# When sibling tolerance meets cannibalism of the dead in spiderlings

Antoine Lempereur, Raphaël Jeanson

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Antoine Lempereur, Raphaël Jeanson. When sibling tolerance meets cannibalism of the dead in spiderlings. Animal Behaviour, 2025, 222, pp.123129. 10.1016/j.anbehav.2025.123129. hal-05381033

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# When sibling tolerance meets cannibalism of the dead in spiderlings

Antoine Lempereur<sup>1,5</sup>, Raphaël Jeanson<sup>*</sup>

Centre de Recherches sur la Cognition Animale (UMR5169), Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, Toulouse, France

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## ARTICLE INFO

**Article history:**

Received 12 September 2024
Initial acceptance 19 November 2024
Final acceptance 2 January 2025
Available online xxx
MS. number: 24-00568

**Keywords:**

Agelena
aggressiveness
lipid
necrophagy
nutritional status
sociality
spider
survival

Social life requires group members to tolerate each other, which implies the presence of effective mechanisms to prevent aggression. The existence of a transient social life brings an additional dimension, as the response to social signals provided by conspecifics varies during ontogenesis. This raises questions about the mechanism driving the switch in the processing of social cues. Spiders are a relevant model for answering these questions, as juveniles are gregarious and tolerant at the start of their development and then behave aggressively towards their conspecifics at more advanced stages. Previous research has shown that spiderlings reared in groups are tolerant of their siblings, but that those maintained alone are cannibalistic. In many taxa, the nutritional status of individuals plays an important role in the expression of intraspecific aggression. We found that in juveniles of the solitary spider *Agelena labyrinthica*, lipid stores decreased over time when starved, but social context did not have any effect. This suggests that differences in energy stores do not cause differences in how individuals interact with each other. We then examined whether spiderlings that tolerated living siblings cannibalized corpses immediately after death or after a 24 h delay. We found that spiders engage in necrophagy at a similar rate, regardless of the age of the corpse. These results suggest that living spiders produce an as-yet-undetermined signal that protects them from cannibalism but disappears immediately after their death, exposing them to pre-dation by siblings.

© 2025 The Author(s). Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Living in society requires its members to show tolerance towards each other and refrain from any aggressive tendencies that might jeopardize the group's stability. The situation becomes more complicated when animals change the nature of their social interactions during ontogeny or in response to variations in environmental conditions, leading to a modification or even a reversal of their reactions towards conspecifics. Indeed, many species of invertebrates and vertebrates pass from a social phase characterized by tolerance and attraction towards conspecifics to a solitary state marked by a range of behaviours from indifference to avoidance or aggression (Baeza et al., 2002; Davis & Kassel, 1975; Mougenot et al., 2012; Sakakura & Tsukamoto, 1999). This variation in social interactions implies changes in communication processes. These may be based on a modification of the cues or signals produced by individuals and/or alterations in how they perceive and respond to them. However, the mechanisms responsible for changes in social interactions in transient social species are not well understood. In addition, a better understanding of the mechanisms responsible for changes in social behaviour could provide

important insights into the evolution of sociality, given that maintaining cohesion between juveniles is considered to be a major route to permanent sociality (Lin & Michener, 1972).

Spiders are relevant models for addressing this question, as all species, over 52 700 known to date, are considered to have a transient social phase. Indeed, juveniles are gregarious and tolerant towards their siblings during their earliest developmental stage before dispersing and behaving aggressively towards their conspecifics as adults (Chiara et al., 2019; Foelix, 2011). In the solitary spider *Agelena labyrinthica*, our previous work has shown that aggressiveness is the consequence, not a cause, of social isolation. Indeed, spiderlings reared alone for 20 days display aggressive behaviours and cannibalize their siblings at a high rate, whereas those reared in groups remain tolerant (Chiara et al., 2019; Mauduit & Jeanson, 2023a, 2023b). In juvenile and adult spiders, as in many other taxa, the propensity to cannibalism is often linked to the level of satiety or the nutritional status (Andrade, 1998; Bilde & Lubin, 2001; Petersen et al., 2010; Roberts et al., 2003; Rypstra, 1986; Samu et al., 1999). One hypothesis is that aggression in pairs of spiderlings reared alone is attributable to differences in energy reserves, which can be assessed by lipid stores, compared with siblings kept in groups. However, there is a limited understanding

<footnote>* Corresponding author.
E-mail address: raphael.jeanson@univ-tlse3.fr (R. Jeanson).</footnote>
of the potential impact of social context on the utilization of fat reserves in arthropods. Indeed, most studies so far have centred around the effects of social interactions on food intake and fat accumulation, rather than on fat consumption. For instance, females of the cockroach *Blattella germanica* eat more food when they are in pairs compared to when they are alone (Holbrook et al., 2000). Similarly, females of the house cricket, *Acheta domesticus*, reared in groups have been shown to store more lipids than those reared in isolation (Gutiérrez et al., 2020). However, studies on vertebrates have shown that the social environment can have a significant impact on metabolic rates of individuals, with energy expenditure varying upwards or downwards depending on the nature of the social bonds in the species and the ecological conditions (Chrétien et al., 2021; Masonjones, 2001). In this context, the first aim of the present study was to investigate whether the early social context influences the depletion of fat content in unfed spiderlings. If lipid reserves vary according to the social context, we predicted that juveniles reared in isolation would have lower lipid reserves because they show higher levels of aggression than their siblings reared in groups.

In spiderlings, tolerance can last a long time, with up to 80% survival after 3 months in pairs of unfed individuals (Lesne et al., 2016). This lack of cannibalism is not associated with an inability to hunt, as spiders can capture and consume flies from their first days outside the egg sac (Mauduit & Jeanson, 2023a). This indicates that effective mechanisms inhibit the expression of aggression against conspecifics, although they are not yet known. The first step to elucidating these processes is to determine whether the expression of tolerance requires siblings to be alive. Contrary to popular belief, spiders do not consume only live prey that they have killed. Indeed, adults of several species can engage in opportunistic scavenging, including eating dead conspecifics (Vetter, 2011, 2013). Juveniles can also feed on dead flies (Vickers et al., 2014) and, in some species, the mother can bring dead prey for her offspring to feed on (Barrantes & Weng, 2009). Our second objective was thus to examine whether juveniles that tolerate living spiderlings would cannibalize dead siblings (i.e. necrophagy). To do this, we killed spiderlings using flash freezing and offered the corpse immediately or after 24 h to a sibling raised in groups (i.e. tolerant towards a living individual). A higher rate of necrophagy after 24 h would suggest the progressive degradation of a hypothetical recognition signal. However, a similar level of necrophagy between treatments would provide indirect evidence of the involvement of a life signal in the expression of tolerance.

# METHODS

## Biological Model

Agelena labyrinthica (Agelenidae) is a web-building spider widely distributed in southern and central Europe. Females lay one to three egg sacs containing an average of 80–120 eggs in late summer. After approximately 20 days of incubation, the spiderlings hatch within the egg sac. This first instar phase lasts for 1 week and ends in a first moult, which takes place inside the egg sac, leading to the second instar. Under natural conditions, second-instar spiderlings spend the winter in the egg sac before emerging the following spring. The average body length (prosoma and opisthosoma) of second-instar spiderlings is 2.5 ± 0.3 mm (mean ± standard deviation, N = 60, Mougenot et al., 2012). We collected a total of 17 egg sacs in southwest France in August and September 2023. The egg sacs were kept in a cooled chamber at 4 °C in the dark to simulate winter diapause until the start of the experiments. When necessary,

the egg sacs were removed from the chamber and manually opened with metal tweezers and a brush to extract the spiderlings. This was done to ensure that the juveniles experienced the same social context before being assigned to different experimental treatments (Chiara et al., 2019). The day of opening was referred to as day 0.

For all the conditions tested in this study, spiderlings reared or tested in groups were always siblings and were never fed during the experiments. When tests involved two spiderlings (either dead or alive), they were always taken from two separate rearing boxes to avoid any familiarity.

## Influence of Age and Social Context on Lipid Reserves and Aggressiveness

Spiderlings from three egg sacs were randomly assigned to one of two treatments. They were introduced either alone (N = 140 individuals) or in groups of four (N = 35 groups) to transparent petri dishes (diameter = 52 mm, height = 13 mm). Spiderlings were sprayed with water three times a week and maintained in a climate-controlled room (25 °C ± 1 °C, 60% RH) under a 12:12 h light:dark cycle (light at 0800 hours). At days 0, 10 and 20, a subset of the dishes from each treatment was sampled and all the individuals in each of these dishes were killed to measure their lipid stores (reared alone: D₀: N = 24, D₁₀: N = 28, D₂₀: N = 28; reared in groups: D₀: N = 28, D₁₀: N = 24, D₂₀: N = 31). The spiderlings were killed by immersion in liquid nitrogen and then stored at a temperature of -80 °C until they were weighed. To measure lipid stores, we first introduced dead spiderlings into Eppendorf microtubes (1.5 ml) which were then placed in an oven (model UFE-500 Memmert, Schwabach, Germany) at 65 °C for 24 h. Afterwards, the corpses were weighed using a microbalance (Mettler Toledo AT21 Balance Scales Comparator D with a precision of 0.001 mg, Greifensee, Switzerland). The corpses were then immersed in microtubes filled with 1 ml of chloroform (Sigma Aldrich). We carried out three baths, one per day, to extract lipids. Once the baths were completed, the corpses were dried again in the oven at 65 °C for 24 h before being weighed a second time. The lipid mass of each spiderling was determined by measuring the change in dry mass before and after chloroform extraction. The percentage fat content was then calculated by dividing the lipid mass by the initial dry mass and multiplying by 100.

On day 20, a subset of spiders that were not used for measuring lipid stores was used to evaluate their aggressiveness. Pairs of spiders that were reared under identical social conditions were introduced to polyvinyl chloride (PVC) arenas (diameter = 22 mm, height = 4 mm), which were covered with transparent Plexiglas lids. We tested 27 pairs of spiders reared alone and 20 pairs of siblings reared in groups. We filmed the pairs for 8 h using a camera (Canon Legria HFG30). For four consecutive days, we then inspected each arena to record deaths. The level of aggression was assessed from the videos considering the first 20 interactions (Chiara et al., 2019). We defined interaction as any encounter between two individuals whose interindividual distance is less than one body length (Mougenot et al., 2012). For each interaction, we assessed whether it was aggressive or not. We considered the following behaviours as aggressive: bite attempt (when one individual raised its legs and lunged forward), chase (when one individual moved rapidly towards the other) or fight (when both individuals attacked and bit each other) (Mauduit & Jeanson, 2023a). Nonaggressive interactions included any contact that did not show signs of aggression, such as encounters between two mobile spiders or prolonged contact (> 1 s) between two immobile individuals. For
each pair, we counted the number of aggressive interactions, which
could range between 0 and 20.

Necrophagy

We aimed to determine whether spiderlings that are normally
tolerant of living individuals cannibalized the corpses of their sib-
lings. We used spiderlings from nine egg sacs that were introduced
either alone (N = 116 individuals) or in groups of five siblings (N =
140 groups) in transparent petri dishes (diameter = 52 mm,
height = 13 mm) for 8 days (hereafter: D<sub>8</sub>) or 20 days (hereafter:
D<sub>20</sub>). At 8 days, spiderlings are active and highly tolerant of living
conspecifics irrespective of the social context experienced during
early development (Chiara et al., 2019; Mauduit and Jeanson,
2023a). When spiderlings reach 20 days of age, those that have
been kept in isolation usually show aggression towards their sib-
lings, while those that have been raised in groups remain tolerant.
Spiderlings were sprayed with water three times a week and
maintained in a climate-controlled room (25 °C ± 1 °C, 60% RH)
under a 12:12 h light: dark cycle (light at 0800 hours).

At 8 or 20 days, spiderlings were randomly assigned to one of
the following treatments.

(1) Treatment Fly: one spiderling reared in a group was tested with a fly (Drosophila melanogaster) killed in liquid nitrogen immediately before the experiment (D<sub>8</sub>: *N* = 29, D<sub>20</sub>: *N* = 29).

(2) Treatment C₀: one spiderling reared in a group was tested with one sibling reared in a group from another dish that was killed in liquid nitrogen immediately before the experiment (D₈: *N* = 29, D₂₀: *N* = 29).

(3) Treatment C<sub>24</sub>: one spiderling reared in a group was tested with one sibling reared in a group from another dish that was killed in liquid nitrogen 24 h before the experiment (D<sub>8</sub>: *N* = 29, D<sub>20</sub>: *N* = 27). In the 24 h before the test, the corpse was kept at room temperature. The duration of 24 h was chosen arbitrarily.

(4) Treatment C<sub>24.2</sub>: one spiderling reared in a group was tested with one sibling reared in a group from another dish that was killed in liquid nitrogen 24 h before the experiment (D<sub>8</sub>: *N* = 29, D<sub>20</sub>: *N* = 29). Before the start of the experiment, the corpse was again immersed in liquid nitrogen. This condition aimed to determine whether any potential difference in necrophagy between the C<sub>0</sub> and C<sub>24</sub> treatments was due to the age of the corpses (24-h-old) or to the fact that they had not been immersed in nitrogen immediately before the start of the experiment.

In addition, we tested pairs of live spiders that were reared either
in isolation (D₀: *N* = 28, D₂₀: *N* = 28) or in groups and collected from
two different breeding dishes (D₀: *N* = 28, D₂₀: *N* = 28).

The behavioural tests were carried out in PVC arenas (diameter = 22 mm, height = 4 mm) covered with transparent Plexiglas lids. For conditions involving a dead stimulus, the live spiders were first introduced into the test arenas. The stimuli (treatments Fly, C₀ and C₂₄.₂) were introduced individually in Eppendorf microtubes (1.5 ml) that were immersed in a container containing liquid nitrogen. After 1 min, the microtubes were removed from the liquid nitrogen, and the stimuli were placed on top of the lids covering the arenas where they were to be introduced.

After a maximum of 2–3 min of thawing, the lids were moved
slightly, and the stimuli were deposited in a random location in the
arenas. The stimuli for the treatment C<sub>24</sub> kept at room temperature
for 24 h, were transferred to Eppendorf microtubes (1.5 ml) and
then introduced into the PVC arena. When two live spiders were

tested, they were introduced one after the other into the test arenas
with a delay of less than a minute.

The arenas were filmed for 8 h with a camera (Canon Legria HFG30). We considered predation to be any contact of the chelicerae with the stimulus for more than 5 s. In some situations, contact with the chelicerae was difficult to see in the videos. We, therefore, considered that any pulling behaviour, where the spider pulled the corpse towards itself using its front pair of legs, was also associated with an act of predation. For each pair, we measured the latencies between the introduction of the live spider and its first contact with the stimulus and the latencies between the first contact between the spider and the first event of predation. In cases where no predation event was observed within 8 h of video recording, we assigned a latency of 8 h and considered this data as censored in the survival analysis (see below). After a period of 24 h, we verified each instance of predation by inspecting each arena under a stereomicroscope (Nikon SMZ1000). Eaten corpses were easily identifiable due to their deformed and transparent appearance.

Data Analysis

We performed all statistical analyses using the software R v4.0.2 (R Development Core Team, 2024). We used a linear mixed model (LMM; function lmer in package lmerTest, Kuznetsova et al., 2017) to compare the fraction of lipid stores (after square root transformation) between spiderlings of different ages and reared under different social contexts with egg sac ID as a random factor. The numbers of aggressive interactions in pairs of 20-day-old spiders reared alone or in groups were compared using a t test. We used a log-rank test with the function survdiff from the survival package (Therneau, 2024) to compare the cannibalism rate in pairs of spiderlings reared alone or in groups. We used a log-rank test to compare latencies before first contact and before predation between treatments in 8-day-old and 20-day-old spiders. We then conducted pairwise comparisons between treatments with the function pairwise_survdiff implemented in the package survminer (Kassambara et al., 2021). The latencies recorded in treatments with two live spiders (pairs of spiders reared in groups or social isolation) were not included in the comparisons because having two mobile individuals does not provide comparable situations to treatments where only one spider was mobile and confronted with an immobile stimulus. However, the survival curves for these conditions were plotted for visual comparisons. We used a Spearman rank correlation test to establish whether there was an association between latencies to first contact and the time between first contact and predation.

Ethical Note

This work complies with current French legislation on the use of
invertebrates in research. We have collected the minimum number
of individuals necessary for our research and we have taken care to
collect them from different field sites to maintain stable pop-
ulations over the years. The social isolation to which the spiders
were subjected is a normal stage in their development.

RESULTS

Influence of Age and Social Context on Lipid Stores and Behaviour

We detected an influence of age (LMM: *F*<sub>1,155.48</sub> = 57.70, *P* < 0.001) but no influence of the social context (LMM: *F*<sub>1,155.14</sub> = 2.48, *P* = 0.12) on the lipid reserves of spiderlings, the interaction between the two factors being nonsignificant (LMM: *F*<sub>1,155.50</sub> = 0.57, *P* = 0.57). After 20 days of starvation, the spiders showed a
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Influence of social context on fat content and behaviour. (a) Fat content (% of dry mass) as a function of the social context and age of spiderlings. (b) The number of aggressive events in the first 20 interactions in pairs of 20-day-old spiders reared alone or in groups. For (a) and (b) the horizontal line in each box represents the median, and the box represents the first and third quartiles. The lower (or upper) whisker extends to the most extreme value located 1.5 times the interquartile ranges from the first (or third) quartile. Different letters above the boxes indicate significant differences. (c) Survival curves of latencies to cannibalism in pairs containing two spiderlings reared alone (Iso/Iso) or in groups (Gr/Gr) for 20 days. Coloured bands indicate 95% confidence intervals. The number of replicates is indicated in parentheses.</figcaption></figure>

reduction of around 50% in their fat stores (Fig. 1a). Pairs of spiderlings kept in groups displayed significantly less aggressive interactions than those maintained alone for 20 days (t test: *t*<sub>39,21</sub> = -5.03, *P* < 0.001; Fig. 1b). Aggressive interactions were observed in 96% (26/27) of pairs composed of spiders reared in isolation compared with 45% (9/20) of pairs containing individuals kept in groups for 20 days (Fig. 1b). Cannibalism was more intense in pairs of 20 day old spiders reared in social isolation than in those reared in groups (log-rank test: χ<sup>2</sup><sub>1</sub> = 7.2, *P* = 0.007). Only 10% of pairs with individuals reared in the group showed cannibalism within 96 h, compared with around 50% in arenas containing two spiderlings reared in social isolation (Fig. 1c).

## Necrophagy

In 8-day-old spiderlings, no significant difference was found in the latencies to consume a dead stimulus between treatments (log-rank test: χ²₃ = 5.2, *P* = 0.20; Fig. 2a). On average, 50% of dead spiderlings were consumed within the first 2 h. In the 20-day-old spiderlings, a significant difference was detected in the latencies to consume a dead stimulus between treatments (log-rank test: χ²₃ = 15.1, *P* = 0.002; Fig. 2d). Flies were consumed faster than dead spiders in all treatments (*P* < 0.007 for all comparisons), but there was no difference in the latency to cannibalize a dead spider (*P* > 0.95). Around half of the dead spiders were eaten within the first hour.

We detected no significant differences between treatments in the latencies before the spider contacted the dead stimulus (log-rank test: χ²₃ = 2.4, *P* = 0.5) for 8-day-old spiderlings (Fig. 2b). There was a significant difference in the latencies to first contact between treatments in 20-day-old spiderlings (log-rank test: χ²₃ = 14.2, *P* = 0.003; Fig. 2e). Specifically, individuals contacted flies and fresh corpses (*C*₀) more rapidly than 24-h-old corpses that had been immersed once (*C*₂₄, *P* = 0.01) or twice in liquid nitrogen (*C*₂₄.₂, *P* = 0.015; Fig. 2e). At both 8 and 20 days, the latencies before contacting either type of spider corpse (i.e. *C*₀, *C*₂₄ or *C*₂₄.₂) followed exponential distributions (see Appendix, Fig. A1).

In 8-day-old spiderlings, the lag times between first contact and predation differed between treatments (log-rank test: χ²₃ = 10.9, *P* = 0.01), with spiders predating flies more quickly than fresh corpses (*P* = 0.007), the other comparisons being nonsignificant (*P* > 0.09; Fig. 2c). In 20-day-old spiderlings, latencies to consuming corpses after first contact also differed between treatments (log-rank test: χ²₃ = 15.1, *P* = 0.0102), with flies being eaten faster than spider corpses (for all comparisons: *P* < 0.007; Fig. 2f). Pooling the data from 8- and 20-day-old young spiderlings, we found no correlation between the latency between first contact with the corpse and the latency between first contact and predation (Spearman correlation test - *C*₀: rho = 0.05, *P* = 0.7; *C*₂₄: rho = 0.19, *P* = 0.16; *C*₂₄.₂: rho = -0.09, *P* = 0.52; see Appendix, Fig. A2). This suggests that the difference in latency before touching the corpse, possibly resulting from a difference in activity, did not influence the propensity to predate.

## DISCUSSION

In this study, we first investigated whether differences in fat storage mediated the effect of social setting on spiderlings' aggressiveness towards their siblings. We found that lipid reserves declined with starvation, with a 50% reduction after 20 days, but we observed no influence of social context. This result allows us to reasonably reject the hypothesis that lower energy reserves trigger aggression in socially isolated spiderlings. This provides evidence that the loss of tolerance is likely driven by changes in communication processes that are unrelated to nutritional status. We then found that spiders readily consumed dead flies or dead siblings. Interestingly, spiderlings reared in groups that are highly tolerant towards live conspecifics quickly consumed dead siblings, with an average of 50% of corpses being predated within 2 h for 8-day-old spiderlings and within 1 h for 20-day-old spiderlings. This difference in the consumption rates of corpses with age resulted from a combination of a shorter time to come into contact with the corpses (Fig. 2b-e), possibly due to increased activity (Chiara et al., 2019), and a slightly higher tendency to eat the corpse upon first contact in
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Survival curves for latencies (a, d) before stimulus predation, (b, e) before first contact with the stimulus and (c, f) between first contact and predation in pairs containing an (a, b, c) 8-day-old or (d, e, f) 20-day-old spiderling. A group-reared spiderling was confronted with either a live conspecific (Gr/Gr condition), a dead conspecific (C₀: fresh corpse; C₂₄: 24 h corpse; C₂₄.₂: 24 h corpse immersed twice in liquid nitrogen), or a dead fly (Fly). Two spiderlings reared in isolation were paired (Iso/Iso). Coloured bands indicate 95% confidence intervals. A total of 27–29 replicates were performed for each condition.</figcaption></figure>
20-day-old spiderlings (Fig. 2c–f). The flies tended to be eaten more quickly, with 80% being eaten within 1 min of first contact on average in the 8-day-old and 20-day-old spiders, compared with 55% for either of the spider corpses. Importantly, we found no difference in the tendency to engage in necrophagy when spiderlings were offered dead spiders that were killed either immediately before the test or 24 h prior. The existence of exponential distributions of contact times with either type of corpse indicates that the contact rate remained constant throughout the trial. This implies that the occurrence of events was independent of the time elapsed since the start of the test (Haccou & Meelis, 1992), suggesting that the attractiveness of the corpses did not vary throughout the 8 h test. Also, the overlap of survival curves before first contact reveals that attractiveness did not vary between fresh and 24-h-old corpses. Similarly, juveniles were just as likely to feed on either type of spider corpse on first contact. Overall, our results indicate that spiderlings that tolerate living conspecifics consumed dead siblings immediately after death and that the probability of engaging in necrophagy did not vary with the age of the corpse.

The onset of necrophagy in otherwise tolerant spiders suggests that death either suppresses cues normally involved in the expression of tolerance or brings cues that prompt predation. In arthropods, changes in communication signals associated with behavioural changes towards dead conspecifics have been studied mainly in social insects, where the removal of corpses is crucial to limit the risk of spreading pathogens that could jeopardize the survival of the colony. In the ant *Myrmica rubra*, the accumulation of fatty acids (e.g. oleic acid) over several hours and days elicits corpse removal (i.e. necrophoresis; Diez et al., 2013). In the termite *Reticulitermes flavipes*, fresh corpses rapidly produce volatile compounds that peak after 30–60 min before declining, but older corpses produce increasing quantities of fatty acids several hours after death (Sun et al., 2017). This difference in the release of chemical substances is associated with changes in behaviour, with the recent corpses being cannibalized while the oldest are buried. In the Argentine ant, *Linepithema humile*, living workers produce two cuticular compounds, the quantities of which are halved in the first 10 min following the death of the individuals (Choe et al., 2009). The loss of a life signal, rather than the release of chemical substances signalling the decomposition of dead ants, seems to be responsible for triggering necrophoric behaviour. In another context, the production of chemical substances also prevents intraspecific predation between living individuals, as in *Locusta migratoria*, where crowded individuals produce a volatile compound that suppresses cannibalism (Chang et al., 2023).

We hypothesized that living juveniles produce cues that protect them from cannibalism and that disappear almost immediately upon death. An argument against the progressive release of chemicals associated with degradation leading to necrophagy is that there was no variation over time, either within 8 h or after 24 h delay, in the rate of cannibalism. To date, we have not identified the nature of these putative cues. Based on work performed in other taxa, we are inclined to consider that chemical cues would be ideal candidates. Male spiders of several species can induce quiescence in females to reduce the risk of sexual cannibalism, with a possible contribution from the release of chemical substances (Becker et al., 2005; Cargnelutti et al., 2023). Acoustic communication is also important in the regulation of interactions in spiders (Barth, 2002). However, the role of vibrations has mainly been studied in the context of mating (e.g. Schüch et al., 1990), but their contribution to the expression of tolerance in gregarious spiderlings or adult social spiders is virtually unexplored. Future studies should therefore

explore the possible cues that disappear when individuals die, prompting siblings to engage in necrophagy when they prefer to starve rather than cannibalize living conspecifics.

## Author Contributions

**Raphaël Jeanson:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Antoine Lempereur:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

## Data Availability

Data are provided as Supplementary Material.

## Declaration of Interest

The authors declare no conflicts of interest.

## Acknowledgments

We thank Z. Benoit-Delaby and E. Mauduit for their contribution to preliminary experiments. We also thank two anonymous references for their comments on our manuscript. This work was funded by the Centre National de la Recherche Scientifique and University Paul Sabatier (Toulouse).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.anbehav.2025.123129.

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**Appendix**

<figure><img src="image_6.png" /><figcaption>**Figure A1.** Survival curves for latencies before contact with the corpse for 8-day-old and 20-day-old spiderlings. These are the same curves as those plotted in Fig. 2b–e. Coloured bands indicate 95% confidence intervals. Each red dashed curve represents the exponential fit to the corresponding experimental data. We fitted the function $\text{Fraction} = a \times e^{-r} \text{ time}$ using the function nls of the stats R package. For 8-day-old spiderlings, we obtained the coefficients $a = 1.11$ and $r = -0.92$ for $C_0$, $a = 1.05$ and $r = -1.03$ for $C_{24}$ and $a = 1.04$ and $r = -0.94$ for $C_{24.2}$. For 20-day-old spiderlings, the coefficients were $a = 1.05$ and $r = -1.20$ for $C_0$, $a = 1.02$ and $r = -0.85$ for $C_{24}$ and $a = 1.09$ and $r = -0.95$ for $C_{24.2}$.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure A2.** Relationships between time elapsed before first contact with a corpse and latencies for predation in treatments (a) C₀, (b) C₂₄ and (c) C₂₄.₁. For each type of corpse, data collected from 8-day-old and 20-day-old spiders were pooled.</figcaption></figure>