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Dear Editor,
We carefully revised the MS “Hard to choose for tiny pests: host-seeking behaviour in Xenos
vesparum triungulins” following the suggestions of both referees:
8Introduction and Discussion have been shortened by omitting not relevant data on Xenos
vesparum cycle;
8we added more information on host-seeking behaviour in other parasitoids;
8we clarified our experimental set-up by adding details on our apparatus and choice trials
(see Methods);
8we omitted Carbon Dioxide trials – even if the role of CO2 in host location may be relevant -
because preliminary: limited sample size, no data about CO2 levels on the wasp comb,
experimental CO2 emission (under 2 mL/min flow) probably too low;
8the Discussion has been re-organized according to the different topics considered (positive
and negative evidences for host-location; the role of stimuli used by Xenos triungulins to
escape from the cephalothorax; gregariousness);
8the Table has been converted to a Figure (see Fig.2).
Due to the preliminary nature of some choice trials here reported, results have been discussed with
caution; therefore, we think that a Short Communication Format could be more appropriate for this
paper.
With best regards
Fabio Manfredini
HARD TO CHOOSE FOR TINY PESTS:
HOST-SEEKING BEHAVIOUR IN XENOS VESPARUM TRIUNGULINS
Fabio Manfredini*Y, Alessandro Massolo# and Laura Beani#
*Dipartimento di Biologia Evolutiva, Università di Siena, Via Aldo Moro 2, 53100 Siena, Italy
#Dipartimento di Biologia Evoluzionistica, Università di Firenze, Via Romana 17, 50125 Firenze,
Italy
ABSTRACT
The 1st instar larvae of strepsipteran parasites, commonly referred as “triungulins”, are the host-
seeking stage: they must locate, invade and successfully develop in the new host, in order to start
their parasitic cycle. A few information are available about the behaviour of Xenos vesparum
triungulins. They emerge in batches from the endoparasitic female infecting Polistes dominulus, the
primary host, and reach the nest through a vector (a foraging wasp or the parasitized wasp itself).
Once there, they have the possibility to penetrate into wasp immatures at different developmental
stages. In this study, we performed preliminary analyses aimed to investigate which cues are
important to direct triungulins movements during their brief stay on wasp nests. In laboratory
conditions we selectively presented different stimuli to Xenos larvae: apparently, the host larva
itself is attractive in an open arena, but not inside a confined space, neither epicuticular compounds
of wasp larvae are able to control triungulins movements. These are more likely oriented by their
gregarious behaviour, whereas light (positive phototaxy) could previously enhance their emergence
via the brood canal opening in the female cephalothorax.
KEY WORDS: gregariousness, host-seeking behaviour, triungulin, infective larva, Strepsiptera,
koinobiont parasitoid.
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INTRODUCTION
Parasites, being a significant portion of total biomass in natural ecosystems and mainly in insect
societies (Hughes et al., 2008), have a great eco-ethological significance. In particular, manipulative
parasites are able to redirect host relationships and to extend the range of its habitat (Lefevre et al.
2009). Xenos vesparum is a castrator “manipulative” parasite (Hughes 2005), which has a great
impact on the small annual society of the paper wasp Polistes dominulus, its primary host (Beani
2006; Hughes 2005; Hughes et al. 2004b). In this host-parasite system, the association between the
two insects is long-lasting and its success is based on the parasite tuning with host development. X.
vesparum penetrates into a P. dominulus immature as 1st instar larva and coexists with it for weeks
or months, depending on the sex of both parasite and host. The intimate association with its host
forces the parasite to adapt to a changing environment: this life style is typical of insect parasitoids,
a large group of insects, primarily in the orders of Hymenoptera and Diptera, whose larvae develop
by feeding in / on the bodies of other arthropods (Brodeur & Boivin 2004; Strand & Pech 1995).
Thus, X. vesparum could be reasonably linked to koinobionts parasitoids, where parasite
development is finely tuned to the development of its holometabolous host, since it manages to
achieve growth and metamorphosis processes (Tanaka 2006; Vinson & Iwantsch 1980).
Together with adult males, 1st instar larvae are the sole free-living developmental stages in
X. vesparum life cycle. They are often called “triungulins” (i.e. three-clawed) for their
morphological similarity to coleopteran campodeiform larvae (Kathirithamby 1989; Kathirithamby
2009; Pohl 1998), although in this species the first two pairs of legs are equipped with disc-like
pulvilli, not with claws, and the last with spines (Fig. 1). They represent the host-seeking stage: they
are extremely motile and resistant to environmental conditions (they may survive for 48 hours in
Petri dishes without food, see Giusti et al.2007, and independently on the humidity conditions, pers.
obs.), although relative humidity has been referred as a critical longevity factor in other species
(Hassan 1939, quoted in Kathirithamby 2009). Moreover, they are particularly kin in penetrating
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into the host and establishing inside without any dramatic consequences neither for themselves nor
for the host (Manfredini et al. 2007a).
Generally, parasitoid infective larvae are the most aggressive among immatures, capable of
ensuring the parasitic success of the species by installing themselves fruitfully on the host. For this
purpose, they are usually very mobile and easily exposed to physical fights with rival conspecifics
(Doury et al. 1995; Giron et al. 2004); moreover, they often have a direct effect on host physiology
through their bite, whereby they inject salivary glands secretions into the host, thus manipulating
both cellular and humoral components of its hemolymph (Doury et al. 1997; Richards & Edwards
2002). Strepsipteran triungulins possess all the morphological features of a successful infective
organism (Fig. 1): antennae, eyes, mouthparts, slender legs, tarsal expansions and long caudal
appendages which allow jumping movements. Almost no information are available about their host-
seeking behaviour and very few is known on the invasion process (Kathirithamby 2001;
Kathirithamby et al. 2003; Maeta et al. 2001). For Xenos triungulins this starts once they have
reached a wasp nest, either by grasping to a foraging wasp (phoretic transport) or by means of a
stylopized wasp which directly visits a nest (Hughes et al. 2003; Vannini et al. 2008). The
“wandering” behaviour among spring colonies, recently described (Beani & Massolo, 2007) in
wasps infected by X. vesparum females, i.e. the source of triungulins, could be interpreted as a
manipulative extension of the usual wasp range by the parasite (Lefevre et al. 2009). In the field the
parasite load ranges from 1 to 4.1 per adult wasp (Vannini et al. 2008) and thus it is presumably
higher at infection. After the emergence of the winged male and the extrusion of the cephalothorax
of the female, a neotenic permanent endoparasite, mating occurs (Beani et al. 2005). Fertilized eggs
start the first steps of segmentation process, but then stop and remain quiescent during winter
diapause (pers. obs.). The following spring, embryogenesis will terminate to give birth to hundreds
of 1st instar larvae, ready for another season of infections and usually released in groups of 10, 20 or
more (pers. obs.). Thus, Xenos females may over-winter inside its host, a hibernating female wasp,
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whereas Xenos males die in summer, as well as their hosts, regardless of their putative caste (Beani
2006).
For many reasons (not just morphology), Xenos triungulins are quite analogous to host-
seeking stages of Dipteran and Coleopteran koinobiont endoparasitoids (Castelo & Lazzari 2004;
Crespo & Castelo 2008; Royer et al. 1999). According to Brodeur and Boivin (2004), in parasitoids
with active larvae the host seeking stage undergoes a chain of “hierarchical steps” to achieve
successful parasitism: it must locate, evaluate and penetrate the appropriate host, evade or overcome
the host immune response and adapt to (or regulate) the constantly changing host environment. Host
seeking larvae promptly respond to different kind of cues, generally divided in three main
categories (Godfray 1994): signals from host microhabitat, which are easy to detect at a distance but
give little reliable information; stimuli indirectly associated with the activity of the host (for
example phonotaxy, long-distance kairomones, CO2 emission, etc.); stimuli from the host itself, i.e.
visual and chemical cues, that are the most reliable ones, often too weak for detection over long
distances, due to the strong selection pressure on hosts to avoid being recognized (for a review on
parasitoids exploiting chemical information, see Fatouros et al. 2008).
In this pioneering study we investigated which short-range cues are important to orient X.
vesparum triungulins during host location on the comb: are P. dominulus larvae per se attractive for
triungulins? In a series of repeated independent experiments in laboratory, we tried to recreate semi-
natural choice trials, focusing on the arrival of triungulins on the nest. We registered the movements
of batches of host-seeking triungulins on a neutral substrate in presence of 3rd- 4th instar wasp
larvae, which were singly placed in the middle of an open arena, and successively inside a confined
and screened space, in order to distinguish chemical from visual signals. In further trials, we
investigated the effect of single stimuli, i.e. epicuticular compounds from wasp larvae vs fly larvae
used as a control. Only in case of a clear-cut effect of wasp stimuli in orienting the movements of
triungulins, we could apply the same technique to assess host selection, as concerns sex,
developmental stage and superparasitism. Although preliminary, this study on Strepsiptera
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represents a good model to analyse host-seeking behaviour among immatures parasitoids, due to
scanty data in literature about the ecology of active 1st instar larvae (Feener & Brown 1997).
METHODS
Study animals
Hibernating clusters of Polistes dominulus Christ were collected in San Gimignanello
(Siena, Italy) at the end of the winter. They were carefully screened to exclude parasitic infections
and placed in groups of 3 individuals inside 20 x 20 x 20 cm Plexiglas cages with sugar, water and
Sarcophaga sp. larvae ad libitum, to allow colonies foundation. Under fixed conditions of 15L/9D
and 28±2°C, Polistes wasps started building first nest cells around 3 weeks after collection. These
animals were the starting pool for laboratory experiments, although additional colonies were
collected during the summer directly from the field. In this case, nests underwent a period of
quarantine to exclude any possibility of natural infection by Strepsiptera.
Our sources of triungulins were overwintered wasps coming from the same hibernating
clusters as above and parasitized by a single (rarely 2) female of Xenos vesparum Rossi. These
females, extruding their cephalothorax through wasp abdomen, released batches of triungulins after
4 weeks at 15L/9D and 28°C. Experiments were carried out in June and July 2008, i.e. the peak of
infection in wasp colonies (Hughes et al. 2003).
Choice trials in laboratory
For preliminary experiments of host location, we selected from different wasp colonies 3rd or
4th instar larvae, i.e. the commonest stages, easier to manipulate than 1st – 2nd instars and not close to
pupation as 5th instar. These developmental stages are easily recognizable for their relatively large
size and not yet melanized (light brown) mouth apparatus. We carefully removed each wasp larva
from its cell with forceps and placed it in the middle of a circular open arena (10 cm wide Petri
dish, covered with filter paper which was changed at each trial). Then, with a thin needle, we
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collected batches of triungulins escaping from one Xenos cephalothorax (i.e. siblings) and we
spreaded them on the paper (around 10 individuals per arena trial). Finally, we recorded their
movements during first 20 minutes of activity with a video camera connected to a
stereomicroscope. We excluded the nest as trial substrate to avoid a possible confounding source of
chemical signals (for a review on wasp cuticular hydrocarbons, already present on the comb, see
Gamboa 2004) and to better follow the movements of triungulins, able of walking and jumping on
filter paper as well as on plastic substrates.
In a second set of experiments, triungulins were offered a choice in a vial between 3rd – 4th
instar wasp larvae and no wasp as control: although Polistes nests are opened, they are often located
under tiles or other shelters, thus a confined space could be appropriate to simulate a natural
condition. We prepared transparent plastic tubes (1 cm wide, 7 cm long) with a small hole in the
middle. The wasp larva was positioned at one extremity of the tube while the other extremity was
left empty. Clusters of 20-30 triungulins were inserted into the tube with a thin needle through the
small hole. A cotton membrane at 1 cm from each extremity prevented triungulins from contacting
the wasp larva and a parafilm over the hole prevented triungulins from escaping. For 27
independent experiments we recorded Xenos larvae position in respect to target and control after 1
hr (time 1) and after 4 hrs (time 2). The central portion of the tube around the entry hole (≈ 1 cm
long) was not considered, being a neutral zone. To control for a possible effect of light, we used the
same transparent tubes as above (deprived of both the wasp larva and the cotton membrane) 90°
oriented to the light source and we recorded triungulins prevalence in light side vs dark side.
Finally, an additional set of stimuli was proposed to Xenos triungulins in order to observe
their susceptibility to wasp cuticular compounds. Single wasp larvae were placed in a glass vial and
washed in 100μl heptane for 30 sec to obtain a solution of larval cuticular waxes, mainly
hydrocarbons (Cotoneschi et al. 2007). We created a 4 field square arena (measuring 10 cm on each
side) where one field was occupied by the stimulus (a drop of hydrocarbon solution from larval
wasp cuticle), the opposite one was a positive control (a drop of hydrocarbon solution from
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immature Sarcophaga sp. fly, obtained following the same procedure as above) and the remaining
fields were negative controls (no stimuli at all). Then we placed clusters of 20-30 triungulins in the
middle of the arena and we recorded their distribution among 4 fields after 20 minutes.
Experimental data were analysed with the computer program SPSS for Windows: we performed
chi-square test and Cohen’s kappa coefficient test.
RESULTS
Preliminary 20-min experiments of host location in open arenas with diffuse lightening suggested
that triungulins were attracted by the presence of living wasp immatures. When we placed batches
of triungulins and one wasp larva inside the arena, they usually managed to approach the target
within the first 5 minutes of activity. Typically they moved towards it not directly, but describing
round trajectories and lifting their body from one side to the other, alternatively. They were able to
jump, by means of their caudal setae. In 5 out of 6 trials, more than 50% of triungulins approached
the larva, but only a few of them began to move on the surface of the larva, whereas the others were
out of our sight under the larva or disappeared from the arena at the end of the trial.
Further choice trials between a putative host and no wasp as control (inside a tube, i.e. a
confined space) showed a rather unexpected pattern (Fig. 2). First of all, we checked for positive or
negative phototaxy. Light really seemed to be an attractive factor: in all 9 trials considered, after 1
hour more than 2/3 of our triungulins were positioned in the side of the tube facing the light source.
For this reason, following trials were carried on by covering the tube. Among 27 trials, a significant
asymmetrical distribution (χ2 test) was observed in 21 trials, either after 1 hour or 4 hours and we
observed a choice for the wasp larva in 13 cases at time 1 and in 14 cases at time 2. Despite the
evidence of a trend to move towards the larva, the difference was not significant at both times (χ2 =
1.18 and 2.32, df = 1, PExact > 0.05). At all, in 15 trials in which triungulins were asymmetrically
distributed, their position in respect to wasp larva and control at time 2 was not different compared
to time 1, which means they tended to maintain the acquired position within the tube for longer time
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spans: the inter-rate agreement was significant (KCohen = 0.609 , P = 0.022). Finally, this set of trials
revealed a trend towards gregariousness for triungulins in regard to their movements within the
tube: irrespectively of their choice for wasp larva or empty tube, more than 2/3 of the members of
each experimental cluster were grouped in 21 trials out of 27, both at time 1 and time 2 (χ2 =8.33 ,
PExact = 0.006).
The use of single stimuli associated to the presence of the host showed that epicuticular
compounds from wasp immatures did not influenced Xenos triungulins, once they were outside
mother’s cephalothorax. In 6 trials with diffuse lightening, triungulins were never clustered around
spots of wasp cuticular compounds but once were significantly grouped in the opposite field with
fly odorant and twice in fields without any signal (PExact<0.001): their overall distribution was
random ((χ2 = 4.96, df =3, ns).
DISCUSSION
Contrary to our expectations due to their short life-span, X. vesparum triungulins do not appear
particularly reactive to host-associated stimuli. Nevertheless, their approaches to the wasp silhouette
in an open arena suggest that they can locate the host. Their peculiar behaviour while directing
themselves towards the target is noticeable: round trajectories and repeated inclinations of the body
seem to respond neither to a visual nor to an acoustic stimulus (in this case we would expect a
straighter displacement). On the contrary, this attitude could be interpreted as a symmetrical
chemio-reception by means of antennae or further sensory organs, hypothetically placed on both
sides of triungulins body. Anyhow, it is difficult to establish whether their movements are the motor
pattern of the infection process or something casual: they jump on the wasp larva, explore it,
sometimes stop there, sometimes leave and go behind later.
The double-choice tests in a confined space, simulating a sheltered nest, did not confirm our
first expectation, i.e. that triungulins are able to exploit chemical cues to redirect their movements
towards the host: they grouped at both extremities of the tube, independently on the
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presence/absence of the wasp larva (see Fig. 2). Then we analysed the influence of single factors,
the cuticle compounds of wasp larvae first, i.e. mainly non-volatile, long-chain hydrocarbons,
which were critical for nest-mate identification in wasps (Gamboa 2004) and were recently
described in P. dominulus larvae too (Cotoneschi et al. 2007). Unfortunately, spots of epicuticular
compounds did not elicit the arrestment of triungulins on the spot, as we would expect based on
similar four-well arena experiments, where chemical stimuli of bee larval food influenced the
movements of Varroa destructor (Nazzi et al., 2004), as well as in several egg parasitoids (Fatours
et al., 2008). Laboratory artefacts are unlikely, due to our simple experimental set-up, nevertheless
cuticular extracts need to be concentrated in future analyses.
Relatively to light as directional cue, we argue that it is probably more important for
promoting triungulins’ exit from mother cephalothorax than for orientating their movements once
outside. We already know that blowing gently at the cephalothorax evokes the emergence of
batches of 1st instar larvae within a few minutes (Kathirithamby 2009), because a light CO2
emission might simulate the presence of a potential host (Guerenstein & Hildebrand 2008); once
activated inside the mother’s body, Xenos larvae may exploit a light cue to find the exit of the brood
canal, i.e. the cephalothorax brood opening (Beani et al. 2005). Maybe they simply react to the CO2
and light stimulus by increasing their activity without taking into account any specific direction
(David Giron, pers. comm.). Thus, “kinesis” (a non-directional stimulus-dependent change) seems
to be more appropriate than “taxis” (a directional change towards gradient stimulus intensity),
nevertheless in these first trials we did not measure changes in the activity of triungulins in presence
or in absence of a stimulus. As a matter of fact, during laboratory infections, clusters of triungulins
normally formed at the end of the abdomen of parasitized wasps (pers. obs.).
It is possible that triungulins perceive the presence of the host but do not take into account
any directional information, due to their direct release on the comb by a vector, a foraging wasp as
well as a stylopized wasp releasing triungulins on different colonies (Beani & Massolo, 2007). If we
consider that Polistes nests are already crowded of wasps at early summer, when most of the
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strepsipteran infections occur, triungulins must have a huge choice and little time to decide. Wasp
larvae are very reactive, provided with mandible, and adults collaborate with them in trying to
remove the annoying tiny pests from the nest. These reactions belong to the first step of insects
defence process against parasitoid invasions, better known as “behavioural defence” (Bailey & Zuk
2008; Schmid-Hempel & Ebert 2003). In insect colonies, behavioural defence represents a key
element of the social immune system, whereby single individuals cooperate to obtain avoidance,
control or elimination of parasitic infections (Cremer et al. 2007). The annual colonies of paper
wasps may be considered as a small “homeostatic fortresses” (Hughes et. al. 2008), where
individuals control the disease due to both their hygienic behaviour and early desertion of the nest
by parasitized inactive adults (Hughes et al, 2004 b).
The dynamic of the process is here complicated by the fact that both Xenos larvae and
Polistes hosts are gregarious (Fig. 2). Regardless of the modality of transportation on wasp nest,
data on parasite prevalence in naturally infected colonies (Hughes et al. 2003; Vannini et al., 2008)
let us suppose that many triungulins (maybe decades) are contemporary present on the same nest,
searching for a suitable P. dominulus larva where to penetrate. Gregariousness – here the tendency
to aggregate in confined as well as in open space – promotes the displacement of batches of
infective larvae through the brood canal and could have evolved to increase parasitism success
when conditions are favourable. Peaks of nest infections on the field happen during the hottest
hours of the day, when most foragers are outside the nest (Ortolani & Cervo 2009): this moment
corresponds also to high metabolic activity of wasp larvae (thus high production of CO2) and
intense light. We cannot exclude any cooperative action between sibling parasites during the
infection process (Costa & Fitzgerald, 1996): a decreased risk of predation is likely to occur in
group. On the other hand, movements in group could reduce host-seeking efficiency, introducing
possible mutual interference among triungulins (Royer et al. 1999); however, no competition has
been observed among triungulins, as confirmed by successful superparasitism by sibling and non-
sibling individuals (Vannini et al., 2008).
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For many aspects, the behavioural patterns of Xenos infective larvae remind us of Meloe
triungulins, even though they belong to different insect orders (Strepsiptera and Coleoptera,
respectively). Also beetle 1st instar larvae are gregarious, in fact they cooperate in forming
aggregations (they move as a unit) and in holding onto vegetation, waiting for a foraging bee that
can bring them to the nest (Hafernik & Saul-Gershenz 2000). The cooperation and chemical
communication exhibited by Meloe triungulins for mutual benefit is so evident (sometimes they
form living bridges with the aim to attract and contact the bee) that some authors suggest that it
transcends aggregation behaviour to become social behaviour (Saul-Gershenz & Millar 2006). In
our case, gregariousness is not such an advanced phenomenon, but it could represent anyhow an
initial step in this direction. Another similarity with beetle 1st instar larvae is the negative
thermotaxy: Xenos triungulins, in fact, tend to move in the opposite direction as respect to a heat
source (pers. obs.).
Proximate explanations for triungulins behaviour are otherwise still to be defined. A
parsimonious hypothesis – based on the peculiar ecology of parasite and host – could be that, once
on the nest, triungulins are more likely to find very easily a host, especially if they do not
discriminate host quality. A few choice trials between large vs small larvae, or healthy vs infected
ones, suggested that triungulins “overwhelmingly entered the first host they encountered” (Hughes,
2003). Based on our previous experiences, there is no evidence of any strict physiological barrier in
host selection mechanisms by X. vesparum, differently from what reported in other host-parasitoid
systems. In the laboratory we managed to successfully infect various developmental stages, naïve or
already parasitized wasps (superparasitism by sibling and non-sibling triungulins is also well
diffused in the field, see Vannini et al. 2008), males which are less parasitized than females in
nature (Hughes et al. 2004a; Hughes et al. 2004b) and even non-primary hosts, as P. gallicus, in
which the parasite development was interrupted (Manfredini et al. 2007b). These findings clearly
show that, in our system, ecological-behavioural barriers are perhaps more important than chemical
and visual signals for a perfect tuning of the parasitoid with the biology of the host.
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ACKNOWLEDGMENTS
We would like to thank Stefano Turillazzi for his methodological suggestions while starting these
experiments, Eugenio Paccagnini for the technological support in SEM analysis, Romano Dallai,
Luciano Lepri, David Giron and David Hughes for all their helpful comments.
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FIGURE CAPTIONS
Figure 1 – Scanning electron micrographs of Xenos vesparum 1st instar larvae. (A) Dorsal view of a
triungulin: note disc-pulvilli on the first two pairs of legs and spines on tarsi in the last pair
(arrowhead). (B) Dorsal view of the cephalic segment with external photoreceptors well evident
(arrows), whereas “Stemmata” (sensu Pohl 1998, meaning ocelli) are here not visible. (C) Ventral
view of the cephalic segment, with mandibles (arrow), jaws (maxillae, Mx) and labium (Lb). (D)
Detail of a disc-pulvillum on the first pair of legs. (E) Two pairs of setae on the last abdominal
segments: caudal appendages (arrowheads) are longer and thicker.
Figure 2 – Double-choice trials by groups of triungulins (active individuals ranging from 15 to 35
for each group) moving inside a tube with one wasp larva at one extremity and no wasp as control.
For both time points (time 1 = 1 hr and time 2 = 4 hrs) the percentage of trials is reported where
triungulins were significantly grouped (chi-square test) towards the wasp larva or the control.
Above each column is the number of observations (out of a total of 27 trials).
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towards larva no preference towards control
% statistically significant movements
time 1
time 2
Figure 2
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