Ahitchhiker’sguidetoacrowdedsyconium:howdofig...
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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: [email protected]
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Functional Ecology 2010, 24, 741–749 doi: 10.1111/j.1365-2435.2010.01696.x
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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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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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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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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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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
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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
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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.
References
Anderson, R.C. (1984) The origins of zooparasitic nematodes. Canadian Jour-
nal of Zoology, 62, 317–328.
Baldwin, J.G., Nadler, S.A. &Adams, B.J. (2004) Evolution of plant parasitism
among nematodes.Annual Review of Phytopathology, 42, 83–105.
Bartholomaeus, F., Davies, K., Ye, W., Kanzaki, N. & Giblin-Davis, R.M.
(2009) Schistonchus virens sp. n. (Aphelenchoididae) and Parasitodiplogaster
australis sp. n. (Diplogastridae) from Ficus virens (Moraceae) in Australia.
Nematology, 11, 583–601.
Binns, E.S. (1982) Phoresy as migration – some functional aspects of phoresy in
mites.Biological Reviews, 57, 571–620.
Blüthgen, N., Mezger, D. & Linsenmair, K.E. (2006) Ant–hemipteran tropho-
bioses in a Bornean rainforest – diversity, specificity and monopolisation.
Insectes Sociaux, 53, 194–203.
Brenner, S. (1974) The genetics ofCaenorhabditis elegans.Genetics, 77, 71–94.
Bretscher, A.J., Busch, K.E. & de Bono,M. (2008) A carbon dioxide avoidance
behavior is integrated with responses to ambient oxygen and food inCaenor-
habditis elegans. Proceedings of the National Academy of Sciences USA, 105,
8044–8049.
Center, B.J., Giblin-Davis, R.M., Herre, E.A. & Chung-Schickler, G.C. (1999)
Histological comparisons of parasitism by Schistonchus spp. (Nemata:
Aphelenchoididae) in Neotropical Ficus spp. Journal of Nematology, 31,
393–406.
Colwell, R.K. (1979) The geographical ecology of hummingbird flower mites in
relation to their host plants and carriers. Recent Advances in Acarology, 2,
461–468.
Colwell, R.K. (1985) Stowaways on the Hummingbird Express. Natural
History, 94, 56–63.
Colwell, R.K. (1986) Community ecology and sexual selection: lessons from
hummingbird flower mites. Ecological Communities (eds T.J. Case & J.
Diamond), pp. 406–424. Harper &Row,NewYork.
Cook, J.M. & Rasplus, J.-Y. (2003) Mutualists with attitude: coevolving fig
wasps and figs.Trends in Ecology and Evolution, 18, 241–248.
Croll, N.A. &Mathews, B.E. (1977)Biology of Nematodes. JohnWiley & Sons,
NewYork.
Curtis, R.H.C. (2007) Do phytohormones influence nematode invasion and
feeding site establishment?Nematology, 9, 155–160.
DeCrappeo, N. & Giblin-Davis, R.M. (2001) Schistonchus aureus n. sp. and S.
laevigatus n. sp. (Aphelenchoididae): associates of native Floridian Ficus
spp. and their Pegoscapus pollinators (Agaonidae). Journal of Nematology,
33, 91–103.
Degenhardt, J., Hiltpold, I., Köllner, T.G., Frey,M., Gierl, A., Gershenzon, J.,
Hibbard, B.E., Ellersieck, M.R. & Turlings, T.C.J. (2009) Restoring a
maize root signal that attracts insect-killing nematodes to control a
major pest. Proceedings of the National Academy of Sciences USA, 106,
13213–13218.
Dicke, M. &Hilker, M. (2003) Induced plant defences: frommolecular biology
to evolutionary ecology.Basic and Applied Ecology, 4, 3–14.
Dunn, D.W., Yu, D.W., Ridley, J. & Cook, J.M. (2008) Longevity, early
emergence and body size in a pollinating fig wasp – implications for
stability in a fig–pollinator mutualism. Journal of Animal Ecology, 77,
927–935.
Farish, D.J. &Axtell, R.C. (1971) Phoresy redefined and examined inMacroch-
eles muscaedomesticae (Acarina:Macrochelidae).Acarologia, 13, 16–29.
Galil, J. & Eisikowitch, D. (1968) Flowering cycles and fruit types of Ficus
sycomorus in Israel.New Phytologist, 67, 745–758.
Garcı́a-Franco, J.G., Martı́nez, B.D. & Pérez, T.M. (2001) Hummingbird
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
DOI: 10.1111/j.1365-2311.2010.01176.x.
Giblin-Davis, R.M., Ye,W., Kanzaki, N., Williams, D.,Morris, K. & Thomas,
W.K. (2006) Stomatal ultrastructure, molecular phylogeny, and description
of Parasitodiplogaster laevigata n. sp. (Nematoda: Diplogastridae), a para-
site of fig wasps. Journal of Nematology, 38, 137–149.
Giblin-Davis, R.M., Center, B.J., Nadel, H., Frank, J.H. & Ramı́rez, B.W.
(1995) Nematodes associated with fig wasps, Pegoscapus spp. (Agaonidae),
and syconia of native Floridian figs (Ficus spp.). Journal of Nematology, 27,
1–14.
Giblin-Davis, R.M., Davies, K.A., Morris, K. & Thomas, W.K. (2003) Evolu-
tion of parasitism in insect-transmitted plant nematodes. Journal of Nema-
tology, 35, 133–141.
Gulcu, B., Hazir, S., Giblin-Davis, R.M., Ye, W., Kanzaki, N., Mergen, H.,
Keskin, N. & Thomas, W.K. (2008) Molecular variability of Schistonchus
caprifici (Nematoda: Aphelenchoididae) from Ficus carica in Turkey.Nema-
tology, 10, 639–649.
Harbison, C.W., Jacobsen, M.V. & Clayton, D.H. (2009) A hitchhiker’s guide
to parasite transmission: the phoretic behaviour of feather lice. International
Journal of Parasitology, 39, 569–575.
Heil, M. (2008) Indirect defence via tritrophic interactions. New Phytologist,
178, 41–61.
Herre, E.A. (1993) Population structure and the evolution of virulence in nema-
tode parasites of fig wasps. Science, 259, 1442–1445.
Herre, E.A. (1996) Factors affecting the evolution of virulence: nematode para-
sites of fig wasps as a case study.Parasitology, 111, S179–S191.
Herre, E.A., Jandér, K.C. & Machado, C.A. (2008) Evolutionary ecology of
figs and their associates: recent progress and outstanding puzzles. Annual
Review of Ecology, Evolution and Systematics, 21, 439–458.
Hong, R.L. & Sommer, R.J. (2006) Chemoattraction in Pristionchus
nematodes and implications for insect recognition. Current Biology, 16,
2359–2365.
Houck, M.A. & OConnor, B.M. (1991) Ecological and evolutionary signifi-
cance of phoresy in the Astigmata. Annual Review of Entomology, 36, 611–
636.
Howard, R.W. & Blomquist, G.J. (2005) Ecological, behavioral, and biochemi-
cal aspects of insect hydrocarbons. Annual Review of Entomology, 50,
371–393.
Huigens, M.A., Pashalidou, F.G., Qian, M.-H., Bukovinszky, T., Smid, H.M.,
van Loon, J.J.A., Dicke, M. & Fatouros, N.E. (2009) Hitch-hiking parasitic
wasp learns to exploit butterfly aphrodisiac. Proceedings of the National
Academy of Sciences USA, 106, 820–825.
Jousselin, E., van Noort, S., Rasplus, J.-Y. & Greeff, J.M. (2006) Patterns of
diversification of Afrotropical Otiteselline fig wasps: phylogenetic study
reveals a double radiation across host figs and conservatism of host associa-
tion. Journal of Evolutionary Biology, 19, 253–266.
Jousselin, E., van Noort, S., Berry, V., Rasplus, J.-Y., Rønsted, N., Erasmus, J.
C. & Greeff, J.M. (2008) One fig to bind them all: host conservatism in a fig
wasp community unraveled by cospeciation analyses among pollinating and
nonpollinating figwasps.Evolution, 62, 1777–1797.
Kaplan, I., Sardanelli, S. &Denno, R.F. (2009) Field evidence for indirect inter-
actions between foliar-feeding insect and root-feeding nematode communi-
ties onNicotiana tabacum.Ecological Entomology, 34, 262–270.
Kjellberg, F., Doumesche, B. & Bronstein, J. (1988) Longevity of a fig wasp
(Blastophaga psenes). Proceedings der Koninklijke Akademie van Wetensc-
happen, 91, 117–122.
Kruitbos, L.M., Heritage, S. & Wilson, M.J. (2009) Phoretic dispersal of
entomopathogenic nematodes by Hylobius abietis. Nematology, 11, 419–
427.
Kumari, R.V. & Reddy, Y.N. (1984) Studies on the association of a new nema-
tode species Schistonchus hispida sp. n. (Aphelenchoidea Nickle, 1971) and
wasp.Proceedings of the Indian Academy of Parasitology, 5, 21–25.
� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Functional Ecology, 24, 741–749
748 A. Krishnan et al.
113
-
Lewis, E.E., Grewal, P.S. & Gaugler, R. (1995) Hierarchical order of host cues
in parasite foraging strategies: a question of context. Parasitology, 110, 207–
213.
Lloyd, J. & Davies, K.A. (1997) Two new species of Schistonchus (Tylenchida:
Aphelenchoididae) associated with Ficus macrophylla from Australia.
Fundamental and AppliedNematology, 20, 79–86.
Lopez, L.C.S., Filizola, B., Deiss, I. & Rios, R.I. (2005) Phoretic behaviour of
bromeliad annelids (Dero) and ostracods (Elpidium) using frogs and lizards
as dispersal agents.Hydrobiologia, 549, 15–22.
Machado, C.A., Jousselin, E., Kjellberg, F., Compton, S.G. & Herre, E.A.
(2001) Phylogenetic relationships, historical biogeography, and character
evolution of fig-pollinating wasps. Proceedings of the Royal Society London
Series B, 268, 685–694.
Marussich, W.A. & Machado, C.A. (2007) Host-specificity and coevolution
among pollinating and nonpollinating New World fig wasps. Molecular
Ecology, 16, 1925–1946.
Mörck, C. & Pilon,M. (2006)C. elegans feeding defective mutants have shorter
body lengths and increased autophagy. BMCDevelopmental Biology, 6, 39–
50.
Mumm, R., Tiemann, T., Varama, M. & Hilker, M. (2005) Choosy egg parasi-
toids: Specificity of oviposition-induced pine volatiles exploited by an egg
parasitoid of pine sawflies. Entomologia Experimentalis et Applicata, 115,
217–225.
O’Halloran, D.M. & Burnell, A.M. (2003) An investigation of chemotaxis in
the insect parasitic nematode Heterorhabditis bacteriophora. Parasitology,
127, 375–385.
O’Halloran, D.M., Fitzpatrick, D.A. & Burnell, A.M. (2006) The chemosenso-
ry systemofCaenorhabditis elegans and other nematodes.Chemical Ecology:
From Gene to Ecosystem (eds M. Dicke & W. Takken), pp.71–88. Springer,
Netherlands.
Owen, J.P. & Mullens, B.A. (2004) The influence of heat and vibration on the
movement of the northern fowl mite (Acari: Macronyssidae). Journal of
Medical Entomology, 41, 865–872.
Peñalver, E., Engel, M.S. & Grimaldi, D.A. (2006) Fig wasps in Dominican
amber.AmericanMuseumNovitates, 3541, 1–16.
Peterson,M.A., Dobler, S., Larson, E.L., Juárez, D., Schlarbaum, T., Monsen,
K.J. & Francke, W. (2007) Profiles of cuticular hydrocarbons mediate male
mate choice and sexual isolation between hybridising Chrysochus (Coleop-
tera: Chrysomelidae).Chemoecology, 17, 87–97.
Pline, M. & Dusenbery, D.B. (1987) Responses of plant-parasitic nematode
Meloidogyne incognita to carbon dioxide determined by video camera-com-
puter tracking. Journal of Chemical Ecology, 13, 873–888.
Poinar, G. Jr, (2003) Trends in the evolution of insect parasitism by nematodes
as inferred from fossil evidence. Journal of Nematology, 35, 129–132.
Poinar, G.O. Jr & Herre, E.A. (1991) Speciation and adaptive radiation in the
fig wasp nematode, Parasitodiplogaster (Diplogasteridae: Rhabditida) in
Panama.Revue Nématologie, 14, 361–374.
Poullain, V., Gandon, S., Brockhurst, M.A., Buckling, A. & Hochberg, M.E.
(2008) The evolution of specificity in evolving and coevolving antagonistic
interactions between a bacteria and its phage.Evolution, 62, 1–11.
Powers, T.O., Neher, D.A., Mullin, P., Esquivel, A., Giblin-Davis, R.M.,
Kanzaki, N., Stock, S.P., Mora, M.M. & Uribe-Lorio, L. (2009) Tropical
nematode diversity: vertical stratification of nematode communities in a
CostaRican humid lowland forest.Molecular Ecology, 18, 985–996.
Proffit, M., Schatz, B., Borges, R.M. & Hossaert-Mckey, M. (2007) Chemical
mediation and niche partitioning in non-pollinating fig-wasp communities.
Journal of Animal Ecology, 76, 296–303.
Ranganathan, Y. & Borges, R.M. (2009) Predatory and trophobiont-tending
ants respond differently to fig and fig wasp volatiles. Animal Behaviour, 77,
1539–1545.
Rasmann, S. & Turlings, T.C.J. (2008) First insights into specificity of below-
ground tritrophic interactions.Oikos, 117, 362–369.
Reddy, N.Y. & Rao, P.N. (1984) Schistonchus racemosa sp. N., a nematode
parasite of wasp (Ceratosolen sp.) associated with the fig, Ficus racemosa L.
Indian Journal of Nematology, 16, 135–137.
Rønsted, N., Weiblen, G.D., Clement, W.L., Zerega, N.J. & Savolainen, V.
(2008) Reconstructing the phylogeny of figs (Ficus, Moraceae) to reveal the
history of the fig pollinationmutualism. Symbiosis, 45, 45–56.
Saul-Gershenz, L.S. & Millar, J.G. (2006) Phoretic nest parasites use sexual
deception to obtain transport to their host’s nests. Proceedings of the
National Academy of Sciences USA, 103, 14039–14044.
Schatz, B., Proffit, M., Rakhi, B.V., Borges, R.M. & Hossaert-McKey, M.
(2006) Complex interactions on fig trees: ants capturing parasitic wasps as
indirect mutualists of the fig–figwasp interaction.Oikos, 113, 344–352.
Schmitt, T., Herzner, G., Weckerle, B., Schreier, P. & Strohm, E. (2007) Vola-
tiles of foraging honeybees Apis mellifera (Hymenoptera: Apidae) and their
potential role as semiochemicals.Apidologie, 38, 164–170.
Singer, M.S. & Stireman, J.O. III (2005) The tri-trophic niche concept
and adaptive radiation of phytophagous insects. Ecology Letters, 8,
1247–1255.
Smadja, C. & Butlin, R.K. (2009) On the scent of speciation: the chemosensory
system and its role in premating isolation.Heredity, 102, 77–97.
Soroker, V., Nelson, D.R., Bahar, O., Reneh, S., Yablonski, S. & Palev-
sky, E. (2003) Whitefly wax as a cue for phoresy in the broad mite,
Polyphagotarsonemus latus (Acari: Tarsonemidae). Chemoecology, 13,
163–166.
Spence, K.O., Lewis, E.E. & Perry, R.N. (2008) Host-finding and invasion by
entomopathogenic and plant-parasitic nematodes: evaluating the ability of
laboratory bioassays to predict field results. Journal of Nematology, 40, 93–
98.
Stamps, W.T. & Linit, M.J. (2001) Interaction of intrinsic and extrinsic chemi-
cal cues in the behaviour of Bursaphelenchus xylophilus (Aphelenchida:
Aphelenchoididae) in relation to its beetle vectors.Nematology, 3, 295–301.
Thompson, J.N. (2009) The coevolving web of life. American Naturalist, 173,
125–140.
Thrall, P.H., Hochberg, M.E., Burdon, J.J. & Bever, J.D. (2007) Coevolution
of symbiotic mutualists and parasites in a community context. Trends in
Ecology and Evolution, 22, 120–126.
Tschapka, M. & Cunningham, S.A. (2004) Flower mites of Calyptrogyne
ghiesbreghtiana (Arecaceae): evidence for dispersal using pollinating bats.
Biotropica, 36, 377–381.
van Tol, R.W.H.M., van der Sommen, A.T.C., Boff, M.I.C., van Bezhooijen,
J., Sabelis, M.W. & Smits, P.H. (2001) Plants protect their roots by alerting
the enemies of grubs.Ecology Letters, 4, 292–294.
Van Homrigh, A., Higgie, M., McGuigan, K. & Blows, M.W. (2007)
The depletion of genetic variance by sexual selection. Current Biology,
17, 528–532.
Vet, L.E.M.&Dicke, D. (1992) Ecology of infochemical use by natural enemies
in a tritrophic context.Annual Review of Entomology, 37, 141–172.
Vet, L.E.M., Sokolowski, M.B., MacDonald, D.E. & Snellen, H. (1993)
Responses of a generalist and specialist parasitoid (Hymenoptera: Eucoili-
dae) to drosophilid larval kairomones. Journal of Insect Behavior, 6, 615–
624.
Vovlas, N., Inserra, R.N. &Greco, N. (1992) Schistonchus caprifici parasitizing
caprifig (Ficus carica Sylvestris) florets and relationships with its fig wasp
(Blastophaga psenes) vector.Nematologica, 38, 215–226.
Vovlas, N. & Larizza, A. (1996) Relationship of Schistonchus caprifici (Aphe-
lenchoididae) with fig inflorescences, the fig pollinator Blastophaga psenes,
and its cleptoparasite Philotrypesis caricae. Fundamental and Applied Nema-
tology, 19, 443–448.
Vovlas, N., Troccoli, A., VanNoort, S. & Van den Berg, E. (1998) Schistonchus
africanus n. sp. (Aphelenchida: Aphelenchoididae) associated with Ficus
thonningii (Moraceae) and its pollinator wasp Elisabethiella stuckenbergi
(Chalcidoidea: Agaonidae). Journal of Nematology, 30, 404–410.
Weiblen, G.D. (2002) How to be a fig wasp. Annual Review of Entomology, 47,
299–330.
Weiblen, G.D. & Bush, G.L. (2002) Speciation in fig pollinators and parasites.
Molecular Ecology, 11, 573–1578.
Zeh, D.W. & Zeh, J.A. (1992) Failed predation or transportation? Causes and
consequences of phoretic behavior in the pseudoscorpion Dinocheirus
arizonensis (Pseudoscorpionida: Chernetidae) Journal of Insect Behavior, 5,
37–49.
Zeng, Y., Giblin-Davis, R.M.&Ye,W. (2007) Two new species of Schistonchus
(Nematoda: Aphelenchoididae) associatedwith Ficus hispida in China.Nem-
atology, 9, 169–187.
Zhao, X., Schmitt, M. & Hawes, M.C. (2000) Species-dependent effects of bor-
der cell and root tip exudates on nematode behavior. Phytopathology, 90,
1239–1245.
Zhao, L.L., Wei, W., Kang, L. & Sun, J.H. (2007) Chemotaxis of the pinewood
nematode, Bursaphelenchus xylophilus, to volatiles associated with host pine,
Pinus massoniana, and its vectorMonochamus alternatus. Journal of Chemi-
cal Ecology, 33, 1207–1216.
Zuckerman, B.M. & Jansson, H.-B. (1984) Nematode chemotaxis and possible
mechanisms of host ⁄ prey recognition.Annual Review of Phytopathology, 22,95–113.
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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