# The spiders that lost their personalities: assessing long-term behavioural individuality in social Stegodyphus dumicola

Lena Grinsted, Raphaël Royauté, Signe Eiserhardt, Virginia Settepani, Niels J Dingemanse, Trine Bilde

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Lena Grinsted, Raphaël Royauté, Signe Eiserhardt, Virginia Settepani, Niels J Dingemanse, et al. The spiders that lost their personalities: assessing long-term behavioural individuality in social Stegodyphus dumicola. Animal Behaviour, 2025, 224, pp.123167. 10.1016/j.anbehav.2025.123167. hal-05055995

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# The spiders that lost their personalities: assessing long-term behavioural individuality in social *Stegodyphus dumicola*

Lena Grinsted<sup>a, *</sup>, Raphaël Royauté<sup>b, c</sup>, Signe Eiserhardt<sup>c</sup>, Virginia Settepani<sup>c</sup>, Niels J. Dingemanse<sup>d, 1</sup>, Trine Bilde<sup>c, 1</sup>

## ARTICLE INFO

**Article history:**

Received 23 July 2024

Initial acceptance 31 October 2024

Final acceptance 20 January 2025

Available online xxx

MS. number 24-00462

**Keywords:**

adaptation

animal personality

behavioural reaction norm

behavioural syndrome

behavioural type

natural selection

plasticity

temperament

Across animal taxa, many species show behavioural individuality, that is, repeatable between-individual differences in behaviour. Numerous studies link behavioural individuality to major ecological and evolutionary processes. Here we conceptualize how such links hinge critically on the often untested assumption that individuality persists over long-term, ecologically relevant time frames. We test this assumption in an emerging model, the social spider *Stegodyphus dumicola*, by assaying individuals every 2 weeks, for a total of nine times over 4 months for three different behavioural phenotypes: boldness and fleeing in solitary settings, and prey capture latency in a social setting. Repeatable differences in boldness and fleeing were initially strong but faded within weeks and failed to predict behaviour later in life. Only fleeing remained moderately repeatable at the end of the study period. Prey capture in a social setting was not repeatable throughout the study period. Our results suggest that care must be taken when attempting to link short-term individuality, that is assumed to represent stable trait values, with ecological and evolutionary outcomes. We discuss the importance of quantifying both long-term repeatability and plasticity of behaviour, by using repeated assays across ontogeny in model organisms, to fully understand links between individuality and ecological and evolutionary processes.

© 2025 The Authors. 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/).

Behavioural individuality, sometimes described as animal personalities or behavioural types, is continuously being documented broadly across animal taxa, from both field and laboratory studies and across all major behavioural domains (Bell et al., 2009; Cabrera et al., 2021; Dochtermann et al., 2019). Recent meta-analyses imply that the existence of such behavioural individuality, defined as repeatable rank-order differences between individuals of the same species, represents a ubiquitous characteristic of most, if not all, animal populations (Bell et al., 2009; Cabrera et al., 2021; Dochtermann et al., 2019; Garamszegi et al., 2012). Furthermore, behavioural individuality has been linked to major ecological and evolutionary processes, such as invasion success (Fogarty et al., 2011), population dynamics (Wright et al., 2019) and speciation

(Ingley & Johnson, 2014). However, the study of repeatable behavioural differences is full of pitfalls, and conclusive documentation of ecological and evolutionary links with behavioural individuality requires robust study designs that test underlying assumptions and account for confounding effects. Responding to worrying trends of suboptimal study designs in scientific publications (Niemelä & Dingemanse, 2018), several guidelines have recently been published to urge researchers to (1) avoid using single, and instead use multiple, behavioural measurements to study individual differences (Dingemanse & Wright, 2020; Niemelä & Dingemanse, 2018), (2) distinguish between genuine individuality versus pseudopersonality that can occur due to correlations with environmental effects (Niemelä & Dingemanse, 2017; Wilson, 2018), (3) avoid biased research designs that prevent a disentangling of plasticity and repeatability (Dingemanse et al., 2010; Dingemanse & Wright, 2020) and (4) carefully consider ontogenetic changes in behaviour when designing the experiment (Cabrera et al., 2021).

<footnote><sup>a</sup> School of the Environment and Life Sciences, University of Portsmouth, Portsmouth, U.K.</footnote>

<footnote><sup>b</sup> Université Paris-Saclay, INRAE, AgroParisTech, UMR EcoSys, Palaiseau, France</footnote>

<footnote><sup>c</sup> Department of Biology, Aarhus University, Aarhus, Denmark</footnote>

<footnote><sup>d</sup> Behavioural Ecology, Faculty of Biology, LMU Muenchen, Planegg-Martinsried, Germany</footnote>

<footnote>* Corresponding author.
E-mail address: lena.grinsted@port.ac.uk (L. Grinsted).</footnote>

<footnote n="1">¹ Shared senior authors.</footnote>
The latter recommendation arose from a meta-analysis showing that most animals subjected to longer-term studies across developmental stages show behavioural consistency within a specific life stage but rarely across stages (Cabrera et al., 2021). This means that, as a rule, behavioural types as established based on a relatively short-term study cannot be expected to represent long-term or life-long behavioural types (Cabrera et al., 2021). This is an important point because often individuality is studied over very short periods of time; for example, over just a few days. Assessing short-term individuality in this way might be relevant if the study predicts that behaviour in a very specific life stage is linked with fitness, and if life stage is controlled for in the experimental design. A problem arises, however, when short-term behavioural types are treated as a fixed and stable trait and correlated with evolutionary or ecological outcomes, without testing the underlying assumption that the behaviour remains repeatable in the long term (Cabrera et al., 2021; Dingemanse & Wright, 2020; Grinsted & Bacon, 2014). This pitfall may result in spurious conclusions about the link between behavioural individuality and major evolutionary or ecological outcomes.

To illustrate this, imagine a behavioural trait of an animal where rank-order differences between individuals are repeatable over the long term (e.g. across several developmental stages of the species), and we wish to test whether individuality correlates with fitness (Fig. 1a). If we assess individuality over a short period and correlate average individual behavioural scores with fitness, we will get the same result whether behaviour was assessed in a juvenile stage (Fig. 1b) or during adulthood (Fig. 1c). However, in many species,

individuality is not consistent across ontogeny (Cabrera et al., 2021) and individual differences that are strongly repeatable in the short term may reshuffle in the long term (Fig. 1d). If we simply assume that behaviour is repeatable in the long term without empirically testing this assumption, we risk reporting erroneous results. In that case, short-term individuality in early adulthood (Fig. 1e) may show very different correlations with fitness than individuality assessed in late adulthood (Fig. 1f). Hence, if studies aim to link short-term behavioural individuality with ecological or evolutionary consequences, then developmental stage must be controlled for in the experimental design, and predictions and results should be reported carefully to specify which life stage is expected to correlate with fitness. Alternatively, the assumption that short-term individuality persists in the longer term must be tested by repeatedly assaying behaviour over as long a period of time as is ecologically relevant (Cabrera et al., 2021).

In this study, we test the assumption that long-term individuality is maintained across ontogeny in the social spider *Stegodyphus dumicola*. Social spiders are cooperative breeders characterized by permanent group living, allomaternal brood care and cooperative hunting, whereas subsocial spiders are temporarily social only as juveniles in their maternal nest before they disperse to live and breed solitarily (Grinsted & Lubin, 2019; Lubin & Bilde, 2007). Over the last decade, both social and subsocial spiders of the genera *Anelosimus* and *Stegodyphus* have become popular for exploring major ecoevolutionary links with behavioural individuality (e.g. personality-based group selection, Pruitt et al., 2017), negative frequency dependent selection (Lichtenstein &

<figure><img src="image_3.png" /></figure>

**Figure 1.** In a hypothetical study linking behavioural individuality with an ecological or evolutionary outcome, the conclusion will be affected by the timing of the behavioural assays if an assumption of long-lasting, rank-order, individual differences is violated. This illustration depicts hypothetical behavioural reaction norms of two different scenarios where behavioural individuality is maintained either (a–c) over the long term, or (d–f) over the short term only. Behaviour is assayed only over a fraction of the entirety of the experimental period in this research paradigm. This illustrates how conclusions reached might differ depending on whether behaviour was assessed (b, e) in the beginning of the study, or (e, f) towards the end of the study. A relevant fitness outcome is here illustrated on a scale from low (purple) to high (yellow). Mean–level behavioural phenotypes in the population decrease similarly over time in (a) and (d). The population is represented by five individuals (in a–f).
Pruitt, 2015; Pruitt et al., 2019), cyclone-induced selection (Little et al., 2019) and adaptive task differentiation (Keiser, Jones, et al., 2014; Pruitt & Riechert, 2011). Often, spiders of any developmental stage are assayed a single time, or multiple times within a few days to determine their personality, stating that behavioural scores are highly consistent over time (Keiser et al., 2015; Keiser, Howell, et al., 2016). Next, a treatment is implemented, such as manipulating the composition of behavioural types within groups. After a longer time period (e.g. several months), the resulting individual and/or group success is then correlated with pretreatment behavioural types (Lichtenstein et al., 2017; Pruitt et al., 2018, 2019). When age or developmental stage of animals is not controlled for, the implicit assumption is that rank-order differences between individuals remain repeatable for more than a week. In other words, it is then assumed that behavioural scores measured at any developmental stage can represent fixed trait values that are maintained during an ecologically relevant time frame during which individuality is hypothesized to affect fitness (Fig. 1).

However, evidence suggests that perhaps behavioural types do not remain fixed over the long term in social and subsocial spiders. In the social spider *S. sarasinorum*, repeatability of boldness drops to just *R* = 0.10 when assessed over a relatively long time period of 51 days (versus *R* = 0.32 over 4 days, Parthasarathy et al., 2019), and the link between boldness and task differentiation in foraging events disappears when controlling for hunger state (Parthasarathy et al., 2022). In the subsocial spider *A. studiosus*, the commonly assessed behavioural trait 'boldness' can vary nearly as much within individuals over the course of a day as it varies between individuals, and is, furthermore, strongly affected by reproductive status (Watts et al., 2015). While the subsocial spider *S. tentoriciola* showed moderate repeatability in boldness over 30 min intervals (*R* = 0.28–0.37), individuality severely weakened after just 1 week (*R* = 0.086, Settepani et al., 2015).

One of the social spiders most used in studies on behavioural individuality is the African velvet spider *S. dumicola* (Keiser et al., 2016; Keiser, Jones, et al., 2014; Keiser, Modlmeier, et al., 2014; Pruitt et al., 2018, 2019), and yet the implicit assumption that short-term individuality is maintained in the long term has to date not been tested in this species. Like other social *Stegodyphus* species, this spider has an annual life cycle where similarly aged juveniles maintain communal nests and forage cooperatively (Kullmann, 1972). After sexual maturation and within-colony mating, breeding females and allomothers care for colony offspring together (Junghanns et al., 2017, 2019). To test for long-term individuality, we assayed 133 juvenile spiders once every 2 weeks for a total of nine times over a 4-month period spanning a large proportion of their approximately 1-year life span. This 4-month period is ecologically relevant as it is the time after juveniles have gained independence from maternal care, where they grow and cooperate in communal tasks like prey capture, and before sexual maturity and the start of the breeding stage (Kraus & Kraus, 1988; Kullmann, 1972).

We assayed three behavioural traits to cover several biologically relevant phenotypes in social spiders. In addition to the classical boldness measure, where spiders receive a puff of air, we also developed an assay for fleeing behaviour, representing an immediate threat anticipated to elicit a stronger antipredator response than boldness. We designed this assay to include a short drop to the ground, which may be more biologically relevant to spiders than receiving a puff of air, as spiders naturally let themselves drop when perceiving an immediate threat. In highly social species, though, individuality might be affected by, or expressed only within, a social context (van Oers et al., 2005) and a key question for cooperative species is whether behavioural individuality affects task participation (Gordon, 1996; Lubin, 1995; Settepani et al.,

2013). Hence, in the third assay we assessed individual prey attack behaviour during group foraging. Boldness and fleeing were measured as latencies to move again after huddling in response to a threat, while prey attack was measured as individual latency to attack a prey item in a group setting. We asked two key questions. (1) Are individual differences in behaviour repeatable long term, through ontogeny? (2) Does an individual's initial behaviour predict its behaviour later in life?

## METHODS

### *Study System and Laboratory Set-Up*

The social spider *S. dumicola*, family Eresidae, occurs in central and southern Africa (World Spider Catalog, 2021) where it forms social groups (colonies) with up to hundreds of colony members (Wickler & Seibt, 1993). Social spiders are cooperative breeders, with female colony members cooperating in prey capture, feeding, web building and brood care (Aviles & Guevara, 2017; Grinsted & Lubin, 2019; Lubin & Bilde, 2007). Although reproductive skew and differences between individuals in the extent to which they participate in group activities tend to occur in social *Stegodyphus* spiders, traditional dominance hierarchies and contest competition appear to be lacking (Beleyur et al., 2015; Grinsted & Bilde, 2013; Grinsted et al., 2014; Junghanns et al., 2017; Parthasarathy et al., 2022; Settepani et al., 2013). Mothers and nonbreeding allomothers regurgitate food for hatchlings and end up allowing juveniles to feed on their bodies (gerontophagy, Junghanns et al., 2017; Seibt & Wickler, 1987). Offspring stay in their natal nest until they mature and breed with their relatives, producing highly inbred colonies with extraordinarily low genetic diversity (Settepani et al., 2014, 2017).

Nine spider colonies were collected in May 2012 from Pongola Game Reserve, KwaZulu-Natal, South Africa (permit no. OP 1896/2012), and transported to the laboratory in pillowcases. Here, their nests were carefully dissected and spiders transferred, with their colony mates, to enclosures where they produced broods in April 2013. Spiderlings were left undisturbed in their natal colony for about 3 months until the end of the maternal period (after gerontophagy, Seibt & Wickler, 1987). We then selected 168 spiderlings and divided these into 28 experimental groups, each containing six familiar individuals, and placed each experimental group in a plastic box (11 × 11 cm and 6 cm high) with a mesh lid. Spiders were kept in a climate room with constant temperature (25 °C) and daily light fluctuation (12:12 h light:dark cycle). Both source colonies and experimental spider groups were fed live flies (*Drosophila melanogaster* and *Musca domestica*) ad libitum and sprayed with water once a week.

Behavioural assays were conducted from August to December 2013. To distinguish individuals, spiders were marked with colour dust only when too small for wet paint to be used safely, and later, in addition to colour dust, a dot of water-based, nontoxic acrylic paint on the abdomen. A pilot study revealed no harmful effects of this marking method. Each time a spider moulted, the colour was reapplied. If two or more spiders in a group had moulted at the same time, small amounts of coloured dust still remained on their bodies, allowing us to track their identity and reapply their colour. Juvenile males and females cannot be distinguished morphologically, but we noted if a spider moulted into a subadult, and later adult, male. Females mature at a slower rate and did not reach sexual maturity during the study period. Social spiders have a strongly female-biased sex ratio and males do not contribute to cooperative prey capture and brood care (Aviles et al., 2000; Bechsgaard et al., 2019). Behavioural individuality is therefore expected to differ between the sexes, and here we focus on the
cooperative sex only. Hence, the nine spiders that turned out to be male during the experimental period were excluded from statistical analyses.

## Behavioural Assays

Three behavioural assays were each performed a total of nine times across 115 days at roughly 2-week intervals (every 13–16 days): solitary boldness, solitary fleeing and prey attack in a social context. Boldness and fleeing aimed to elicit behavioural responses to threats of varying intensity: a puff of air mimicking an approaching avian predator (boldness) versus dropping to the ground upon perceiving an immediate threat of predation (fleeing). Boldness is the behavioural trait most commonly used in studies on spider behavioural individuality (Parthasarathy et al., 2019, 2022; Pruitt et al., 2017; Settepani et al., 2015). Fleeing was intended to mimic situations in nature where spiders naturally let themselves drop to the ground upon encountering an immediate threat of predation (Edmunds, 1974). Boldness and fleeing were assayed in a solitary, standardized setting to exclude any effects of environmental or social stimuli. As our aim was to draw broadly applicable conclusions, we also assayed the spiders in a social context. Social spiders capture prey cooperatively in a group-spun web and so we included a behavioural assay of prey attack in a social group to test for replicability across several behavioural domains. The participation in cooperative prey attack is of critical ecological importance for social spiders, and so the presence of individuality in this behaviour could have profound consequences for the evolution of task differentiation in spiders (Beleyur et al., 2015; Grinsted & Bacon, 2014; Grinsted & Bilde, 2013; Grinsted et al., 2019, 2020, 2022; Lubin, 1995; Parthasarathy et al., 2022; Settepani et al., 2013, 2015).

### Boldness: latency to move after perceiving a threat

Boldness assays were conducted in a solitary context in a Petri dish with sawdust. It was carried out immediately after the fleeing assay (see below). Boldness was recorded by administering a puff of air from an infant ear-cleaning bulb directed towards the prosoma of the spider (Riechert & Hedrick, 1993), after which spiders reacted by huddling. Boldness was recorded as the latency to resume movement and move one body length, measured to the nearest full second. Spiders that were quicker to resume movements were considered bolder than spiders with higher latencies. If a spider had not resumed movement within 10 min it was given a boldness score of 600 s (18 cases out of a total of 913 observations). We provide results from analyses that included the full data set, as boldness scores of 600 s occurred in <2% of all observations. Excluding these observations did not change our conclusions (see Supplementary Material for model results). After this assay, spiders were returned to their experimental groups.

### Fleeing: latency to move after being dropped

Fleeing assays were conducted in a solitary context. An individual was removed carefully, using soft forceps, from its experimental group, and then dropped into a new environment, a Petri dish filled with wood dust, from a height of 10 cm, after which individuals immediately huddled. Fleeing was recorded as the latency to resume movement and move one body length, measured to nearest full s. If a spider had not resumed movement within 10 min it was given a fleeing score of 600 s (50 cases out of a total of 913 observations). As above, fleeing scores of 600 s represented only a small fraction of total observations and removing these observations did not affect our conclusions (see Supplementary Material for model results). After this assay, while still in the Petri dish, the boldness assay was conducted (see above).

### Prey attack: latency to attack in cooperative hunting

Prey attack assays were conducted in a social context and were never conducted on the same day as the boldness and fleeing assays. A live prey item (either a fruit fly, *D. melanogaster* or a house fly, *M. domestica*, according to the body size of the spiders) was introduced into the middle of the capture web spun by each experimental group of six spiders. Fruit flies were used only when spiderlings were deemed too small to handle a house fly. The species of fly was expected not to affect individual differences in prey capture behaviours as all Dipterans are considered easy and nondangerous prey (Grinsted et al., 2022; Sharpe & Avilés, 2016). We recorded the latency for each spider in the group to attack the prey to nearest full s. The group was observed for a maximum of 10 min. If no spider had attacked within 10 min, prey attack was deemed unsuccessful and excluded from data analyses (25 cases out of 219 prey attack sessions). If an individual spider had not attacked within 10 min of a successful prey attack event it was given a prey attack score of 600 s (five cases out of a total of 530 observations). As above, prey attack scores of 600 s represented only a small fraction of total observations and removing these observations did not affect our conclusions (see Supplementary Material for model results).

## Statistical Analyses

All analyses were performed using R version 3.6.2 (R Core Team, 2021) and the Bayesian package brms (Bürkner, 2017).

We excluded males ($N_{individuals} = 9$) from all analyses as females are the cooperative sex in social spiders (Lubin & Bilde, 2007). Exclusion of males combined with mortality after the set-up of experimental groups but prior to the first behavioural testing resulted in $N_{individuals} = 133$ at test 1. Spiderling mortality naturally occurs in an experiment spanning several months, resulting in $N_{individuals} = 131$ at test 3; $N = 98$ at test 6; $N = 55$ at test 9.

We used two different approaches to address our two research questions as described below. We did not test for behavioural syndromes (correlated behavioural traits) because our focus was on the temporal stability of among-individual variance and repeatability. Furthermore, we found strong random slope components in all three behaviours, meaning that we could point to no single behavioural values to correlate and behavioural correlations would therefore change strongly over time (Mitchell & Houslay, 2021).

### Are individual differences in behaviour repeatable long-term?
(Question 1)

To address this question, we used an individual reaction norm approach (Dingemanse et al., 2010). We assessed temporal changes in the magnitude of between-individual differences by fitting random regressions (Schaeffer, 2004) across all nine behavioural measures, for each of the three behavioural traits. Thereby we assessed whether the among-individual variance in behaviour increased, decreased or remained stable over the 4-month period (reaction norm approach, see below).

### Does initial behaviour predict behaviour later in life? (Question 2)

We addressed this question using a sequential approach. We calculated eight separate repeatabilities, each based upon two behavioural measurements of the ca. 130 spiders, namely the first and then one of the eight subsequent measurements. The resulting repeatabilities thus represent increasing time intervals. We did this for each of the three behavioural traits (sequential approach, see below). Because we are using a Bayesian framework, multiple testing is not an issue in the same way as it would be if using frequentist approaches (Scott & Berger, 2006; Sjölander & Vansteelandt, 2019).
## Long-term repeatability: the reaction norm approach

We used random regression analyses to estimate the pattern of variation in individual reaction norms (Dingemanse et al., 2010). For each behaviour, we fitted a model with ‘days since first sampling’ as a fixed effect and random intercepts of ‘colony of origin’, ‘experimental group’ and ‘individual’. Due to mortality, not all individuals were measured the same number of times. Thus, we added each individual’s ‘last day of sampling’ as an additional fixed effect to prevent bias in the estimated reaction norms (van de Pol & Verhulst, 2006). We added random slopes for the effect of ‘day’ to estimate the correlation between individual intercepts and slopes.

For each behaviour, we specified two versions of the described model: one where we left-centred ‘day’ so that the first day of measurement represented the model’s intercept (i.e. setting the first day as 0 on the X axis), and a second model where ‘day’ was instead right-centred with the last day of measurement representing the model’s intercept (i.e. setting the last day as 0 on the X axis and expressing each measurement as the deviation from this intercept). Models assumed normal distribution of errors, which we confirmed by visual inspection of residual distributions.

This approach enabled us to estimate the variation among individuals ($\sigma_{ID}$) at two separate points in time (the beginning versus the end of the experimental period) while controlling for additional sources of variation. This is due to the inclusion of random slopes for individuals over time: the value for among-individual variance will then depend on where it is estimated along the X axis (Schielzeth & Nakagawa, 2022; Xu et al., 2023).

Specifically, we estimated the following parameters in addition to $\sigma_{ID}$: the population-average change in behaviour with time (fixed effect of ‘day’), variation among experimental groups ($\sigma_{group}$) and variation among source colonies ($\sigma_{colony}$), the variation in individual slopes ($\sigma_{slope}$), the residual variation ($\sigma_{R}$) and the slope-intercept correlation ($r_{ID*slope}$). These values were estimated as standard deviations, which we squared to convert to variances. We then calculated repeatability as: $\sigma_{ID}^2 / (\sigma_{ID}^2 + \sigma_{R}^2)$ for both left-centred and right-centred models to compare repeatability at the beginning and at the end of the behavioural sampling within the average experimental group and source colony. Following Royauté and Dochtermann (2021), we then estimated the difference between left- and right-centred repeatability by taking the difference in posterior distributions ($\Delta R = R_{starting} - R_{ending}$). We based our inference on whether the 95% credible intervals (CI) for these estimates overlapped with 0. Because in some cases credible intervals had minor overlap with 0, we also report the probability of direction of the effect ($P_{mcmc}$, Makowski et al., 2019 a; Makowski, Ben-Shachar, Chen, et al., 2019). This metric indicates the probability of an effect’s existence and is calculated as the proportion of posterior estimates of the same sign as the median. Higher $P_{mcmc}$ values indicate more certainty in the direction of the effect (positive or negative).

In addition, the sign and magnitude of $r_{ID*slope}$ indicate how fast between-individual differences in behaviour decline ($r_{ID*slope} < 0$ resulting in a ‘fanning in’ pattern) or increase ($r_{ID*slope} > 0$ resulting in a ‘fanning out’ pattern) over the 4-month period. We based our inference on whether the 95% credible intervals for these estimates overlapped with 0.

Importantly, our models assumed that the residual variance was stable over time. We tested this assumption by comparing the Widely Applicable Information Criterion (WAIC) values (Watanabe, 2010) between the described models and additional models where we allowed the residual standard deviation to change as a function of days (modelled on a log scale). These more complex models did not perform better than the original models and had comparable point estimates values (see Supplementary Material, Tables S1 and S2). We therefore based our interpretation on the models assuming homogeneous residuals over the sampling period.

## Predicting behaviour later in life: sequential approach

Our first approach modelled changes in among-individual variance over time. However, rank-order differences between individuals can persist despite among-individual variance increasing or declining over time. We therefore tested whether each repeat measure could be predicted by the first behavioural measure. To do so, we fitted mixed effect models on pairs of measurements with increasing time from the first measurements. This resulted in nine different models (a model with assay 1 and 2 only, with assay 1 and 3 only, and so on) for each of the three behaviours tested. We fitted each model with the same structure as described above but excluded the random slopes terms as random slopes cannot be estimated with only two data points per individual. This approach allowed us to estimate the correlation (expressed as a repeatability value, $R = \sigma_{ID}^2 / [\sigma_{ID}^2 + \sigma_{R}^2]$) between the first measure and later measures. Repeatability values close to zero imply that an individual’s behaviour assayed at the focal point in time is not predicted by initial behaviour. Whether two repeatability estimates differ significantly from one another can be inferred from the overlap or lack of overlap of their 83% credible intervals, a threshold that is equivalent to $\alpha = 0.05$ (Austin & Hux, 2002; MacGregor-Fors & Payton, 2013; Royauté & Dochtermann, 2021).

All models described above were fitted with four chains, with a minimum of 7000 iterations with 5000 warmup iterations and a thinning interval of 2. All models had satisfactory convergence diagnostics ($\hat{R} < 1.05$, effective sample sizes >400) along with normal distributions of errors (Stan Development Team, 2024). We used default flat priors as more informative ones (Gaussian (0,10) and Gaussian (0,100)) did not influence our results.

### *Ethical Note*

Spiders were not harmed in this study. A small number of spider colonies were collected from a field site with high abundance of this species, with no resulting effect on the local spider population. Spiders were kept under conditions in the laboratory that mimicked their natural settings and were fed ad libitum. When manipulating the animals, spiders were handled carefully and swiftly to reduce stress. The behavioural assays conducted were designed to induce only short-term stress and fear responses (minutes). As soon as assays had been conducted, spiders were returned carefully to their colonies, minimizing periods of disturbance and stress. Animals were collected with local permits (KwaZulu-Natal Permit no. OP 1896/2012) and formal ethical approval was not required for invertebrate studies.

## RESULTS

### *Are Individual Differences in Behaviour Repeatable Long Term?*

Our results showed a strong decline of individuality over time in boldness (Fig. 2a,d), as implied by a negative correlation between intercepts and slopes of individual reaction norms (Dingemanse et al., 2010; Nussey et al., 2007; $r_{ID*slope}$, 95% credible interval (CI): -0.99 [-1.00, -0.96]; Table S3). The decline in individuality was mainly attributable to the erosion of among-individual variance (Fig. S1) while spiders rapidly became bolder over the 4-month period ($\beta$, 95% CI: -0.63 [-0.89, -0.37]; Table S3). There was substantial repeatability in boldness at the start ($R = 0.41$, 95% CI: [0.29, 0.52]; Table S3) but a total lack of repeatability at the end of the 4-month period ($R = 0.03$, 95% CI: [0.00, 0.09]; Tables S3 and S4).

The results for fleeing mirrored the patterns from boldness of individuality fading over time, while latencies to resume movement generally decreased (Fig. 2b,e). Repeatability for fleeing decreased more slowly than boldness, though, dropping from $R = 0.46$ in the
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Raw data (a–c) and estimated individual reaction norms (d–f) for the three behaviours: (a, d) boldness (latency to recover from a puff of air); (b, e) fleeing (latency to recover from a drop); and (c, f) prey attack (latency to attack a prey item in a group setting). In (d–f), each line represents a linear model fitted to a given individual's behavioural data. Random noise is applied to scatterplots (a–c) to better visualize clustered observations.</figcaption></figure>

beginning to *R* = 0.23 at the end of the sampling period (Δ*R*, 95% CI: 0.24 [−0.03, 0.49]; *P*<sub>mcmc</sub> = 0.95; Tables S3 and S4).

Prey attack behaviour was repeatable neither at the start nor at the end of the 4-month period (*R* start, 95% CI: 0.03 [0.00, 0.13] versus *R* end, 95% CI: 0.04 [0.00, 0.20]; Fig. 2c,f, Tables S3 and S4).

*Does Initial Behaviour Predict Behaviour Later in Life?*

Our sequential approach revealed that the first boldness mea-
sures could predict boldness assayed up to 2 weeks later (R = 0.47,
95% CI: [0.29, 0.62]; Fig. 3a) but not behaviours assayed 4 weeks to
4 months later (each R < 0.01; Fig. 3a, Table S5). Equally, the initial
fleeing measures could predict later behaviour only within the first
4 weeks, where R > 0.27, and not the remaining weeks, where
R < 0.07 (Fig. 3b, Table S5). An individual's initial prey attack
behaviour also failed to predict behaviours later on (R gener-
ally < 0.05; Fig. 3c, Table S5).

DISCUSSION

We found that social *S. dumicola* spiders showed strong, short-
term behavioural individuality, but much weaker long-term in-
dividuality. Between-individual differences in both boldness and
the related fleeing behaviour were highly repeatable in the begin-
ning of the experiment, when young juveniles were assayed in 2-
week intervals (*R* = 0.41 and *R* = 0.46 respectively). However, at
the end of the 4 months boldness was no longer repeatable
(*R* = 0.03), while the repeatability of fleeing behaviour had also
reduced substantially, but was still moderate (*R* = 0.23). Partici-
pation in prey attack was repeatable neither in the short nor in the
long term (*R* ≤ 0.04). This is consistent with studies in the closely
related social spider species *S. sarasinorum* showing that, while the

same group members may engage in prey attack over 2–3 days
(Settepani et al., 2013), hunger state, not behavioural individuality,
is the main determinant of prey capture behaviour (Parthasarathy
et al., 2022). Hence, personality-based task differentiation is un-
likely to occur in social spiders.

Importantly, we also found that the initially measured behav-
iours were unable to predict behaviours assayed more than 4 weeks
later. In other words, behaviours as recorded from a single mea-
surement early on did not correlate with behaviours measured later
on. This was true for all three behaviours assayed (R ≤ 0.07 for
pairwise comparisons of first behavioural scores with all other
scores at >4 weeks in boldness, fleeing and prey attack). This
finding is important, as it suggests that even with moderately long-
term repeatable behaviours, such as fleeing in our study, a single
assay, or a few assays covering a short timescale, are insufficient to
establish a consistent behavioural score, or personality, of an in-
dividual. As discussed in the introduction (Fig. 1), links between
individuality and major evolutionary and ecological outcomes will
be affected by the frequency and timing of behavioural assays.

Hence, our results strongly support previous recommendations
to establish individuality using multiple, rather than single, assays
(Dingemanse & Wright, 2020; Niemelä & Dingemanse, 2018) and to
study the development of individuality in a chosen study organism
across ontogeny (Cabrera et al., 2021). Furthermore, quantifying not
only long-term repeatable differences, but also how plasticity
changes over time, is needed to establish genuine links between
behavioural individuality and fitness. This is because both an in-
dividual's behavioural type and plasticity can be under selection,
and it is important to design experiments to disentangle both ex-
planations (Araya-Ajoy et al., 2015; Dingemanse et al., 2010;
Dingemanse & Wolf, 2010; Niemelä et al., 2021). For example, a
potential correlation between initial behaviour (reaction norm
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Results taken from linear mixed models showing that initial behaviour failed to predict behaviour later in life, leading to a decline in repeatability over time. Data points at each time point result from always comparing the phenotypic scores from the initial behavioural assay with those from the later assay; that is, comparing assay 1 with each of assays 2 through 9. (a–c) Behavioural repeatability ($R = \sigma_{ID}^2 / [\sigma_{ID}^2 + \sigma_{R}^2]$); (d–f) among-individual variance ($\sigma_{ID}^2$); (g–i) residual variance ($\sigma_{R}^2$) for the three behaviours: (a, d, g) boldness (latency to recover from a puff of air); (b, e, h) fleeing (latency to recover from a drop); (c, f, i) prey attack (latency to attack a prey item in a group setting). Thick error bars represent the 83% credible intervals for the posterior distribution of variance and repeatability estimates, thin error bars indicate 95% credible intervals. Inference is based on overlap of 83% credible intervals.</figcaption></figure>

intercept) and age-dependent plasticity (reaction norm slope) can give the impression that selection acts on initial behaviour, when selection may instead actually act on an individual's behavioural plasticity (Stamps & Groothuis, 2010; Wuerz & Krüger, 2015).

Boldness is a commonly used metric for individuality, or personality, in spiders, and has in *S. dumicola* been linked with ecological outcomes such as the emergence of colony-level individuality (collective personality) that affects group hunting and defence behaviours (Hunt et al., 2018; Lichtenstein et al., 2019; Modlmeier et al., 2014; Pruitt et al., 2018; Wright et al., 2016), disease transmission (Keiser, Howell, et al., 2016; Keiser, Wright, et al., 2016) and social organization and task differentiation (Keiser, Jones, et al., 2014; Lichtenstein et al., 2017). A common procedure is to categorize spiders as ‘bold’ or ‘shy’ if they show boldness scores in the range of 0–200 s and 400–600 s, respectively (Hunt et al., 2018; Pruitt et al., 2019). Our results, again, suggest that categorizing individuals as shy versus bold based on a single, or a few, assays should be treated with caution. Indeed, boldness scores were not repeatable in the long term in our study. Boldness scores reshuffled substantially over time and spiders

generally became ‘bolder’ over time so that 100% of spiders would have been categorized as ‘bold’ during the later stages of the juvenile period in our study, as all spiders expressed boldness scores <200 s at the end (Fig. 2a). It should be noted, however, that our boldness assays were always performed immediately after our fleeing assay, and so we cannot rule out that our boldness scores have been affected by the preceding assay. Both boldness and fleeing assessed responses to threats of varying degrees, and both recorded latencies to resume movement. Hence, perhaps our fleeing results, which are not affected by immediately preceding manipulations, are more directly comparable to boldness as reported in other studies. However, even in our fleeing assays we saw a decrease in mean-level latencies over time and the vast majority of spiders ended up with latencies <200 s at the end (Fig. 2b).

Mean-level changes over time are common in both animals and humans (Roberts et al., 2006; Stamps & Groothuis, 2010; Wuerz & Krüger, 2015) and do not preclude the persistence of individuality if rank-order differences between individuals are maintained. For example, in humans, substantial mean-level changes occur at various stages throughout our life span in several personality traits,
while between-individual differences persist and even become stronger with age (Roberts & DelVecchio, 2000; Roberts et al., 2006). Thus, personality assays conducted early in people's lives can predict individual personality decades later, despite substantial mean-level changes over time (Roberts & DelVecchio, 2000). In S. dumicola, however, early behaviours failed to predict any behaviours later than 2–4 weeks after. Our results showed that rank orders were reshuffled over time while boldness, fleeing and prey attack behaviours converged and individuals displayed increasingly similar behavioural phenotypes. A reduction in repeatability of behaviour with increasing time interval is a common phenomenon because an individual assayed over a longer time span is more likely to differ in state, in developmental stage and in the expression of the underlying set of genes affecting the phenotype (Bell et al., 2009; Roberts & DelVecchio, 2000; Stamps & Groothuis, 2010). A slight decrease in repeatability over time does not preclude the presence of long-term individuality, nor does it preclude the possibility of individuality to affect ecological and evolutionary processes (Stamps & Groothuis, 2010). In contrast, the strong erosion of repeatability that we document here puts into question the potential for selection to act on individual differences in S. dumicola.

A key question is whether social spider populations harbour heritable variation in behaviour as a requirement for evolutionary repercussions (Dingemanse et al., 2010; Dingemanse & Réale, 2005; Dingemanse & Wolf, 2010). The presence of behavioural individuality in a species does not necessitate a genetic, heritable component. Repeatable between-individual differences in behaviour can emerge in populations of genetically identical individuals, for example, due to stochastic between-individual differences in experience and internal state combined with costs of flexibility and benefits of behavioural niche partitioning (Bergmüller & Taborsky, 2010; Dall et al., 2004). However, when a behavioural phenotype is highly plastic and entirely determined by environment rather than genotype, there are no genes coding for behavioural individuality for selection to act on (Dingemanse & Réale, 2005). The application of quantitative genetics has confirmed the existence of heritable suites of genetically correlated behaviours in bird (Duckworth & Kruuk, 2009; van Oers et al., 2004), insect (Han & Dingemanse, 2017; Santostefano et al., 2017) and fish (Bell, 2005; Dingemanse et al., 2019) models. However, recent comparative population genetics analyses imply this may not be the case in social spiders (Settepani et al., 2017). Social spiders reproduce with close relatives within their group, and colonies experience frequent extinction and colonization events (Settepani et al., 2014). Strong effects of genetic drift therefore result in extremely low individual and population genetic variation (Lubin & Bilde, 2007; Settepani et al., 2017). It is therefore unlikely that heritable genetic differences between individuals in behavioural types can develop and be maintained (Settepani et al., 2016).

Our findings lead to the question of whether adaptive mechanisms underpin the observed changes in repeatability of individual behaviours over time. One explanation invokes the asset protection principle (Clark, 1994; Sih et al., 2015). This principle predicts that individual differences in risk-taking behaviours result from adaptive behavioural regulation in response to individual variation in expected reproductive fitness (residual reproductive value RRV, Wolf et al., 2007). Individuals with high RRV should be shy in order to protect their reproductive assets compared to individuals with low RRV, which have little to lose from boldly risking predation while acquiring new resources (Wolf et al., 2007). An implicit prediction, experimentally confirmed in wild passerine birds (Nicolaus et al., 2012), is that individuals should plastically upregulate, versus down-regulate, boldness as an adaptive response to decreased versus increased RRV (Dingemanse & Wolf, 2010). This means that adaptive behavioural plasticity is expected to lead to the

erosion of individual repeatability over time via negative feedback loops between state and behaviour, when risky (bold) behaviour increases RRV and cautious (shy) behaviour leads to a loss of RRV (Sih et al., 2015). As a consequence, random environmental effects causing initially repeatable individual differences in state (e.g. in RRV, Wolf et al., 2007), and thus in risk taking (e.g. boldness), are predicted to cause convergence among individuals in state and behaviour over time when negative feedback loops take place (McElreath et al., 2007; Sih et al., 2015). The resulting predicted pattern of fanning-in (Sih et al., 2015) is closely matched by our data (Fig. 2). Thus, one potential explanation of our results is that the initial behavioural repeatability we observed resulted from uncontrolled environmental variance (Dingemanse et al., 2010) inducing pseudorepeatability (Westneat et al., 2011); that is, the appearance of behavioural individuality where none exists (Niemelä & Dingemanse, 2017), which subsequently faded over time.

An alternative explanation for the patterns of variation we observed is that the initial assays captured genuinely repeatable individual variation in responses to novelty, but that all spiders quickly habituated to the stimulus, thereby increasingly hiding any repeatable differences in novelty responses over time. We would argue against this habituation explanation for two main reasons. First, in a study on a related social spider, S. sarasinorum, moderate repeatability of boldness was recovered based on a high number of assays (six) conducted across a relatively short time period (21 days; R = 0.32), while the same number of assays (six) conducted over a much longer time scale instead produced much lower repeatability (51 days; R = 0.10; Parthasarathy et al., 2019). Second, a meta-analysis conducted across taxonomic groups and covering a range of behavioural phenotypes suggested that repeatability estimates are generally not affected by the number of times animals are assayed (Bell et al., 2009). Together, this strongly suggests that age or development, not habituation, causes behavioural individuality in social spiders to greatly weaken or entirely evaporate.

The way both mean-level behavioural phenotypes and between-individual behavioural differences change over an ecologically relevant time frame, during which selection is predicted to act, is crucial for understanding whether and how evolution has shaped individuality and the interplay between repeatability and plasticity of behaviour. In an emerging model for studying the link between behavioural individuality and major ecological and evolutionary consequences we find that short-term assessed behaviour is not stable in the long-term and does not correlate with behaviour later on. Hence, testing the underlying assumption that short-term behaviour can be used as a fixed trait, and is a reliable proxy for longer-term personality must be tested across ontogeny in model organisms. This is an important lesson for future studies aiming to link behavioural individuality with selection in any organism.

## Author Contributions

**Lena Grinsted:** Writing – review & editing, Writing – original draft, Visualization, Investigation. **Niels J. Dingemanse:** Writing – review & editing, Writing – original draft, Supervision, Investigation. **Raphaël Royauté:** Writing – review & editing, Visualization, Investigation, Formal analysis, Data curation. **Signe Eiserhardt:** Methodology, Investigation, Conceptualization. **Trine Bilde:** Writing – review & editing, Supervision, Project administration, Methodology, Conceptualization. **Virginia Settepani:** Methodology, Conceptualization.

## Data Availability

All data and code for analyses are available on the Open Science Framework's project for this article, at https://osf.io/xr8mb/.
Declaration of Interest

The authors declare that there are no competing interests.

Acknowledgments

Research permits were provided by KwaZulu Natal (permit no. OP 1896/2012). L.G. was supported by The Leverhulme Trust (Early Career Fellowship: ECF-2016-080). N.J.D. was supported by the German Science Foundation (grant IDs: DI 1694/1-2 & DI 1694/5-1).

Supplementary Material

Supplementary material associated with this article is available, in the online version, at https://doi.org/10.1016/j.anbehav.2025.123167.

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