Animal Behaviour 183 (2022) 41e50

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Animal Behaviour
journal homepage: www.elsevier.com/locate/anbehav

The effects of prenatal predator cue exposure on offspring substrate

preferences in the wolf spider Tigrosa helluo
Hailey Shannon, Dylan Kutz, Matthew Persons*
Department of Biology, Susquehanna University, Selinsgrove, PA, USA

a r t i c l e i n f o
Prey may gain fitness benefits from recognizing predators at an early age. Predator cues perceived by
Article history: gravid prey have the potential to prime offspring to alter their behaviour in the presence of predation
Received 12 January 2021 threats after birth. Wolf spiders (Araneae: Lycosidae) provide an opportunity to explore this phenom-
Initial acceptance 15 March 2021 enon since females carry their eggsacs prior to eclosion and, after emergence, carry spiderlings for days
Final acceptance 18 August 2021 as they disperse. During maternal care, mothers may prenatally expose offspring to predator odour cues
Available online 24 November 2021 and potentially influence their posteclosion dispersal when in the presence of these cues. Spiderlings of
MS. number: A21-00026R2 the wolf spider Tigrosa helluo are eaten by adults of the wolf spider Pardosa milvina. We measured dif-
ferences in substrate preferences of spiderlings produced from egg-carrying T. helluo females that had or
Keywords: had not been exposed to cues of adult P. milvina. Over a 15-day period we counted the number of
anticipatory plasticity
offspring climbing on substrates previously walked on by P. milvina, crickets or blank control substrates.
intraguild predation
For the first 10 out of 15 days, spiderlings of predator-exposed mothers climbed on Pardosa substrates
kairomone
maternal effect
significantly more compared to control mothers. We found no significant spiderling preference in time
predator recognition spent on cricket-cued or control substrates across treatments and no difference in chemical cue substrate
prenatal priming preference of the mothers while spiderlings dispersed. Among wolf spiders, vertical climbing and contact
with predator-cued substrates can induce adaptive tonic immobility (freeze response) that has been
associated with increased survival in the presence of live predatory lycosids. Spider predator cues may
prime spiderling antipredator behaviour either directly through prenatal exposure in the eggsac or
indirectly by modifying the mother's behaviour prior to eclosion, but we found no evidence of mother
substrate preference influencing spiderling distribution during dispersal.
© 2021 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Prey benefit from recognizing predators as early as possible to
deploy appropriate antipredator defences. Parents of some species
may convey information about predation risk in the environment
to their offspring before birth, resulting in anticipatory parental
effects or adaptive transgenerational plasticity in offspring
(reviewed in Uller et al., 2013). Offspring may also assess this risk
directly from predator cues themselves. For some invertebrate
species with polyphenic traits, the presence of predator-associated
chemical cues can work transgenerationally to induce morpho-
logical changes that reduce predation (Agrawal et al., 1999; Gilbert,
1966; Van der Stap et al., 2007; Weisser et al., 1999). For example,
the herbivorous rotifer Brachionus calyciflorus exhibits posterolat-
eral spine development when exposed to predation risk of a
carnivorous rotifer (Gilbert, 1966), and the proportion of winged
morphs of the pea aphid Acyrthosiphon pisum increases in
* Corresponding author.
E-mail address: persons@susqu.edu (M. Persons).
populations exposed to predatory beetles (Weisser et al., 1999).
Predator cues are also capable of prenatally modifying the devel-
opmental timing of some species. Eggs of the red-eyed treefrog,
Agalychnis callidryas (Warkentin, 2000), and the spitting spider
Scytodes pallidum (Li, 2002) are both known to shorten embryonic
development time after being exposed to predator cues in the
environment. Presumably, these responses reduce the probability
of egg predation in both species.
The ability of embryos to detect and respond to predatory
threats either directly (through their egg membrane) or indirectly
(through maternal cues) may impart significant fitness advantages
to offspring. If offspring can be primed as embryos about relevant
predation threats in the environment at the time of birth, young
may use appropriate behavioural responses immediately upon
emergence from the eggs. Several studies have found that, in ver-
tebrates, maternal prenatal exposure to predator cues can modify
the antipredator behaviour of offspring (Bestion et al., 2014;
Giesing et al., 2011; St-Cyr et al., 2018; St-Cyr & McGowan, 2015).
For example, Atherton and McCormick (2015) found that heart

https://doi.org/10.1016/j.anbehav.2021.10.019
0003-3472/© 2021 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
42 H. Shannon et al. / Animal Behaviour 183 (2022) 41e50
rates of embryonic cinnamon clownfish, Amphiprion melanopus,
increased upon exposure to conspecific alarm cues (CACs) com-
bined with predator odour of the dottyback Pseudochromis fuscus,
suggesting innate recognition of CACs and possible modifications in
antipredator responses across the egg membrane. This phenome-
non has also been noted in some invertebrates. Predator-naïve
crickets (Gryllus pennsylvanicus) exhibited adaptive antipredator
behaviours including protracted freezing or tonic immobility re-
sponses, reduced speed and increased refuge seeking when their
gravid mothers were exposed to chemical cues of various wolf
spider species compared to crickets whose mothers were not
exposed to such cues (Storm & Lima, 2010). These induced anti-
predator responses among embryos may be the result of innate
recognition systems, priming or accelerated learning processes by
offspring. In many cases it remains unclear, however, the extent to
which predator cues may modify prey behaviour directly through
prenatal detection by the embryo or indirectly through maternal
effects.
Viviparous species’ physical or behavioural modifications from
prenatal exposure to predator cues may be more likely to be
shaped by maternal effects compared to oviparous species, as
offspring of the former can only gather environmental informa-
tion as embryos indirectly though maternal signals, such as hor-
mones. Oviparous species, particularly those species that
transport their eggs outside of the body, could provide more op-
portunities for both maternal and direct embryonic effects via
exposure of offspring prenatally to relevant predator information.
This is the case among wolf spiders (Araneae: Lycosidae) since
females carry their eggsacs with them during incubation, help
open the eggsac and carry the young for days to weeks after
eclosion (Walker et al., 2003). Their mobile maternal care may
therefore expose offspring to different predator cues prior to and
after their emergence. Wolf spiders are also particularly sensitive
and receptive to chemical cues, putatively silk and excreta, from
other predatory lycosids. They use multiple sensory modalities
while detecting predators including visual (Lohrey et al., 2009),
vibratory (Lohrey et al., 2009; Sitvarin et al., 2016), acoustic
(Lohrey et al., 2009), airborne chemical (Schonewolf et al., 2006)
and especially substratum-borne chemotactile information
(Okuyama, 2015; Persons et al., 2001, 2002; Persons & Rypstra,
2001; Riechert & Hendrick, 1990; Sitvarin et al., 2016). Taken
together, these features make wolf spiders an especially inter-
esting subject for examining prenatal predator effects on post-
emergent spiderling dispersal behaviour.
Freezing or tonic immobility is the primary defensive behaviour
exhibited by wolf spider prey in the presence of cues from preda-
tory heterospecific lycosids, and the chemical cues alone are suffi-
cient to induce this response even in the absence of a live predator
(Bell, Rypstra, & Persons, 2006; Hoefler et al., 2008; Lehmann et al.,
2004; Persons et al., 2002; Taylor et al., 2005). Chemical cue-
induced tonic immobility likely increases survival by making the
prey visually and/or vibratorily cryptic to predatory wolf spiders
since wolf spider sensory systems are highly movement sensitive
(Barnes et al., 2002; Persons et al., 2001; Persons & Rypstra, 2001).
Wolf spider foraging time in a patch (i.e. giving-up time) is
strongly associated with sensory information about prey (Persons
& Uetz, 1996, 1997a, 1998). Prey that spend more time moving
cause wolf spider predators to stay in a given foraging patch longer;
therefore, tonic immobility induces wolf spider predators to move
more quickly out of a foraging area and therefore reduce predation
risk (Persons & Uetz, 1997b). Lycosid predators also use movement-
based tracking of prey to assess prey density in a patch, which may
be particularly important in areas where sibling spiderlings from a
dispersing eggsac may be concentrated (Persons & Uetz, 1998). This
movement-based assessment of a foraging patch occurs among
male and female spiders of different ages (juveniles, adults) and
hunger states (Persons, 1999; Persons & Uetz, 1999).
Predator chemical cue-induced freeze responses occur among
both adult spiders and early-instar spiderlings. Silk and excreta
from predatory wolf spiders can both prime early-instar spider-
lings to show reduced movement (Eiben & Persons, 2007) and
influence their dispersal behaviour after eclosion from the eggsac
(Persons & Lynam, 2004). Exposure to silk and excreta cues from
larger predatory spiders also induces vertical movement re-
sponses in prey spiders. Vertical movement responses increase
survival in the presence of a live predatory lycosid compared to
movements on the ground or horizontally (Folz et al., 2006;
Persons et al., 2002; Rypstra et al., 2007). However, it remains
unknown whether predator chemical cue exposure while still in
the eggsac can influence or prime spiderling antipredator re-
sponses postemergence.
Here we measured differences in substrate preference among
spiderlings produced from egg-carrying Tigrosa helluo (hereafter
Tigrosa) females that had or had not been exposed to chemotactile
cues (i.e. silk and excreta; hereafter ‘cues’) of adult female Pardosa
milvina (hereafter Pardosa). We chose Tigrosa as the subjects and
Pardosa as the predator due to their asymmetric intraguild preda-
tory relationship. Adult Tigrosa consume adult Pardosa, which, in
turn, can consume early-instar Tigrosa (M. Persons, personal
observation). Pardosa under predation risk produce fewer eggsacs
(Persons et al., 2002), introducing possible confounds into inter-
preting predator cue effects. In our design the Tigrosa spiderlings
may perceive Pardosa cues as a predation threat, but the Tigrosa
female mother should perceive the Pardosa cue as a potential food
cue rather than a predation threat (Persons & Rypstra, 2000). This
should provide some mitigation to the potential issue of low eggsac
production with maternal exposure to predatory cues. If predator
cues act prenatally to modify prey behaviour, we predicted that
spiderlings that eclosed from eggsacs with mothers exposed to
predator cues would show enhanced freeze responses and greater
vertical climbing than eggsacs from unexposed mothers. Increased
freeze responses and climbing should result in more spiderlings
accumulating on elevated predator-cued substrates if the eggsac
was previously exposed to these cues.
METHODS
Spider Collection and Maintenance
We collected 33 adult female Tigrosa from agricultural fields
near Susquehanna University, Selinsgrove, Snyder County, Penn-
sylvania, U.S.A. during SeptembereOctober 2014. Spiders were
individually housed in opaque white round plastic dishes (11.5 cm
in diameter and 8 cm in height) with an approximately 1.5 cm layer
of peat moss to serve as a source of moisture and burrowing sub-
strate. We assumed that many of the females had already been
mated in the field at their time of collection; the 33 females used for
the study all produced eggsacs as a result of such mating. Spiders
were fed an ad libitum diet of subadult banded crickets, Gryllodes
sigillatus, approximately every 5 days until they produced an egg-
sac. The adult female Pardosa used as sources of predator cues for
this experiment were also collected at the same time and from the
same agricultural fields near Susquehanna University, Selinsgrove,
Snyder County, Pennsylvania, U.S.A. Pardosa were maintained in
small translucent plastic containers (8 cm in diameter and 5 cm in
height Solo® cups) with a damp peat moss substrate. Female Par-
dosa were maintained on a diet of first- and second-instar
G. sigillatus nymphs ad libitum approximately every 5e6 days.
We also watered the peat moss for all spiders ad libitum for the
duration of the experiment.
 H. Shannon et al. / Animal Behaviour 183 (2022) 41e50
Ethical Note
We adhered to the ASAB/ABS Guidelines for the ethical treat-
ment of animals in research. We made efforts to ensure adequate
care and maintenance of all spiders used throughout this study
from the time of collection through the remainder of their natural
lives under laboratory conditions.
Stimulus Substrate Preparation
We maintained field-mated adult female Tigrosa in individual
containers until eggsac production, after which we randomly
assigned females to one of two substrate treatments: a control
substrate of moistened fresh peat moss devoid of predator cues
(N ¼ 16), or a Pardosa-cued (hereafter ‘predator-cued’) substrate
containing moistened peat moss with chemical cues from an adult
female Pardosa (N ¼ 17).
The predator substrates were set up by placing a well-fed adult
female Pardosa in a clean, ethanol-sanitized deli dish (11.5 cm
diam., 8 cm height) with an approximately 1.5 cm layer of fresh
peat moss in it. Adult female P. milvina are approximately
5.27 ± 0.42 mm (mean ± SD) in length (N ¼ 15). This female
remained in this container laying down silk and excreta cues for 5
days. Every 5 days the Pardosa was fed and placed on fresh peat
moss in a new, cleaned and sanitized container. Female Pardosa
used as stimuli were transferred to and fed in an empty container
that was not being used for substrate treatments. While being fed,
they remained in these feeding containers for approximately
30 min. After feeding, the empty container was cleaned and sani-
tized. This feeding procedure was done to minimize potential
confounding influences of cricket chemical cues that may mask or
interact with Pardosa cues. Once a Pardosa was removed from the
feeding container, it was always placed in a new container with
fresh peat moss (devoid of any cues) to lay down cues; she was used
continuously in such a manner until death. The predator substrate
was discarded at the end of this 5-day preparation period if there
were no Tigrosa females ready to be placed on the cues.
Tigrosa females that were selected for the predator substrate
treatment were placed in the deli dishes containing the silk of
Pardosa after a Pardosa female had been in the container for 5 days.
The females in the control treatment were placed on a 1.5 cm layer
of clean, fresh peat moss (devoid of cues) in a sanitized deli dish.
The Tigrosa females in both the predator substrate and the control
substrate treatments had their substrates changed every 5 days.
This was to ensure that cues for the experiment did not become
‘stale’ since aged wolf spider cues are known to influence the
response of heterospecific lycosids (Barnes et al., 2002). Each time
their substrates were changed, Tigrosa females were fed two or
three small crickets. Care was taken to ensure that the crickets were
consumed promptly and that feeding occurred on the substrate
that was being discarded to minimize eggsac exposure to cricket
cues. Eggsac-carrying females assigned to the predator substrate
treatment were maintained on the substrate for 20 days, shortly
before the general Tigrosa eggsac eclosion window (M. Persons,
personal observation). They were switched on day 20 to a cue-free
substrate of peat moss until emergence from the eggsac. This was
done to ensure that spiderling exposure to Pardosa cues occurred
only within the eggsac since Tigrosa spiderlings eclose between 21
and 25 days after eggsac construction.
Although separately exposing the mother and eggsacs to pred-
ator cues would experimentally separate maternal effects (sans
yolk provisioning) from direct effects, this was not possible since
maternal care is required to produce viable young. Spiderlings
cannot exit the eggsac without the mother opening it, and the
eggsac often requires rotation early during development to prevent
43
egg agglutination (Colancecco et al., 2007; Marchetti & Persons,
2020). Furthermore, there is evidence that longer spiderling con-
tact time with the mother and siblings produces bolder and more
exploratory spiderlings once dispersed (Punzo & Alvarez, 2002)
and that these spiderlings have improved nervous system devel-
opment and do better on spatial learning tasks (Punzo & Ludwig,
2002). This suggests that the early social contact with their
mother and siblings between eggsac eclosion and dispersal can be
an important time of spiderling behavioural development. For this
reason, females were kept with their eggsac at all times while being
maintained within their respective chemical cue treatment and
spiderlings were allowed a natural dispersal from their mothers.
Stimulus Cup Preparation
We measured the response of Tigrosa spiderlings to one of three
cups that had previously been perfused with odour and tactile cues
associated with either an adult female Pardosa (spiderling pred-
ator), banded cricket nymph (cricket cues) or a control cup without
cues from either a cricket or spider. Substrate cues were collected
by covering small plastic containers (souffle  cups 8 cm in diameter
and 5 cm in height) with masking tape to provide a tractable sur-
face for Pardosa. Each tape-covered cup was then inverted and
placed into an opaque white round plastic dish (11.5 cm in diameter
and 8 cm in height) that contained approximately 1.5 cm of
moistened peat moss. Into these containers we placed either one
adult female Pardosa, one banded cricket of approximately equal
size as an adult female Pardosa (0.025 g), or nothing at all to pro-
duce the predator, cricket and control substrates, respectively.
These substrate cups stayed in the deli dishes for 5 days gathering
cues before being placed into an arena. If the cup was not needed
once these 5 days had passed, then it was removed from the deli
dish, carefully wiped down with dry paper towels and placed in a
new deli dish of the same cue type (e.g. cricket-cued cups went
back into a deli dish containing a cricket), but with a new cue source
(i.e. a different Pardosa or cricket). We did not use solvents to clean
the container because of possible confounds including residual
odours and unknown reactions with the masking tape. We also
wanted to simulate more natural conditions whereby silk may
become disturbed and degraded over time. Our 5-day deposition
period was also used to standardize the age and quantity of the cues
(i.e. time since deposition) on the cups when placed in the testing
arenas.
When needed, we placed one 5-day aged chemically cued cup of
each substrate (Pardosa, cricket, control) into the plastic test arena
(described below) with forceps to minimize handling and avoid the
possible removal or contamination of the substrate; substrates
were swapped every 3e5 days. When swapping the substrates, the
old chemically cued cup that had been in the arena was wiped off
carefully and thoroughly with dry paper towel to remove silk and
cues that may have been laid down by the Tigrosa spiderlings or the
mother, and placed back into a deli dish containing the same sub-
strate type it originally came from (e.g. cricket-cued cups went back
into a deli dish containing a cricket) to refresh the experimental
cues. Spiderlings were exposed to chemically cued cups from two to
five different adult female Pardosa during the dispersal assay of this
experiment.
Trial Protocols
Behaviour trials were run using the Tigrosa spiderlings, begin-
ning 2e5 days after eggsac eclosion. Spiderlings sometimes took 2
days to fully exit the eggsac. One day after the last spiderlings
eclosed, we removed them from their mother and recorded the
number of spiderlings in the clutch. After being counted, the
44 H. Shannon et al. / Animal Behaviour 183 (2022) 41e50

spiderlings were allowed to remount their mother. Female Tigrosa
produced between 61 and 220 spiderlings per egg sac (mean ± -
SD ¼ 125 ± 42). We found no difference in the number of spider-
lings in the control treatment compared to the predator-cued
Pardosa treatment (two-sample t test: t31 ¼ 1.503, P ¼ 0.145;
mean ± SD: control 110.1 ± 29 spiderlings per eggsac; Pardosa-cued
treatment ¼ 130.7 ± 42 spiderlings per eggsac).
After the spiderlings were fully resettled, each mother and
clutch were then transferred to a large, circular plastic arena
(Tupperware® containers 20 cm in diameter and 8 cm in height)
with a lid that had been cleaned and sanitized with ethanol. In each
container two wetted cotton wads were placed in the centre to
provide moisture for the mother and spiderlings. Three inverted
chemically cued cups (8 cm in diameter and 5 cm in height)
wrapped in blue masking tape were placed equidistant (centres
approximately 10 cm apart) from each other along the edges of the
container (Fig. 1). Each cup had previously contained either an adult
female Pardosa, an approximately 0.6 cm long banded cricket
nymph or nothing as described above. Thus, each testing arena
contained three cups with chemical cues associated with a pred-
ator, a potential food item and a control substrate. We recorded the
number of spiderlings on each cup once daily for 15 days. Spider-
lings on the mother, the lid, the bottom of the container or the
cotton used to provide water were not counted as being on any
substrate. We also recorded the mother's position once daily during
the 15-day period by noting the section of the arena (control,
cricket, predator-cued cup) in which the majority of her body was
positioned. If the majority of the mother's body was not clearly in
one section, she was not considered to be in a particular section.
Statistical Analysis
between day 4 and 13 because not all females had spiderlings
dispersing prior to day 4, and by day 14, spiderlings were travelling
to the top of the testing arena rather than on the substrates. Dis-
tributions for each mother's substrate experience treatment across
days 4e13 also showed absolute kurtosis and skewness values
below 2.0 and were therefore assumed to not deviate significantly
from a normal distribution. Distributions in each combination of
measures by day also were approximately normal within days 4e13
(ShapiroeWilk test using residuals). Because dispersal from the
mother is strongly related to the age of the spiderling posteclosion,
we were careful to standardize testing of spiderlings based on days
posteclosion.
We used two-way ANOVAs on days 4, 7, 10 and 13 to measure
effects of spiderling climbing over subsequent days to determine
how long spiderlings exhibited a cup preference based on the
mother's substrate. For two-way ANOVAs, mother's substrate
(predator or no predator) and cup odour cues (control, cricket,
predator) were independent variables with number of spiderlings
on each cup as the dependent variable. We also measured the
number of times the mother was on each of the three cup types
over a 15-day period and analysed the data using a generalized
linear mixed model (GLMM). The effects of maternal treatment
group (predator or control) and time on the probability of mothers
choosing either Pardosa-, cricket- or control-cued cups were
examined in this model, with mother identity (ID) as a random
effect. Models were run separately for each of the three cue cup
choices, with a binomial distribution where the response of interest
(the mother's selection of either the Pardosa, cricket or control cup)
was coded as 1 and all other responses were coded as 0. This
allowed us to note potential patterns in the cue choices of mothers
and, subsequently, examine whether mothers' cue choices were
similar to those of their offspring.

We used a three-way within-between subjects mixed model

ANOVA to measure the effect of the mother's substrate experience
(between factor), cup (between factor) and day (repeated measure)
on the number of spiderlings per mother climbing on each cup over
a 10-day period (days 4e13 of the total 15-day experimental
period). We measured the number of spiderlings per replicate only
RESULTS
Maternal substrate exposure treatments significantly influenced
spiderling substrate choice after eclosion. We found a strong and
highly significant interaction between the mother's substrate

(a) (b)

Pardosa
Pardosa cues Cricket cues
cues

No cues No
Cricket cues
cues

Figure 1. (a) Diagram illustrating the 20 cm diameter testing arena in which Tigrosa helluo spiderlings (small black spiders) and their mother (large dark grey spider) were placed
following eggsac eclosion. Mothers and eggsacs were previously exposed to either Pardosa cues for 20 days (N ¼ 17) or no cues (N ¼ 16). Three inverted plastic souffle  cups were
wrapped with blue masking tape. One cup served as a control and was unmanipulated (No cues). Another cup had a single adult female Pardosa (Pardosa cues, dotted) or cricket
(Cricket cues, striped) walk on the cups for 5 days, after which the cup was replaced with fresh cues. At the time of Tigrosa introduction there were no cues present anywhere in the
arena other than the cups. The mother's positions and the number of spiderlings on each cup type were recorded daily for 15 days. (b) Photo of testing apparatus with mother and
spiderlings superimposed to show relative size of test subjects to the container. See Methods for details.
 H. Shannon et al. / Animal Behaviour 183 (2022) 41e50
treatment and cup substrate choice (Table 1), as well as the
mother's substrate treatment and day (Table 1). There was also a
highly significant three-way interaction between maternal sub-
strate treatment, cup and day on the mean number of Tigrosa spi-
derlings on the three cup types (Table 1, Fig. 2a, b, c) and a highly
significant mother substrate*cup interaction on the number of
spiders on each cup type (Table 2).
During day 4, Tigrosa spiderlings from mothers previously
exposed to chemical cues of Pardosa climbed on predator-cued cups
significantly more than mothers that had not been exposed to
Pardosa cues (Table 2, Fig. 2a). Significant mother substrate*cup
interactions were also found on days 7 and 10 but not on day 13
(Table 2, Fig. 2b, c, d). Spiderlings continued to appear more
frequently on the predator-cued cups when their mother was
exposed to these cues while carrying the eggsac. This pattern
continued until day 12, but by day 13, there were no significant
differences between mother substrate type and cup treatment
(Table 2, Fig. 2d, Fig. 3). Mothers did not demonstrate a strong
preference towards cue cups matching their respective treatments
when total counts of mothers' cup choices were examined (Fig. 4).
There was no significant effect of day on the probability of mothers
choosing any of the cue cups (Table 3). There was also no significant
effect of mothers' substrate treatment (control or Pardosa sub-
strate) on the probability of mothers choosing either predator- or
control-cued cups, but mothers’ substrate treatment was signifi-
cant for the probability of mothers choosing cricket-cued cups
(Table 3). Based on the GLMM model, this effect was driven by
control treatment mothers being less likely to choose cricket-cued
cups (z ¼ 3.182, P ¼ 0.001), but a post hoc Tukey test revealed no
significant difference in choosing cricket cups between the treat-
ment groups (P ¼ 0.797).
DISCUSSION
Prenatal exposure to predator cues significantly influenced
spiderling substrate choice, but the effect diminished over time,
resulting in no differences by day 13. Spiderlings with mothers
exposed to Pardosa cues were significantly more likely to be found
climbing on the predator-cued substrates than on the control or
cricket substrates. This same pattern was not observed among
offspring from mothers exposed to control substrates. There was
also no significant association between the mothers’ treatment and
the substrate she was located on/near throughout the 15-day trial
period. This evidence suggests that Tigrosa spiderlings modified
their cue choice behaviours posteclosion after exposure to potential
predatory cues while within the eggsac, and that this behaviour
was independent of maternal behaviours posteclosion.
Table 1
Three-way within-between subjects ANOVA for the number of spiderlings on one of
three treated cup substrates across days 4e13 of a 15-day experiment
Treatment df F P Power
Mother substrate 1 0.034 0.8569 0.053
Cup substrate 2 0.619 0.5484 0.136
Cup substrate*mother substrate 2 13.429 0.0002 0.996
Day effect 9 4.110 0.0002 0.996
Day*mother substrate 9 4.521 0.0001 0.998
Cup substrate*day 18 1.210 0.2572 0.801
Cup substrate*day*mother substrate 18 2.361 0.002 0.993
Mother substrate consisted of eggsac-carrying female Tigrosa that were placed on
substrates with or without Pardosa predator chemical cues present during the
eggsac incubation period. Cup substrate consisted of unmanipulated control cups or
those previously walked on by a cricket or predatory Pardosa for 4 days. Bolded
values are significant with a ¼ 0.05. Power is the probability of rejecting the null
hypothesis when, in fact, it is false (probability of avoiding a type II error).
45
Freinschlag and Schausberger (2016) found that spider mites
whose gravid mothers were exposed to cues from predatory mites
exhibited a preference for leaf sites where predators were less
likely to be encountered compared to site selection for unexposed
mothers. This demonstrates how prenatal learning or priming that
results in site selection away from predator-rich environments can
enhance offspring fitness. In our study, the Tigrosa spiderlings
appeared more frequently on substrates with predator cues. Such
behaviour seems maladaptive since it would result in spending
more time in areas where predators are present or had visited
recently. We propose a few possible explanations for this seemingly
counterintuitive response.
There were significantly more spiderlings on the Pardosa-cued
cups when the mother and eggsac had previously been exposed to
Pardosa cues, but for spiderlings with mothers not previously
exposed to Pardosa cues, we found no increased association with
the Pardosa-cued cups. This increase likely reflects a tonic immo-
bility response in these spiderlings when contacting Pardosa cues.
In other wolf spider species, contact with chemical cues from larger
wolf spider species induces long periods of immobility and slow
walking, which, in turn, results in longer periods spent in areas
with these predator cues (Barnes et al., 2002; Bell et al., 2006;
Lehmann et al., 2004; Persons et al., 2001; Persons & Rypstra, 2001;
Rypstra et al., 2016). Tonic immobility and slower movement of
spiders on these substrates results in significantly increased sur-
vival in the presence of live predatory wolf spiders compared to
prey spiders that lack access to this chemical information (Barnes
et al., 2002; Bell et al., 2006; Hoefler et al., 2008; Lehmann et al.,
2004; Persons et al., 2001; Rypstra et al., 2017).
Tonic immobility is adaptive since it renders prey visually and
vibratorily cryptic for wolf spider predators. The amount of time
prey spend moving is directly related to both the probability of
lunge responses of wolf spider predators as well as the length of
time the predators choose to spend in the area (Persons & Uetz,
1997b). In fact, for some species, the primary determinant for
how long wolf spiders search for prey in an area before leaving is
the perception of moving prey rather than capturing prey in an area
(Persons & Uetz, 1996, 1997a). Wolf spiders also use prey move-
ment frequency to assess prey density in an area and remain in
areas based on that information (Persons & Uetz, 1998). Therefore,
cohorts of dispersing spiderlings will significantly reduce the
probability of a nearby wolf spider attacking them or staying in the
area if they freeze on substrates with cues from these predators.
Spiderlings of another wolf spider species (Rabidosa rabida) are
also preyed upon by adult Pardosa. These spiderlings reduce activity
when exposed to Pardosa chemical cues, and this response in-
creases survival in the presence of a live Pardosa predator, partic-
ularly when the spiderlings have had previous experience with
Pardosa cues (Eiben & Persons, 2007). In our experiment, spider-
lings from mothers that were maintained on Pardosa cues may
similarly be reducing activity, thus resulting in much longer contact
time with predator cues than cricket cues and an increased number
of counts on these substrates over time.
Reductions in activity among young after embryonic exposure
to predators are known among other predatoreprey systems.
Young embryonic salamanders that were exposed to predatory
cues while in their egg stage showed reduced activity after emer-
gence (Mathis et al., 2008; To  th & Hettyey, 2018). When gravid
mothers of the predatory mite Phytoseiulus persimilis were exposed
to an intraguild predator (Amblyseius andersoni) and their offspring
were introduced to cues from these same predators, the offspring
showed reduced activity on these predator-cued substrates and
spent more time with the predator cues compared to offspring from
gravid mothers that had not been exposed to an intraguild predator
(Seiter & Schausberger, 2015). The authors suggest that the
46 H. Shannon et al. / Animal Behaviour 183 (2022) 41e50

12 12
Day 4 Day 7
10 10

8 8

6 6

4
Mean number of spiderlings

2 2

0 0
Pardosa Cricket Control Pardosa Cricket Control

16 12
14 Day 10 Day 13
10
12
8
10
8 6
6
4
4
2
2
0 0
Pardosa Cricket Control Pardosa Cricket Control
Cup substrate

Figure 2. Two-way interaction between maternal substrate treatment and cup on the mean number of Tigrosa spiderlings on the three cup types for days 4, 7, 10 and 13 of a 15-day
experiment. Error bars indicate ± 1 SE; grey bars ¼ spiderlings from control treatment mothers; black bars ¼ spiderlings from Pardosa treatment mothers. Maternal substrate
treatment consisted of eggsac-carrying female Tigrosa that were placed on substrates with or without Pardosa predator chemical cues present during the female's incubation period.
Cup substrate consisted of unmanipulated control cups or those previously walked on by a cricket or predatory Pardosa for 4 days.

Table 2 spiderlings of another lycosid species, R. rabida, demonstrated

Two-way ANOVA results for the number of spiderlings on one of three cups during
enhanced freeze responses and increased survival rates. These
days 4, 7, 10 and 13 of a 15-day experiment
spiderlings became sensitized rather than habituated to predatory
Day Treatment df F P Power wolf spider cues, but this same sensitization did not occur with
4 Mother substrate 1,36 9.493 0.0064 0.843 cricket cues. A similar, albeit pre-eclosion priming effect may have
Cup substrate 2,36 4.588 0.0168 0.745 caused the predator-cue preference we observed.
Mother*cup 2,36 18.105 0.0007 0.973
The high count of Tigrosa spiderlings from predator-exposed
7 Mother substrate 1,40 0.845 0.369 0.136
Cup substrate 2,40 0.708 0.499 0.157 mothers on the Pardosa substrate could also be a by-product of an
Mother*cup 2,40 10.964 0.0002 0.992 escape response. Contact with predator cues can initiate a dispersal
10 Mother substrate 1,40 2.099 0.163 0.268 or ballooning response (Moran & Hurd, 1994). Wolf spiders
Cup substrate 2,40 0.4317 0.432 0.181 exposed to predator cues of other lycosids also significantly in-
Mother*cup 2,40 4.158 0.023 0.701
13 Mother substrate 1,40 0.036 0.850 0.054
crease vertical climbing responses, which, in turn, increase survival
Cup substrate 2,40 0.464 0.632 0.118 in the presence of a live predator (Persons et al., 2002). These re-
Mother*cup 2,40 2.724 0.078 0.498 sponses would explain the additional time spiders spent associated
Mother substrate consisted of eggsac-carrying female Tigrosa that were placed on with the top or sides of predator-cued cups. Dispersal can be an
substrates with or without predator cues present during the eggsac incubation adaptive response to predator exposure and has been noted in birds
period. Cup substrate consisted of unmanipulated control cups or those previously (Payo-Payo et al., 2018) and a few invertebrates (Henry et al., 2010;
walked on by a cricket or predatory Pardosa for 4 days. Bolded values are significant Otsuki & Yano, 2014), and, in certain cases, it can be mediated
with a ¼ 0.05. Power is the probability of rejecting the null hypothesis when, in fact,
it is false (probability of avoiding a type II error).
through prenatal exposure to predator cues. For instance, prenatal
exposure to predator cues (Bestion et al., 2014) or maternal stress
cues (Meylan et al., 2002) in some lizards greatly increases the
offspring from exposed mothers showed increased learning dispersal probability of their offspring. Since spiderlings were
compared to offspring from unexposed mothers, where the former maintained in closed containers away from airflow during the
could better differentiate between latent (chemotactile cue only) monitoring phase of this experiment, they were unable to balloon
and high (presence of a live predator) risk and more often selected as they might under natural field conditions. Further experimen-
predator-cued sites as a result of this learning (Seiter & tation is needed to determine whether spiderling ballooning/
Schausberger, 2015). We did not expose Tigrosa embryos to a live dispersal behaviour was indeed altered as a result of prenatal
Pardosa threat, so such an association between a live predator and predator cue exposure. This would seem an adaptive response of
its cues could not have been learned by our subjects; however, juvenile Tigrosa to avoid intraguild predation in Pardosa-laden en-
Eiben and Persons (2007) showed a priming effect on learning vironments as it could result in movement away from predator-rich
where, after 24 h of exposure to Pardosa (predator) chemical cues, locations.

16
14
Average number of spiderlings 12
10
0
1 2
Figure 3. The average number of spiderlings climbing on a Pardosa substrate or cup over a 15-day experimental dispersal period (N ¼ 33 clutches). Dashed grey line ¼ spiderlings
from control treatment mothers; solid black line ¼ spiderlings from Pardosa treatment mothers. Error bars represent the standard error for each day. Maternal substrate treatment
consisted of eggsac-carrying female Tigrosa that were placed on substrates with or without Pardosa predator chemical cues present during the female's incubation period.
Female Tigrosa fed a diet of Pardosa prefer substrates with
Pardosa cues (Persons & Rypstra, 2000). Since spiders used in this
study were wild-caught females in Pardosa-rich habitats, these
mothers may have had prior feeding experience with Pardosa and
may have been attracted to these substrates. Moreover, exposure
to Pardosa-cued substrates in the laboratory may have enhanced
mothers' field-acquired preferences for these cues. Spiderlings
from this treatment group could therefore have been noticed
more often near or on the Pardosa-cued cups as they dispersed
close to their mothers who spent more time near these cups. In
general, wolf spider mothers show increased activity and more
directional movements when encumbered by spiderlings
compared to when they are not (Bonte et al., 2007). This could
provide one significant maternal behavioural effect on spiderling
dispersal. However, our data do not support this explanation for
why more spiderlings were on the Pardosa-cued cups as we did
not see a significant relationship between mother location and
spiderling cue choice. We noted, for instance, that dispersing
mothers maintained on control substrates tended to be observed
on the Pardosa substrate treatments more often than were Par-
dosa-cued mothers (Fig. 3) while spiderlings from Pardosa-cued
mothers spent more time on Pardosa cues than offspring of con-
trol mothers, which, if anything, would bias spiderling movement
against our observed patterns. Additionally, while spiderling cup
choice was influenced by mother treatment group, treatment
group did not significantly influence the probability of mothers
choosing control or Pardosa cups (Table 3). These findings suggest
that the mother's substrate choice is incongruent with that of her
offspring, so we think that spiderling substrate choice was driven
by behavioural differences as a result of pre-eclosion priming to
the Pardosa cues and not maternal behaviour posteclosion. Pre-
vious research has shown that Pardosa spiderlings delay dispersal
when lycosid predator cues are present (Persons & Lynam, 2004),
but this effect only occurred over the first 6 days. Delayed
dispersal may be adaptive in areas with higher predator densities
since it allows spiderlings to maintain protection from their
mothers and also avoid areas of higher prey density; however,
spiderlings cannot delay dispersal indefinitely as their yolk re-
serves are depleted and siblings shift from affiliative behaviours to
H. Shannon et al. / Animal Behaviour 183 (2022) 41e50 47
3 4 5 6 7 8 9 10 11 12 13 14 15
Day
aggression or even cannibalism as hunger levels increase
(Anthony, 2003; Roberts et al., 2003).
It remains possible that preference for Pardosa cues from
offspring of predator-exposed mothers may not be indicative of
antipredator behaviour. While Pardosa will prey upon juvenile
Tigrosa under laboratory conditions (Balfour et al., 2003; Rypstra &
Samu, 2005), Pardosa's impact on young Tigrosa under field con-
ditions may not be extremely strong (Balfour et al., 2003). Predation
of Pardosa by Tigrosa is, however, well documented (Barnes et al.,
2002; Persons et al., 2001), and there is a chance that juvenile
Tigrosa could have identified Pardosa cues as potential prey cues.
During interactions among intraguild predators, the degree of size
asymmetry is generally a good indicator of predator and prey roles
between wolf spider species (Rypstra & Samu, 2005). Tigrosa spi-
derlings in our experiment would not have been able to success-
fully feed on the adult Pardosa that generated the cues they were
detecting, as Pardosa were at least 10 times larger than the Tigrosa
spiderlings. However, reproductive cycles of T. helluo and P. milvina
overlap throughout the year (Rypstra & Samu, 2005), and so the
possibility of a Tigrosa spiderling locating a smaller Pardosa spi-
derling in a Pardosa-laden area remains. Depending on the time of
year, a Tigrosa spiderling could benefit from spending time in Par-
dosa-rich habitats, potentially locating a suitable food source
(early-instar Pardosa), but they would also be under some level of
predation threat by adult Pardosa. Other studies have shown that
chemically mediated antipredator responses among wolf spiders
tend to be influenced by the quantity of predatory lycosid cues
present rather than the size of the predator that produced them
(Persons & Rypstra, 2001). Additionally, woodfrog tadpoles, Lith-
obates sylvaticus, have demonstrated differential responses to
predatory cues based on what time of day they were conditioned to
those odours as eggs and what time of day they were tested as
tadpoles (Ferrari et al., 2010). This demonstrates that prey may have
quantitative and temporally sensitive distinctions in predatory
threat levels.
Interestingly, there did not seem to be a preference for cricket-
cued cups by spiderlings of either treatment group even though all
Tigrosa mothers were maintained on crickets throughout the
experiment. Quesada and Schausberger (2012) observed a case of
48 H. Shannon et al. / Animal Behaviour 183 (2022) 41e50

70 embryos still in the egg, or, more likely, after eclosion but before
emergence from the eggsac. Lycosids emerge from the egg and
60 moult while still in the eggsac (Trabalon et al., 2018). As such they
Number of observations

50
40
30
20
10
0 preferences for conspecific calls played to them prior to hatching
Pardosa Cricket Control
Cup substrate
Figure 4. Total number of observations of mothers on each of three different cup
substrates over a 15-day period. Grey bars ¼ control treatment mothers; black
bars ¼ Pardosa treatment mothers. Mother substrate consisted of eggsac-carrying fe-
male Tigrosa that were placed on substrates with or without Pardosa predator chemical
cues present during the female's incubation period. Cup substrate consisted of un-
manipulated control cups or those previously walked on by a cricket or predatory
Pardosa for 4 days.
prenatal learning in a predatory mite (Neoseiulus californicus) in
which protonymphs demonstrated a preference for prey that was
consumed by their gravid mothers. Tigrosa also demonstrates
preferences for cues of recently consumed prey (Persons & Rypstra,
2000). If the Tigrosa spiderlings from our present study were so
primed to display preferences towards their mother's diets, spi-
derlings from both treatment groups should have been observed
most often on the cricket substrate, as all mothers were fed crickets
during this experiment. However, this was not the case for either
treatment group; instead, spiderlings demonstrated a preference
for the treatment environment to which they were exposed while
in their eggsacs. While care was taken to minimize spiderling
exposure to cricket cues while in the eggsac, these young had
limited exposure to cricket cues prior to eclosion either directly
when crickets were introduced into experimental housing con-
tainers for their mothers to consume, or indirectly through silk and
excreta from their mothers. Our results suggest that Tigrosa spi-
derlings biased their cue choice posteclosion on environmental
treatment cues that they were exposed to while in the eggsac and
that their cue choices did not appear to be influenced by maternal
diet.
It remains unclear when predator cues mediate spiderling re-
sponses while still in the eggsac since recognition could occur with
spend part of the same instar inside the eggsac as they do after
eclosion. These spiderlings would presumably have similar sensory
abilities immediately after their first moult as they do after emer-
gence. Given longer time to develop sensory organs for detection of
odourants after the first moult, recognition while still in the egg
seems less likely. However, some animals, such as cuttlefish, can
fully respond or habituate to and/or learn associations with pred-
atory stimuli while in the egg (Mezrai et al., 2020; Romagny et al.,
2012). Bobwhite quail, Colinus virginianus, chicks also display
(Carlsen & Lickliter, 1999), and some frog species display prefer-
ences for novel odourants they have been exposed to while in the
egg stage (Hepper & Waldman, 1992). Presumably, these abilities to
respond to outside stimuli may extend to embryos of numerous
species that are sufficiently developed prior to hatching. Given that
direct contact with the substrate is not possible while still in the
eggsac, it seems likely that predator cues were detected primarily
or exclusively via airborne volatiles. At least under some conditions,
wolf spiders can detect airborne cues associated with prey (Persons
& Rypstra, 2000), predators (Schonewolf et al., 2006) and pro-
spective mates (Searcy et al., 1999). It is also possible that some
unknown cue, perhaps a vibratory signal or an incidental move-
ment pattern, could be conveyed to offspring by the mother
directly, or that silk and/or predator excreta cues attached to the
mother or eggsac provided a chemotactile rather than an airborne
source of information for spiderlings upon emergence. Such a
chemotactile mechanism would not require the use of predator
airborne cues or maternal signals. However, such a cue would
require an extremely short predator imprinting period among
spiderlings since spiderlings from the Pardosa-cued maternal
treatment would have only about 24 h of additional Pardosa che-
motactile cues available to shift their responses compared to the
maternal control treatment.
In conclusion, our findings indicate that pre-eclosion embryonic
and/or first-instar experiences of lycosids are capable of inducing
behavioural changes in spiderlings after eggsac emergence. Pre-
natal exposure to predator cues may be correlated with the use of
elevated antipredator behaviours such as freezing, vertical climbing
and/or ballooning in predator-dense environments or, alternatively,
adaptive searching for prey posteclosion. Further study is needed to
identify the particular antipredator and/or prey selection behav-
iours that are being exhibited and to examine the effects that
different predator cues or exposure windows may have on this

Table 3
Results of the generalized linear mixed model on the effects of mothers’ (T. helluo) substrate treatment group (control or Pardosa substrates) and day on mother cue cup choice
(Pardosa, cricket or control cups)

Cue cup choice Main and fixed effects Estimate z c2 df P

Pardosa Treatment 0.376 1 0.540

Day 3.752 1 0.053
Intercept 0.696 2.029 0.042
Pardosa 0.096 0.199 0.842
Cricket Treatment 5.296 1 0.021
Day 1.805 1 0.179
Intercept 0.983 3.182 0.001
Pardosa 0.116 0.257 0.797
Control Treatment 0.099 1 0.753
Day 0.247 1 0.619
Intercept 1.558 4.110 3.96e-05
Pardosa 0.146 0.279 0.780

Values for treatment group and day represent the main effects of the model as examined with an ANOVA. Values for intercept and Pardosa are representative of the estimates
(and associated values for the estimates) for the relative impact of treatment type (control and Pardosa substrates) on the odds of choosing the respective cue cup, as generated
with the linear mixed effect model. Bolded values are significant with a ¼ 0.05.
 H. Shannon et al. / Animal Behaviour 183 (2022) 41e50
response. It is clear that anticipatory behavioural plasticity in wolf
spiders occurs pre-eclosion, which may provide additional adaptive
priming and/or learning benefits to species that carry eggs and/or
offspring prior to dispersal.
Author Contributions
Hailey Shannon: Conceptualization, Methodology, Validation,
Formal Analysis, Investigation, Data Curation, Writing-original
draft, Writing, Review and Editing, Visualization. Dylan Kutz:
Investigation, Writing, Review and Editing. Matthew Persons:
Conceptualization, Methodology, Validation, Formal Analysis,
Investigation, Resources, Data Curation, Writing, Review and Edit-
ing, Visualization, Supervision, Project Administration, Funding
acquisition.
Declarations of Interest
None.
Acknowledgments
This research was supported by a research partners student
summer assistantship and a faculty grant. We thank Amanda
Abrams, Andrea Mobley, Orvil Grunmeier, James Kelly and Sara
Ramezzana for help feeding and collecting the spiders used in this
study. We also thank Ann Rypstra, Alexander Berry, Jake Godfrey
and Alexander Salazar for their feedback on the manuscript as well
as two anonymous referees.
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