural History in Materials and methods)

within which the nematodes and wasps develop provides an

ideal situation for the development of a passenger–vehicle

relationship since the passenger must necessarily disperse to

find another suitable syconium. Nematodes have also been

found in fossil associations with fig wasps (15–45 mya),

indicating a long evolutionary association (Poinar 2003;

Peñalver, Engel & Grimaldi 2006). Parasitodiplogaster and

Schistonchus are phoretic on the pollinating fig wasp; how-

ever, Parasitodiplogaster is parasitic on its wasp host (Poinar

&Herre 1991; Herre 1993, 1996), while Schistonchus is a plant

parasite feeding onmale or female floral tissue within which it

can also induce tissue hypertrophy (Vovlas, Inserra & Greco

1992; Giblin-Davis et al. 1995; Center et al. 1999). Parasito-

diplogaster occurs in New World, African and Australian

figs (Bartholomaeus et al. 2009) while Schistonchus also

occurs in European and Australasian figs and its association

with figs is presumed to have a more ancient origin (Giblin-

Davis et al. 1995; Giblin-Davis et al. 2006). The tritrophic

interaction between fig, wasp, and nematode is thus a highly

specific one, and therefore lends itself to an examination of

questions regarding the processes that select for and maintain

such specificity. Furthermore, although plant-parasitic nema-

todes and phytophagous insects are extremely speciose, the

tritrophic interactions between nematodes, plants and insects

have scarcely been studied (Kaplan, Sardanelli & Denno

2009).

Female pollinating wasps are appropriate and reliable

phoretic vehicles for the nematodes since the males of polli-

nating and non-pollinating wasps are usually wingless and die

within their natal figs, and female non-pollinating fig wasps

do not enter a fig once they have exited their natal syconium

(see Section on Natural History inMaterials and methods for

details). The generalisation that non-pollinating wasps do not

enter the syconium is true for most fig species; however, there

are some exceptions (see Section onNatural History inMate-

rials and methods for details). Nematodes also do not usually

enter fig syconia via ovipositors of the externally-ovipositing

wasps since nematodes have been found only in one species of

non-pollinator parasitising a dioecious fig species (Vovlas &

Larizza 1996). Therefore, a fig nematode must typically seek

out only a female pollinator for a ride into the next fig

syconium. Conditions within the fig syconium are extremely

crowded; hundreds of galls containing male and female polli-

nating and non-pollinating wasps are very closely packed

together, and nematodes must therefore make decisions at

short range about the appropriate phoretic vehicle. Given

that there is also high species specificity between figs and their

nematodes (Poinar & Herre 1991; Gulcu et al. 2008), such

that each of the 700+ fig species is predicted to have a unique

species of Schistonchus nematode (Giblin-Davis et al. 1995;

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742 A. Krishnan et al.

107

Gulcu et al. 2008; Bartholomaeus et al. 2009), we hypothes-

ised that this specificity has led to each Schistonchus species

developing specific attraction to the only vehicle that can

reliably allow it to disperse between fig syconia, i.e. the female

pollinating wasp.

Since nematodes show chemotaxis to specific host chemi-

cals (Hong & Sommer 2006; O’Halloran, Fitzpatrick &

Burnell 2006; Zhao et al. 2007; Rasmann & Turlings 2008),

we investigated the role of whole insects, cuticular hydrocar-

bons, and volatiles of potential vehicles in attracting plant-

parasitic Schistonchus nematodes of Ficus racemosa. In this

first-ever study of the chemical ecology of fig nematodes, we

specifically (i) determined whether the nematodes used short-

range or long-range cues in locating potential vehicles, (ii)

determined whether they preferred pollinating over parasitic

wasps and if they preferred one sex over the other, and (iii)

identified which chemical classes, volatile vs. non-volatile

cues, were useful in determining preference differences among

the species and between the sexes.

Materials and methods

N A T U R A L H I ST OR Y O F T H E F I G– W A S P – N E M A T O D E

T R I T R O P H I C SY S T E M

In the fig brood-site pollination mutualism, the nursery is the fig

syconium (globular enclosed inflorescence) in which seeds are pro-

duced and pollinators breed. In typical monoecious figs, pollinating

foundress female wasps enter the syconium at the pollen-receptive

or B-phase, pollinate some female flowers, and also oviposit into

other flowers resulting in galls (Galil & Eisikowitch 1968). The

foundresses die shortly after pollination and oviposition, and their

offspring develop within the galled flowers during the interfloral or

C-phase. The non-pollinating wasps [Hymenoptera: Agaonidae

(paraphyletic)] do not enter the fig syconium but oviposit into the

syconium from the outside, using long ovipositors (Proffit et al.

2007; however, there are some exceptions; see Cook & Rasplus

2003; Herre, Jandér & Machado 2008). The parasites could be

flower gallers, parasitoids, cleptoparasites or inquilines (Cook &

Rasplus 2003; Ghara & Borges 2010) and have variable impacts on

the fig and fig wasp mutualism (Herre, Jandér & Machado 2008)

since they breed within the syconium at the expense of seeds and

pollinator progeny. Male wasps are usually wingless, emerge first,

and mate with freshly eclosed females. Female pollinators collect

pollen from freshly dehisced male flowers and leave the syconium

through an exit hole prepared by the cooperative efforts of pollina-

tor males who die within their natal syconium (D-phase or wasp

emergence ⁄ dispersal phase). The winged parasitic females usuallyexit the syconia at the same time as the female pollinators through

the exit hole prepared by male pollinators. The juvenile or dispersal

stage plant-parasitic nematodes of figs enter the dispersing female

pollinating wasps (in the D-phase syconium) and are carried in the

abdomen (‘abdominal folds’) or in the hemocoel into a receptive

stage fig syconium (B-phase) where the nematodes disembark from

the body of the foundress female wasp and feed on tissues of plant

origin particularly floral ones (Reddy & Rao 1984; Vovlas, Inserra

& Greco 1992; Giblin-Davis et al. 1995; Vovlas & Larizza 1996;

Vovlas et al. 1998). Thus nematodes need to disperse between

ephemeral syconia in order to continue their life cycles.

S T U D Y S I T E A N D S P E C I E S

Fig syconia of the monoecious Ficus racemosaL. in wasp-dispersal or

D-phase were collected in and around the Indian Institute of Science

campus in Bangalore, India (12�58¢N 77�35¢E). The syconia were cutopen to collect pollinators (Ceratosolen fuscicepsMayr) and non-pol-

linating fig wasps (Apocryptophagus testaceaMayr, Apocryptophagus

fusca Girault, Apocryptophagus agraensis Joseph, Apocrypta west-

woodi Grandi and Apocrypta sp. 2) which mostly constitute the fig

wasp community available in Ficus racemosa at this site (Proffit et al.

2007; M. Ghara, Y. Ranganathan and R.M. Borges, personal obser-

vations). Wasps were dissected in distilled water and examined under

a microscope for the presence of nematodes. Apocryptophagus

stratheni Joseph which also occurs in this community was too rare to

be thus examined. Nematodes were found only in female C. fusciceps

and were identified as Schistonchus racemosa Reddy & Rao. Nema-

todes were only observed in the lumen of the fig syconia, and were

often observed performing nictation behaviour; i.e. the dispersing

stages stand up on their ‘tails’ and engage in waving their bodies. This

is a typical behaviour exhibited by nematodes for transmission to a

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-

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

different species with pollinator and non-pollinator numbers in a

syconium ranging from zero to several hundred in a highly stochastic

manner (M. Ghara, A. Krishnan and R.M. Borges, unpublished

data). Nematodes were present only in those figs into which female

pollinating wasps had entered.

C H O I C E AS S AY S

Nematodes were collected from the lumen of opened D-phase fig

syconia. Assays were designed to score the behaviour of individual

nematodes, rather than sets of nematodes, since the numbers of nema-

todes within syconia were highly variable. Some syconia were devoid

of nematodes. Choice assays with individual nematodes were carried

out in six-well plates (12Æ5 cm · 7Æ5 cm, each well = 3Æ6 cm diame-ter). The number of nematodes used for each choice assay was

variable (24–80) based on availability; the actual numbers used are

given in the Results section. Each well was half-filled with 1Æ6%agarose in a buffer containing 1 mM CaCl2, 1 mM MgSO4, and

50 mM potassium phosphate (pH 6Æ0) (after Brenner 1974). Thesolidified agarose was layered with 2% BaSO4 suspension in the same

buffer. The BaSO4 layer formed a background on which nematodes

left a clear trail as they moved on the plate. Since fig wasp nematodes

are much smaller (�400 lm length,�13 lmwidth; Vovlas & Larizza1996) than the model nematode Caenorhabditis elegans (�1400 lmlength, �80 lm width; Mörck & Pilon 2006), their tracks could notbe directly visualised on agarose alone. Therefore BaSO4 was used, as

it is known to be an inert, non-toxic compound, was easily available,

and convenient to handle. All choice assays were conducted with

competing wasps or competing extracts rather than against solvent

controls, since the objective of this study was to determine nematode

choices in chemically crowded environments and thus to replicate

natural conditions as much as possible. All choice assays were

conducted in the dark to mimic natural conditions and to prevent

variation in ambient light from interfering with the experiments. The

translation of laboratory nematode assays to natural conditions is

vital (Spence, Lewis & Perry 2008).

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Nematode dispersal between fig syconia 743

108

D I S T A N C E R ES P O N SE AS S A Y

Male and female pollinator wasps were collected and freeze killed

at )20 �C overnight. Two wasps (one male and one female) wereplaced at different distances (10 mm, 5 mm or 3 mm) on oppo-

site sides of a central point in each well (Fig. 1a). In this and in

all subsequent two-choice assays, the positions of the wasps were

interchanged between trials to counter direction biases in nema-

todes. Single nematodes were picked up from figs using a single

bristle from a brush mounted on an insect pin, placed between

the wasps, and left in darkness for 2 h. At the end of this time,

the assay plates were inverted and either stored at 4 �C to immo-bilise the nematodes and scored later, or scored under a light

microscope immediately for nematode choices. The nematode was

deemed to have made a choice if trails led from the central point

to a particular wasp. Wells which contained no trails or in which

trails led to both wasps were classified as ‘no choice’. A distance

response curve was plotted and the appropriate distance for

further experiments was determined to be 3 mm (see Results).

W H O L E W A S P C H O I C E A SS A YS

Whole wasp choice assays were conducted by giving the nema-

todes choices between males and females of pollinator wasps

and females of pollinating (C. fusciceps) and non-pollinating

wasps (A. testacea and Apocrypta sp. 2). Apocrytophagus agraen-

sis and A. fusca were not available at the time of these experi-

ments and Apocrypta westwoodi was too rare to be used. Only

freshly eclosed wasps were collected by opening D-phase figs, as

they would be most suitable as phoretic vehicles at that time. In

all experiments, wasps were placed 3 mm from the central point

where the nematode was placed (Figs. 1a,b). The assay was

carried out in the same manner as described above.

W A S P V O LA T I L E C H O I C E A SS A YS

Wasps (male and female pollinators, and female non-pollinators

of A. testacea and Apocrypta sp. 2) collected from D-phase figs

were placed in )20 �C and freeze-killed 3 h before the assay,and were stored at )20 �C until the assay was performed. Twoslits were made in the agarose at a distance of about 2Æ5 mm oneither side of the central point (Fig. 1b) to prevent diffusion of

non-volatile cues from the wasps towards the nematodes. The

wasps were placed outside the slits, and the assay was carried

out as described earlier.

W A S P C U T I C U L AR H Y D R O C A R B ON C H O I C E AS S A YS

Cuticular hydrocarbon extracts of wasps (male and female pollina-

tors, and female non-pollinators of A. testacea and Apocrypta sp. 2)

collected from D-phase figs were made by adding 400 lL of pentaneto twenty live wasps of each species in a glass vial. The vials were

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 theywere used. Each vial containing the cuticular hydrocarbon extract

from twenty wasps was re-suspended in 50 lL of pentane, and usedfor five assays. Into each agar-filled well, 10 lL each of two differentextracts were added such that the extracts were separated by approxi-

mately 3 mm. The pentane was allowed to evaporate, and the spread

of the extracts were traced out with pin pricks on the surface of the

BaSO4 (Fig. 1c). A single nematode was placed at a central point

between the two extracts and left in darkness for 2 h, after which the

choice of the nematode was scored as above. In all cases, nematodes

were noted to have sampled both extracts before making their final

choice; the assay would have been unacceptable had this not held

true.

(a) (b)

(c) (d)

Fig. 1. Experimental set up for nematode

assays. Single wells of the six-well plate

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 hydro-

carbon choice assays between male pollina-

tor ⁄ female non-pollinators (X) and femalepollinators (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 mmdistance.

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744 A. Krishnan et al.

109

C A F E T E R I A C H OI C E A SS A Y S

In order to mimic nematode choice of a vehicle in the crowded

syconial environment, cafeteria assays were conducted. Each cafeteria

choice assay was carried out with eight freeze-killed wasps (one polli-

nator female, one pollinator male, two non-pollinator males and four

non-pollinator females) arranged in a ring around a central point in

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

each of A. testacea and A. fusca (both labelled fNP2). The male non-

pollinating wasps were picked randomly and belonged to Apocrypta

sp. 2, A. testacea or A. fusca. The random selection of non-pollinator

males was due to the fact that fig syconia can contain unpredictable

numbers of non-pollinator males with some syconia being devoid of

males; thus availability of fresh males was the limiting factor which

led to the adoption of this experimental design. The positions of the

wasps were varied between trials to counter direction biases. Each

wasp was placed 3 mm from the central point (Fig. 1d). The nema-

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 nematodewere scored.

D A T A A N A LY S I S

The data from all two-choice assays were analysed using chi-square

tests conducted with the software package STATISTICA (Tulsa, Okla-

homa, USA). All assays labelled ‘no choice’ were excluded from the

analyses. In most of the cafeteria choice assays, the nematode was

seen to have mademore than one choice; therefore a weighted scoring

system was used to analyse the data. For convenience, based on the

maximum number of choices made by a nematode within each assay

(recorded post hoc) and the least common multiple (LCM) of this

maximum number, each assay was given a total of twelve points,

which was divided equally among the different choices made by the

nematodes. For example, if in a well, nematode trails led to three

different wasps, then the twelve points were divided by three and a

score of four points was awarded to each wasp. The total number of

points scored by the female pollinators, male pollinators, male non-

pollinators and female non-pollinators was recorded and compared

(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

D I S T A N C E R ES P O N SE AS S A Y

The distance response assays carried out using male and

female pollinators indicated that the nematodes were able to

sense the presence of the wasps at a distance of 3 mm. When

placed at a distance of 5 mm (n = 12) or 10 mm (n = 12),

the nematodes were unable to make a choice and remained

stationary. At a distance of 3 mm from the wasps, 75%

(n = 20) of the nematodes made a choice andmoved towards

one of the wasps. All assays described in this study therefore

used this distance.

W H O L E W A S P C H O I C E A SS A YS : S PE C I E S A N D SE X

P R E F E R EN C E

On being given choices of whole wasps, a significantly larger

percentage of nematodes chose female pollinators over male

pollinators (v2 = 4Æ54, P = 0Æ033) or female non-pollinators(fNP1 Apocrypta sp. 2: v2 = 8Æ05, P = 0Æ004; fNP2 A. testa-cea: v2 = 6Æ76,P = 0Æ009; Fig. 2).

W A S P V O LA T I L E C H O I C E A SS A YS : S PE C I E S A N D SE X

P R E F E R EN C E

When nematodes were presented with only volatile cues, a

significantly larger percentage of nematodes chose female

pollinators over male pollinators (v2 = 4Æ41, P = 0Æ035),female non-pollinators of Apocrypta sp. 2 (fNP1: v2 = 6Æ37,P = 0Æ012) and A. testacea (fNP2: v2 = 6Æ53, P = 0Æ011)(Fig. 2).

W A S P C U T I C U L AR H Y D R O C A R B ON C H O I C E AS S A YS :

S P E C I E S A N D S E X PR E F E R E N C E

In cuticular hydrocarbon choice assays, a significantly larger

number of nematodes chose female pollinator extracts over

female non-pollinator extracts (fNP1 Apocrypta sp. 2:

v2 = 9Æ85, P = 0Æ002; fNP2 A. testacea: v2 = 10Æ70,P = 0Æ001; Fig. 2). In choice tests between male pollinatorand female pollinator cuticular hydrocarbons, there was no

difference between the number of times male pollinator

Fig. 2. Responses of nematodes to whole wasp, volatile and cuticular

hydrocarbon cues when given choices between female pollinators

(fP), male pollinators (mP), females of non-pollinatorApocrypta sp. 2

(fNP1) and females of non-pollinatorA. testacea (fNP2). The choices

(excluding ‘no choice’ responses) were analysed using chi-square tests.

n.s. = non-significant difference (P > 0Æ05); *P < 0Æ05; **P <0Æ01; n includes nematodes exhibiting no choice.

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Nematode dispersal between fig syconia 745

110

extracts were chosen over female pollinator extracts

(v2 = 0Æ0, P = 1Æ0, Fig. 3). However, a significantly largernumber of nematodes chose the male pollinator extracts over

female non-pollinator (Apocrypta sp. 2) extracts (v2 = 25Æ30,P = 0Æ0001, Fig. 2).

C A F E T E R I A C H OI C E A SS A Y S: S PE C I ES A N D S EX

P R E F E R EN C E

Most of the nematodes in the cafeteria choice assays made

more than one choice, and trails leading to several wasps were

observed in these assays. The observed scores were very

different from the expected scores (Fig. 3), with the highest

attraction score being obtained by the female pollinator,

followed by the male pollinator, male non-pollinator and

female non-pollinator. The observed scores were significantly

different from the expected scores (v2 = 826Æ3, P < 0Æ0001,d.f. = 3) and the nematodes preferred whole female pollina-

tors over other wasps.

Discussion

In the chemically complex and physically crowded environ-

ment of the syconium of F. racemosa, where there are a maxi-

mum of fourteen possible choices (seven females and seven

males belonging to pollinator and non-pollinator wasp taxa),

the nematode S. racemosa was particularly attracted to

female pollinating wasps over other tested taxa, via cues from

intact female bodies, or their volatiles and cuticular hydrocar-

bons. Nematodes could not distinguish, however, between

the cuticular hydrocarbons of male and female pollinators.

Yet, they were more attracted to the cuticular hydrocarbons

of the male pollinator compared to those of female non-poll-

inators. Nematodes were only responsive to chemical cues of

potential vehicles at short distances. Thus, passenger nema-

todes are able to use short-range chemical cues to find their

reliable vehicle, i.e. female pollinators, with which they have

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& deBono 2008). In tritrophic interac-

tions, entomopathogenic soil nematodes also respond to

belowground volatiles such as b-caryophyllene emitted byinfested roots (van Tol et al. 2001; Rasmann&Turlings 2008;

Degenhardt et al. 2009). Plant-parasitic nematodes also ori-

ent to specific phytohormones such as auxins (Curtis 2007).

Free-living nematodes, on the other hand, are more sensitive

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

nematodes enables them to distinguish between host plant

species (Zuckerman & Jansson 1984; Zhao, Schmitt & Hawes

2000) and host insects (Hong & Sommer 2006). Similarly, we

found that Schistonchus nematodes of F. racemosa were able

to distinguish between female pollinators and female

non-pollinators using cuticular hydrocarbons and volatiles.

Furthermore, they were able to distinguish between male and

female pollinators when whole wasps and volatile cues were

provided to them, but were unable to distinguish the pollina-

tor sexes based on cuticular hydrocarbons. This may be

because (1) cuticular hydrocarbons of male and female fig

wasps are similar since they serve as species recognition sig-

nals as occurs in some insects (Howard & Blomquist 2005;

Smadja & Butlin 2009), or (2) the cuticular hydrocarbons of

the sexes are different as also occurs in insects (Howard &

Blomquist 2005; Peterson et al. 2007; Van Homrigh et al.

2007), but the nematodes may lack sensitivity to these differ-

ences. The fact that nematodes chose cuticular hydrocarbons

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.

It is noteworthy that the percentage of non-responding nema-

todes was the least when whole wasps were offered compared

to choices made when volatiles or cuticular hydrocarbons

alone were used (Fig. 2). This might mean that whole wasps

provide a complete set of cues (volatiles and cuticular

hydrocarbons) including those that were not tested in these

experiments, or that nematodes need a hierarchy of cues for

complete response (Lewis, Grewal & Gaugler 1995). Only

future experiments will be able to distinguish between these

possibilities. The distance response assay demonstrated that

nematodes necessarily need close-range cues (at distances of

at least 3 mm) in order to respond within such a chemically

crowded environment. In the case of volatiles, these could be

long-chain carbon compounds (up to C29) which have low

volatility but can still be recovered in the volatile headspace

around insects (Schmitt et al. 2007).

Fig. 3. Observed and expected scores for female pollinators (fP),

male pollinators (mP), male non-pollinators (mNP) and female non-

pollinators (fNP) in the cafeteria choice assay. The observed and

expected distribution of scores were significantly different

(v2 = 826Æ3,P < 0Æ00001, n = 40, d.f. = 3).

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Functional Ecology, 24, 741–749

746 A. Krishnan et al.

111

There are many reasons why nematodes specialise on

pollinating fig wasps as vehicles. Within the pollinators,

nematodes should also use only females as vehicles since

pollinator males are wingless and usually die within the fig

(Weiblen 2002). While winged males occur in some species of

Old World non-pollinating wasps, whose females enter fig

syconia for oviposition (Herre, Jandér &Machado 2008), the

males themselves do not enter syconia, and hence would be

considered dead-ends as vehicles into new fig syconia. There-

fore, males of all species of fig wasps are inappropriate

vehicles and should be avoided by nematodes. Indeed, nema-

todes have not been reported in male fig wasps in any study

(Vovlas, Inserra & Greco 1992; Vovlas et al. 1998; Poinar &

Herre 1991; DeCrappeo &Giblin-Davis 2001).

There are many reasons why nematodes do not use non-

pollinating figwasps as vehicles. Firstly, from an evolutionary

perspective, since the mutualism between figs and pollinating

fig wasps is very old (70–90 million years), and nematodes are

also an ancient taxon (Poinar 2003), there has probably been

sufficient time for a specific tritrophic relationship between

figs, pollinators and nematodes to have evolved, as also

revealed by fossil nematodes in this system (Poinar 2003;

Peñalver, Engel & Grimaldi 2006). Indeed, nematodes seem

to be highly co-evolved with and species-specific to figs and

fig pollinators (Poinar & Herre 1993; Giblin-Davis et al.

2003; Gulcu et al. 2008; Bartholomaeus et al. 2009). Of the

numerous nematode species identified within the fig system,

whether of the entomopathogenic Parasitodiplogaster or of

the plant-parasitic Schistonchus, almost all have been

recorded to use only the fig pollinator as vehicle or host

(Kumari & Reddy 1984; Reddy & Rao 1984; Vovlas, Inserra

&Greco 1992; Vovlas et al. 1998; Herre 1993, 1996; Poinar &

Herre 1993; Lloyd & Davies 1997; DeCrappeo & Giblin-

Davis 2001; Zeng, Giblin-Davis & Ye 2007). There is to date

only one record, in the domesticated dioecious fig Ficus

carica, of the nematode Schistonchus caprifici also found in a

non-pollinating fig wasp Philotrypesis caricae (Vovlas &

Larizza 1996). The age of the relationship between figs and

parasitic fig wasps is, however, unknown. Since the species-

specificity of pollinators to the figs is higher than that of the

non-pollinators (Weiblen & Bush 2002; Marussich & Mach-

ado 2007; but see Jousselin et al. 2006, 2008), nematodes

should be selected to evolve or co-evolve with pollinators

rather than non-pollinator wasps, and to specifically adapt to

pollinators as phoretic vehicles. This should be especially true

for plant-parasitic nematodes such as Schistonchus since they

parasitise specific syconial tissues.

Secondly, an important constraint that selects for species-

specificity in this tritrophic interaction is that the timing of

the development of wasps (vehicles) and nematodes (passen-

gers) must coincide since the eclosed wasps and their phoretic

nematodesmust exit the fig syconium at the same time. There-

fore, development synchrony between vehicle and passenger

must also evolve, and this constitutes an important evolution-

ary necessity before phoresy can arise in a nematode system

(Giblin-Davis et al. 2003; Baldwin, Nadler & Adams 2004).

In the F. racemosa system presented in this paper, there is

considerable inter- and intraspecific variation in non-pollinat-

ing wasps with regard to the timing of their oviposition into

the syconiumduring its developmental cycle (Ghara&Borges

2010). This variation may prevent the evolution of precise

matching of development time between fig-specific nematodes

and non-pollinating wasps.While pollinator females live for a

short time (24–72 h) in several fig species examined (Kjell-

berg, Doumesche & Bronstein 1988; Dunn et al. 2008; [24 h

recorded in Ghara & Borges (2010)]), the non-pollinator

species have extended and variable life spans of up to 27 days

in some cases (Ghara & Borges 2010). The precise and short

life span of the pollinator female can also drive nematodes to

preferentially match their developmental time with that of the

pollinating female compared to other wasp species within the

syconia. Furthermore, the evolution of specific innate mecha-

nisms to recognise vehicles would preclude the need for learn-

ing (Vet et al. 1993; Huigens et al. 2009) which is certainly

selected against when the lifespans of vehicle and ⁄or passen-ger are short, as in this case.

Thirdly, nematodes should choose pollinator wasps over

non-pollinator wasps because in many fig species, as in our

study system, only the pollinating wasps enter the syconium

through the ostiole while the non-pollinating wasps oviposit

from the outside (Cook & Rasplus 2003; Herre, Jandér &

Machado 2008). This means that nematodes that use female

pollinators as vehicles can disembark into the next syco-

nium through the entire body of the pollinator. Nematodes

that enter a female non-pollinator could only enter the fig

through the narrow ovipositor. Moreover, in such non-

pollinating species, oviposition is often interrupted, being

disturbed by predatory ants and other external parasites

(Schatz et al. 2006; Ranganathan & Borges 2009). In the

few fig systems where non-pollinating wasps also enter the

syconium (Cook & Rasplus 2003; Herre, Jandér & Mach-

ado 2008), nematodes may be expected to also develop

passenger–vehicle relationships with them. While nematodes

have been found in the ovipositor of one parasitic wasp

within a dioecious fig species (Vovlas & Larizza 1996), they

have not been found in the parasitic wasps of our monoe-

cious fig system or elsewhere. Whether there is a difference

in nematode loads and nematode strategies between dioe-

cious and monoecious fig species is worth investigating,

because in dioecious figs, pollinators can breed only in male

fig trees and not in female fig trees (Cook & Rasplus 2003).

While pollinating fig wasps that enter female figs will die

without progeny (Cook & Rasplus 2003), nematodes that

are transported into such figs by female pollinators are

doomed to reproduce without any chance of dispersal.

Thus, it is intriguing to hypothesise that a nematode within

a male fig syconium should hedge its bets and attempt to

even enter non-pollinating fig wasps if by this strategy the

chances of getting dispersed to another male fig are

enhanced. If future studies reveal that plant-parasitic nema-

todes are found in parasitic non-pollinating wasps in other

fig systems, including monoecious species, we also predict

that they will only be found in those systems where there is

high species-specificity between parasitic wasps and figs.

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Functional Ecology, 24, 741–749

Nematode dispersal between fig syconia 747

112

Thus this study, which is the first on the chemical ecology

of nematodes in the mutualism between figs and fig wasps,

illustrates how nematodes choose their appropriate ride to

another fig syconium within their chemically complex and

physically crowded environment. The study also provides

new testable hypotheses in this very exciting area of species

co-evolution and species-specific interactions within a

tritrophic framework.

Acknowledgements

This research was funded by theMinistry of Environment and Forests, Govern-

ment of India. We thank R. Yettiraj for fig collection, C.M. Brijesh for the

BaSO4 idea, D. Dey for the six-well plates, V.V. Ramamurthy and S. Ganguly

of the Indian Agricultural Research Institute (IARI), New Delhi, for help with

nematode identification, Pablo Castillo for assistance with the nematode litera-

ture, Judie Bronstein, Allen Herre, Finn Kjellberg, Katy Prudic and Rob

Raguso for comments on the manuscript, as well as Mahua Ghara and

Yuvaraj Ranganathan for enthusiastic support.

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Received 26August 2009; accepted 5 December 2009

Handling Editor: Scott Carroll

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Functional Ecology, 24, 741–749

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