Although aspects of an individual’s state are well-known to influence the expression of behavior, it is still unclear how elements of state affect consistent among-individual differences in behavior. With binary, irreversible elements of state, such as mating status, there may be optimal behavioral phenotypes before and after mating, with individuals often prioritizing mate acquisition before and resource acquisition after. Yet, limited plasticity may prevent optimal behavior in both contexts. Additionally, it remains largely unknown if some consistencies in neural or physiological traits may limit the ability of the organism to respond to state changes. In this study, we investigated how changes in a binary state variable, mating status, affected both the mean expression and among-individual variation in behavior and web structure of the redback spider, Latrodectus hasselti. Furthermore, we explored the role of biogenic amines in potentially mediating individual differences in behavior and web structure. We found that mated females were overall more aggressive than virgin females and also built webs structured primarily for capturing prey rather than safety. We also found that individual differences in behavior and web structure were maintained across mating statuses, which indicates the stability of these traits and may drive personality-specific state-dependent fitness trade-offs. Finally, we found that aggressive spiders had higher central nervous system dopamine levels. Interestingly, web structure was often correlated with a catabolite of tyramine (N-acetyltyramine), suggesting that variation in amine catabolism, and not the concentrations of the amines themselves, may drive individual differences in some traits.
Our results demonstrate that although individuals show plasticity in response to changes in state, specifically mating status, individuals also maintain among-individual differences across this state change. Thus, aggressive individuals before mating will tend to be aggressive after. This maintenance of individual differences across state may drive differential fitness benefits before and after mating for different behavioral phenotypes. Furthermore, we show that biogenic amines and their catabolites are related to individual differences, thus identifying a potential mechanism underlying consistent variation.
The state-dependent nature of behavior has long been a focus of behavioral ecology (McNamara and Houston 1996). Recently, there has been an interest in understanding how variation in individual state contributes to individual differences in behavioral and other traits. Yet, research on this topic has been narrow in scope, with a few studies investigating how state variables such as body condition (David et al. 2012; DiRienzo and Montiglio 2016a; DiRienzo and Montiglio 2016b) and pathogens (Coats et al. 2010; Kekäläinen et al. 2014; DiRienzo et al. 2016) affect individual differences. These studies have shown that not only do state variables often affect the mean trait expression in a population, but they also impact the level of among-individual differences in these traits. Yet, the state variables in these studies are all studies which are continuous in nature (e.g., body condition and pathogen exposure), highlighting the need to study events that result in a binary change in state, such as before and after mating. Such changes in state likely have large implications for the mean, variance, and among-individual consistency of behavioral tendencies expressed by a population. Furthermore, although there is extensive research on the hormonal mechanisms of behavior (Wingfield et al. 1987; Adamo et al. 1995; Stevenson et al. 2005; Clotfelter et al. 2007; Aonuma and Watanabe 2012), there is comparatively less work on the role of neurohormones, specifically the biogenic amines, in mediating consistent individual differences in behavior (but see Jones et al. 2011; DiRienzo et al. 2015). Collectively, integrated studies are needed to address whether individual differences are consistent across state changes while concurrently investigating the proximate mechanisms that may be responsible for these stable patterns of behavior.
Many animals, both male and female, may mate only once, possibly twice, in a breeding season or even within their lifetime (Larsen 1991; Andrade 1996; Bergström et al. 2002). Thus, such an event may represent a demarcation between different stages of the individual’s life cycle, whereby prior to mating, the organism invests in behaviors and traits that promote mate acquisition and/or attraction, yet post mating, there is a change such that the individual (typically female) now must invest in behaviors that focus on increasing reproductive output, as well as offspring quality and success. Such changes in behavior in response to mating status have been studied in wasps and moths, where after mating, females will become less responsive to male sexual signals (Anton et al. 2007; Ruther et al. 2007). This response is thought to be adaptive in that it reduces female effort towards mate searching and allows more time to search for oviposition sites (Ruther et al. 2007). Generally, studies investigating behavioral changes across a mating event have focused on changes in sexual signaling (e.g., birds: Amrhein et al. 2002, wasps: Ruther et al. 2007), while overlooking other traits that may be relevant after mating, such as increased foraging effort (but see Fauvergue et al. 2008). Thus, studies need to consider a broader range of traits in order to understand the potential adaptive significance associated with phenotypic changes in response to mating status. For example, before mating, females may spend increased time and effort assessing mates in order to pick a male with good genes or who will provide some form of direct benefit (Moore 1994; Møller and Alatalo 1999). Yet, after mating, the females may spend increased time foraging in order to increase offspring quality or number (Blount et al. 2002; Herberstein et al. 2002; Isaac et al. 2010).
While animals will exhibit trait plasticity in response to changes in state (Dill 1983; Nonacs 2001; Salomon 2009; Katz and Naug 2015), the question remains whether among-individual differences in behavior and other traits are maintained across changes in state. Although it has not been addressed in a mating context, researchers have shown that many organisms indeed show individual behavioral consistency from juvenile to adulthood (crickets: Fisher et al. 2015, marmots: Petelle et al. 2013, squid: Sinn et al. 2008), but this is not always the case (Petelle et al. 2013; Wuerz and Krüger 2015; Wexler et al. 2016). In the context of before/after mating, limits in trait plasticity may prevent individuals from displaying optimal phenotypes across state changes (Sih et al. 2004), thus driving the maintenance of among-individual differences throughout adulthood. Such carryover of individual differences coupled with correlations among similar traits (e.g., foraging aggression and precopulatory sexual cannibalism) may result in different relative fitness benefits for each behavioral phenotype before and after mating. For example, aggressive individuals may make poor mating choices, either by cannibalizing males prior to mating (Arnqvist and Henriksson 1997; Johnson and Sih 2005) or choosing a low-quality male, but possibly offset this cost via superior resource acquisition or increased protection from predators after mating. This, in turn, could have evolutionary implications as limited plasticity may aid in maintaining among-individual differences via personality-specific state-dependent fitness effects. In other words, non-aggressive phenotypes may maximize fitness before mating, while aggressive phenotypes may maximize fitness after mating.
A related, and more general, question is regarding what mechanism(s) underlie consistent individual differences both within and across contexts (Réale et al. 2010). A range of mechanisms has been proposed, the two most common being variation in metabolic and hormonal mechanisms. While the former has received mixed support (Timonin et al. 2011; Le Galliard et al. 2013; Myles-Gonzalez et al. 2015), the latter has a comparatively longer history demonstrating correlations between hormones and mean behavioral tendencies. In invertebrates, the biogenic amines, such as serotonin (5HT), dopamine (DA), octopamine (OA), and tyramine (TA), act as neurohormones, neurotransmitters, and neuromodulators and have been shown to be related to a range of behaviors, including aggression (Adamo et al. 1995; Stevenson et al. 2005), antipredator behavior (Miyatake et al. 2008; Nishi et al. 2010; Jones et al. 2011), and sociality (Wada-Katsumata et al. 2011; Aonuma and Watanabe 2012). Thus, variation in the expression and/or concentrations of these biogenic amines could drive the among-individual differences in behavior that are observed in a host of invertebrate taxa (Pruitt et al. 2008; Fisher et al. 2015; DiRienzo and Montiglio 2016a). Furthermore, if there are limits to the extent that the underlying neural architecture and aminergic systems can respond to changes in state, then among-individual differences would be expected to be maintained if there is indeed individual variation in the associated aminegeric system. Thus, biogenic amines are a primary target for investigating the proximate mechanism of individual differences in invertebrate behavior over time and across changes in state.
Widow spiders make an ideal system to study how behavioral plasticity and individual differences respond to changes in mating status. In many widows, particularly redback spiders (Latrodectus hasselti), females may mate only once or twice in their lifetime, and one mating provides enough sperm to produce thousands of offspring (Andrade 1996, ND pers. obs). Thus, mating represents a change in state, and one would predict a difference in pre/postmating behavior. Before mating, spiders may focus on mate acquisition and safety to ensure that they have some reproductive output, then on increased aggression and foraging after mating in order to maximize that output. Such changes may manifest themselves both in the individual’s behavioral expression and in the structure of its web. In the western black widow, Latrodectus hesperus, females are known to vary in their foraging aggression, such that some are more likely to attack a prey cue than others (DiRienzo and Montiglio 2016a). These individual differences in foraging behavior extend to individual differences in web structure, where more aggressive spiders build webs that include more gumfooted lines, which are long, sticky lines which aid in prey capture (Zevenbergen et al. 2008). In addition, less aggressive spiders invest more in denser webs with more structural lines, presumably to increase safety (Blackledge et al. 2003; DiRienzo and Montiglio 2016b). Finally, biogenic amines have been previously shown to be related to a range of arachnid behaviors (Jones et al. 2011; DiRienzo et al. 2015) and thus make an ideal target for studies on mechanisms of individual differences within and across states.
In this study, we use the redback spider, L. hasselti, to assess how individual behavior and web structure vary in response to differences in mating status. We conducted web building and foraging assays repeatedly on both virgin and non-virgin females. We also tested a subset of virgins who were later mated in the lab before and after mating. Furthermore, we assayed central nervous system biogenic amine and catabolite levels via high-performance liquid chromatography. At a species level, we predicted that virgin spiders will be less aggressive and build safer webs (e.g., denser, more structural lines) relative to non-virgins. We also predicted not only that both virgins and non-virgins would express consistent among-individual differences in both behavior and web structure, but also that females would express consistencies in behavior and web structure before and after mating. Finally, we predicted that mated spiders would have higher DA and OA levels, as these two biogenic amines are commonly associated with aggression in invertebrates (Adamo et al. 1995; Stevenson et al. 2005). Furthermore, given this common amine-behavior association, we predicted a positive relationship between aggressive behavior and the associated web characteristics (e.g., gumfooted lines) and DA and OA levels.
Materials and methods
We used field-caught redback spiders collected in the summer of 2015 in Fukuoka, Japan. We collected both immature and mature female and male spiders. The spiders were brought back to the laboratory at Hokkaido University (Sapporo, Japan), where they were provided with individual containers (7 cm high, 9-cm diameter) that were lightly sanded to facilitate web construction and a unique ID number. The spiders were provided two Acheta domesticus crickets per week (both approximately the same body size as the spiders) and were maintained at 27 °C on a 12:12 light to dark photoperiod.
All spiders were maintained on the standard conditions until the immature spiders matured. This occurred within one to two molts. All spiders that matured in the lab were therefore virgins, while those collected as adults in the field had previously mated (all laid at least one egg case while in the laboratory). After the immature spiders matured, all females were placed into individual web building containers (see web assay methods) and were allowed to build a web for 7 days. After 7 days, we assessed their aggression towards a prey cue nine times: three times within a day, 3 days in a row (see behavioral assay methods). After the third day of behavioral trials, we removed the spider from the web and placed it back in its home container. Each spider was then fed two crickets and allowed to feed for 5 days before starting the next round of behavioral and web assays. This sequence was repeated three times for all spiders; thus, they all built three webs and had their behavior assayed 27 times.
In order to experimentally test if differences in behavior and web structure were affected by mating status, we mated a subset of virgin female spiders (n = 7). The number was limited due to the few males present in the field and sexual cannibalism occurring after each mating trial. The mating occurred after the first round of web and behavioral assays. The virgin spiders were left on their web building structures, and a male was placed on the web, opposite the female’s retreat. Twenty-four hours later, the females were removed from their webs, placed in their home container, and fed two crickets. All matings were successful as indicated by the fact that the females immediately laid an egg case once placed back in their home containers. In total, we used 23 females that were mature in the field and 33 virgin females who matured in the lab (seven of which were later mated).
Spider web assessment
To assess the web structure, we provided spiders with a standardized structure on which to build. All spiders were transferred to a skeletonized cardboard box (L 24.5 × W 19 × H 10 cm). The box had three walls and all but 3 cm of the top removed, thus leaving a rectangular cardboard frame with the back, bottom, and the portion of the top walls remaining. The bottom and back walls were covered in black paper in order to ease the counting of individual web components. This structure provided a shelter along with a frame on which to build their web. This box was placed inside a plastic container (L 40.7 × W 28.5 × H 185 cm), and the spider was given 7 days to construct a web. These methods have been previously used to assess web structure in black widow spiders (DiRienzo and Montiglio 2016b; Montiglio and DiRienzo 2016). After 7 days of web construction, we removed the box and counted the total number of structural and gumfooted lines connected to the floor of the box. In order to get a simple measure of the level of building in three dimensions, we also counted the number of lines connected to the top rim of the box. In order to reduce any handling effects, the webs were assessed on the first day of the behavioral trials, but after the behavioral trials themselves were conducted.
After all behavioral trials, the spider was removed and we gathered the web by winding it onto a plastic rod. The webs were later weighed using a Mettler Toledo XS3DU microbalance. In the North American congener, L. hesperus, web weight is highly correlated (R 2 = 0.8) with web density (measured as the amount of reflectance from an illuminated web) and thus provides a measure as to the level of overall web investment (denser vs. sparse webs) (Blackledge and Zevenbergen 2007).
Spider foraging aggression assay
After being allowed to build a web for 7 days, we assessed spider aggression towards a prey cue. In order to remove any effect of prey behavior (DiRienzo et al. 2013), we used a standardized vibrating mechanism (Classical Silicone Vibrator, Liler, Shenzhen, China) (Grinsted et al. 2013). Attached to the vibrating mechanism was a 10-cm-long plastic cable tie, which allowed us to apply the vibratory cue in specific locations of the web while also reducing the intensity of the vibrations and risk of damaging the web. The vibrator provided 1-s-long pulses at 100 Hz separated by approximately half second-long periods of reduced frequency. This vibratory pattern and frequency are similar to the vibrations produced by houseflies caught in a spiderweb (Walcott 1963) and the frequency that has been shown to elicit a response in other species of spiders (Parry 1965). Spiders respond to the prey cue as they would to a prey trapped in their web. They rapidly approach the cue, then rotate 180° and quickly apply sticky silk in an attempt to subdue the apparent prey item. This response directly relates to prey capture success as individuals who attack the vibratory cue more often also capture more live prey in predator-prey interactions (unpublished data). The prey cue was applied three times, once near the shelter (within 2 cm), once at the end farthest from the shelter, and once in between. If the spider did not build in the predetermined location, the observation was left blank. The cue was held to the web for 15 s at each location, with 10 s separating each application. We recorded if spiders did or did not attack the prey cue in the 15-s interval as a binary response, and if they attacked, if they subsequently retreated back to their shelter as a binary response. The cue application was applied in a random order (e.g., near, far, between; far, between, near). This assay was repeated 24 and 48 h later. Previous work with L. hesperus revealed that females do not habituate to repeated application of the prey cue (DiRienzo and Montiglio 2016a; DiRienzo and Montiglio 2016b). All behavioral trials were conducted between 10:00 and 13:00 and in a randomized order within days in order to minimize any influence of diel rhythms. All assays were conducted by ND. It was not possible to be fully blind to mating status as spiders often had an egg case, thus indicating they were not a virgin.
Biogenic amine analysis
In order to measure biogenic amine levels, we used whole CNS measurements, similar to those that have been previously employed in other invertebrate systems (ants: Punzo and Williams 1994; Aonuma and Watanabe 2012, crickets: Nagao and Tanimura 1988; Nagao and Tanimura 1989; Puiroux et al. 1990; Iba et al. 1995). Furthermore, our analysis focuses on the primary biogenic amines (OA, TA, DA, 5HT) and their catabolites. One week after the behavioral trials were finished, we froze and dissected the spiders. We froze each spider quickly using liquid N2 and then dissected out the central nervous system (CNS) in an ice-cold saline solution (233 mM NaCl, 6.8 mM KCl, 8 mM CaCl2, 5.1 mM MgCl2, 10 mM HEPES, pH 7.8) (Höger et al. 1997). The tissue was collected into a glass homogenizer and homogenized in 50 μl of ice-cold 0.1 M perchloric acid containing 5 ng of 3,4-dihydroxybenzylamine (DHBA, Sigma, St. Louis, MO, USA) as an internal standard. After centrifugation of the homogenate (0 °C, 15,000 rpm, 30 min), we collected 40 μl of supernatant. We then measured the biogenic amines using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). The HPLC-ECD system was composed of a pump (EP-300, EICOM Co., Kyoto, Japan), an auto-sample injector (M-510, EICOM Co., Kyoto, Japan), and a C18 reversed-phase column (250 mm × 4.6 mm internal diameter, 5-μm average particle size, CAPCELL PAK C18MG, Shiseido, Tokyo, Japan) heated to 30 °C in the column oven. A glass carbon electrode (WE-GC, EICOM Co.) was used for electrochemical detection (ECD-100, EICOM Co.). The detector potential was set at 850 mV vs. an Ag/AgCl reference electrode, which was also maintained at 30 °C in a column oven. The mobile phase containing 0.18 M chloroacetic acid and 16 μM disodium EDTA was adjusted to pH 3.6 with NaOH. Sodium-1-octanesulfonate at 1.85 mM as an ion-pair reagent and CH3CN at 8.40% (v/v) as an organic modifier were added into the mobile-phase solution. The flow rate was kept at 0.7 ml/min. The chromatographs were acquired using the computer program PowerChrom (eDAQ Pty Ltd., Denistone East, NSW, Australia). The supernatant of the sample was injected directly onto the HPLC column. After the acquisition, we processed the chromatographs using PowerChrom to obtain the level of biogenic amines by the ratio of the peak area of the substances to the internal standard DHBA. We used a standard mixture for quantitative determination that contained amines, precursors, and catabolites. Twenty compounds at 100 ng/ml each were dl-3,4-dihydroxy mandelic acid (DOMA); l-β-3,4-dihydroxyphenylalanine (DOPA); l-tyrosin (Tyr); N-acetyloctopamine (Nac-OA); (−)-noradrenaline (NA); 5-hydroxy-l-tryptophan (5-HTP); (−)-adrenaline (A); dl-octopamine (OA); 3,4-dihydroxybenzylamine (DHBA, as an internal standard); 3,4-dihydroxy phenylacetic acid (DOPAC); N-acetyldopamine (Nac-DA); 3,4-dihydroxyphenethylamine (DA); 5-hydroxyindole-3-acetic acid (5-HIAA); (Nac-TA); N-acetyl-5-hydroxytryptamine (Nac-5HT); tyramine (TA); l-tryptophan (Trp); 3-methoxytyramine (3-MTA); 5-hydroxytryptamine (5-HT); and 6-hydroxymelatonin (6-HM). Nac-OA, Nac-DA, and Nac-TA were synthesized by Dr. Matsuo (Keio University, Japan). All other substances were purchased from Sigma. Nac-OA was not measured as we were unable to measure the associated peak in many samples. A sample chromatogram is provided in the supplementary materials. All dissections and HPLC analyses were done by HA who was blind to the spider’s mating status.
Relationship between mating status, web structure, and behavior
We assessed the role of mating status on behavior and web structure using generalized linear mixed models. These models included attack behavior, retreat behavior, web weight, and the number of gumfooted, structural, and rim lines as response variables, each of which was analyzed independently. The main effects for attack and retreat behaviors included mating status as a categorical variable (virgin, mated in the field, mated in the lab), spider weight, distance at which the prey cue was presented, and assay number within the week (e.g., 1, 2, or 3). Spider weight and distance were both centered to a mean of 0 and a standard deviation of 1. Models for the different web components had the same fixed effects minus those related to the behavioral assay (e.g., distance and assay number within week). Individual ID was included as a random effect in all models. Attack and retreat behaviors were modeled with binomial error distributions and logit links. The web weight was modeled with a Gaussian error distribution, while the rest of the web components were count data and thus modeled with Poisson error distributions. Models using a Poisson error distribution included an observation-level random effect (OLRE) to account for overdispersion (Harrison 2014). We calculated both the proportion of variance described by the fixed effects within the model (marginal R 2) and the proportion of variance described by both the fixed and random effects (conditional R 2) values for all models following Nakagawa and Schielzeth (2013). All models were fit using the lme4 package (Bates et al. 2013) using the statistical program R (R Core Team 2015).
Consistency of behavior across and within mating statuses
We calculated the adjusted repeatabilities of all behavioral and web traits following the methods of Nakagawa and Schielzeth (2010). Overall, repeatabilities were calculated from the variance components of the fully parameterized models described in the “Relationship between mating status, web structure, and behavior” section. We calculated the repeatabilities within each mating status by fitting identical models, minus the main effect of mating status, to the appropriate subset of data (e.g., only virgin spiders). These models were fit, and the repeatabilites were then calculated from the variance components. We calculated 95% confidence intervals through parametric bootstrapping procedures (number of simulations = 1000) (Bates et al. 2013). Repeatability values were deemed to be significant if their confidence interval did not overlap 0.
We tested for differences in biogenic amine levels in spiders which mated in the field and in virgin spiders using linear models. Our analysis focused on the biogenic amines that are known to have behavioral effects in invertebrates (OA, DA, TA, and 5-HT) and their catabolites (Roeder 2005). Thus, the models contained one of the biogenic amines or associated catabolites as the response and mating status (virgin vs. mated field) and weight (centered) as the main effects.
Influence of biogenic amine concentrations on web structure and behavior
We assessed the relationship between behavior, web structure, and biogenic amine and amine catabolite levels using generalized linear mixed models. These models not only used the same main effect structure as the models used to assess mating status (attack and retreat behaviors: mating status, spider weight, prey cue presentation distance, assay within a week; web components: mating status, spider weight) but also included the measured levels of one of the biogenic amines (centered) as a main effect. Given that different biogenic amines can have similar effects on behavior (e.g., OA and DA), we fit each biogenic amine in a separate model and then used an information criteria (AIC) model comparison in order to determine which biogenic amine best explained the measured behavior/web structure. Given that the biogenic amines and catabolites varied in their relative concentrations, their values were centered to a mean of 0 and standard deviation of 1 prior to fitting. We included a null model, which did not contain a main effect of a biogenic amine, in all comparisons. We conducted separate comparisons for the biogenic amines (OA, TA, DA, and 5HT) and their catabolites (Nac-TA, Nac-DA, and Nac-5HT). We also calculated the delta AIC for all models, as models within two delta AIC from the top model are deemed to be statistically indistinguishable from each other (Burnham and Anderson 2003; Richards 2005), and the Akaike weights, which describe the probability of the model being the best fit relative to the other models in the set. We used parametric bootstrapping (number of simulations = 1000) to determine the 95% confidence intervals of the fixed effects. Those with confidence intervals that do not overlap 0 can be determined to be reliable above or below 0 depending on the direction of the effect.
Relationship between mating status, web structure, and behavior
We found overall that spiders which had mated in the field were more aggressive (ß = 3.162 ± 0.697, p < 0.001) and less likely to retreat after attacking the prey cue (ß = −1.077 ± 0.679, p < 0.001) (Table 1) relative to virgin spiders, suggesting a more risk-prone strategy. These behavioral differences were mirrored in the web structure: Females which mated in the field generally built webs structured for prey capture rather than safety as indicated by the greater number of gumfooted lines (ß = 3.566 ± 0.686, p < 0.001) (Table 2) but fewer lines connecting to the rim (ß = −0.577 ± 0.133, p < 0.001) and lighter webs (ß = −1.191 ± 0.391, p = 0.002) relative to virgin spiders (Table 3). Spiders that were experimentally mated in the laboratory showed almost identical behavioral and web patterns as those which had mated in the field (attack: ß = 2.413 ± 0.509, p < 0.001; gumfooted: ß = 1.797 ± 0.831, p = 0.031; rim: ß = −0.946 ± 0.145, p < 0.001; web weight: ß = −0.916 ± 0.360, p = 0.011) (Tables 1, 2, and 3). The only exception was that those who mated in the laboratory exhibited no difference in the likelihood of retreating relative to virgin spiders (ß = −0.024 ± 0.585) (Table 1).
Consistency of behavior across and within mating statuses
We found significant repeatabilities both across and within treatment groups in all behavioral and web measures (Table 4) except for retreat behavior and the number of gumfooted lines built by the lab-mated females. Overall, the repeatability values ranged from moderate (e.g., ~0.35 for retreat behavior) to extremely high (e.g., ~0.8 for the number of structural and gumfooted lines) (Bell et al. 2009).
Influence of biogenic amine concentrations on web structure and behavior
When comparing the biogenic amines, we found that field-mated spiders had higher levels of dopamine (ß = 75.98 ± 27.56 ng/CNS) and Nac-DA (ß = 140.15 ± 56.82 ng/CNS) relative to virgin spiders. There were no differences in the other measured biogenic amines or catabolites. See supplementary materials for full model outputs (Tables S1, S2).
Our model comparison indicated that biogenic amines were related to several of the behavioral and web measurements (Table S3). Attack behavior was best explained by the model containing dopamine (delta AIC to next model = 0.0, weight = 0.89) and Nac-DA (delta AIC to next model = 3.4, weight = 0.80), even after correcting for mating status. Overall, spiders with higher dopamine and Nac-DA levels were more likely to attack (DA: ß = 1.343, 95% CI = 0.485–2.497; Nac-DA: ß = 1.160, 95% CI = 0.357–2.062) (Fig. 1, Table S4). Dopamine also best explained the number of structural lines (delta AIC to next model = 3.0, weight = 0.66) such that spiders with more dopamine build fewer structural lines (ß = −0.649, 95% CI = −1.358–(−0.146)) (Table S5). The number of rim lines was best explained by two models, with the best fit containing dopamine (delta AIC to next model = 1.4, weight = 0.49) and the second best fit, although statistically indistinguishable, containing octopamine (delta AIC to next model = 1.3, weight = 0.24) and Nac-TA (delta AIC to next model = 3.1, weight = 0.75) (Table S3). The effects of DA, OA, and Nac-TA were all negative such that spiders with higher concentrations built fewer lines connecting to the rim of the web building structure (DA: ß = −0.150, 95% CI = −0.306–(−0.027); OA: ß = −0.120, 95% CI = −0.238–0.018; Nac-TA: ß = −0.163, 95% CI = −0.284–(−0.054)) (Fig. 2, Table S6). Note, though, that the 95% CI for OA did slightly overlap 0, suggesting a weak relationship with the number of rim lines. Finally, web weight was best explained by solely Nac-TA (delta AIC to next model = 3.1, weight = 0.69) (Table S3), such that spiders with greater Nac-TA levels built lighter webs (ß = −0.420, 95% CI = −0.715–(−0.123) (Table S7).
Our results show that virgin females were overall less aggressive than spiders that had previously mated in the field or those which were mated in the laboratory. These differences extended to web structure, where virgin females built webs that would likely increase safety (e.g., increased density with more structural lines that are firmly anchored to the ground, together creating a defensive cloud of silk) (Blackledge et al. 2003; Blackledge and Zevenbergen 2007), relative to either of the mated groups. Virgin females and those who previously mated in the field displayed high levels of repeatability in all behavioral and web characteristics. Furthermore, females which we experimentally mated in the laboratory also displayed high levels of repeatability across their change in state (pre/post mating) (Bell et al. 2009). In terms of biogenic amines, the mated group of females had higher levels of DA and Nac-TA than virgins. Finally, at a species level, several biogenic amines also displayed relationships with behavior and web structure. DA and Nac-DA positively correlated with aggressive behavior and DA negatively with some web components (structural, rim lines), while Nac-TA negatively correlated with other web components (rim lines, web weight).
Trait plasticity in response to mating status has been found in a range of organisms, both male and female, including olfactory behavior in moths and parasitic wasps (Anton et al. 2007; Ruther et al. 2007), singing activity in nightingales (Amrhein et al. 2002), foraging in parasitoid wasps (Fauvergue et al. 2008), and ejaculate allocation in Drosophila (Zevenbergen et al. 2008; Lüpold et al. 2010). Collectively, these are examples of organisms who after mating are either decreasing the expression of traits that are relevant to attracting mates (Anton et al. 2007) or increasing the expression of traits that are relevant to acquiring resources (Herberstein et al. 2002; Isaac et al. 2010). Here, redback spiders express plasticity in traits that likely affect safety and foraging success, with the focus on the former occurring before mating, as indicated by the lower levels of aggression and increased number of structural lines, and the latter after mating as indicated by the increased aggression and number of gumfooted lines. Mated Latrodectus spiders can lay anywhere from 6 to 10 egg cases in their lives, if not more, assuming prey is available (Shulov 1940; Downes 1985, personal observation). Yet, egg cases can often range from 10 to 30% of a female’s body mass (unpublished data), highlighting the need for increased prey capture after producing an egg case in order to maximize reproductive output. Given that gumfooted lines increase prey capture, these changes in web structure and aggression likely aid in increasing resource acquisition (Blackledge and Zevenbergen 2007; Zevenbergen et al. 2008). This increase in aggression after mating mirrors the results found by Watts et al. (2015), who found that boldness behavior in the subsocial spider, Anelosimus studiosus, was higher in brooding vs. non-brooding spiders (Watts et al. 2015). Interestingly, we found that females who were virgins when they arrived in the lab, but were subsequently mated, displayed behaviors and web structures after mating similar to females who previously mated in the field. This highlights that the observed differences between virgin females and those who mated in the field are not simply a by-product of age or environmental differences and indeed represent a plastic response to the change in mating status and the associated increase in energetic requirements.
Overall, the consistent among-individual differences were not affected by mating status. Both virgins and those who had mated in the field displayed high levels of repeatability in all behavioral and web traits. Previous studies have investigated how condition affects behavior and web structure in the western black widow. DiRienzo and Montiglio (2016) demonstrated that changes in body condition, imposed on the spiders by altering food availability, did not affect the repeatability of behavior or web structure. Most importantly, despite the small sample size, we found significant repeatability in aggressive behavior and all web traits in spiders who were experimentally mated in the lab. Thus, among-individual differences are maintained across these major changes in state. Our results suggest that among-individual differences are stable in widow spiders, regardless if the change in state is gradual (e.g., body condition) or in this case immediate (mating status). The stability in among-individual differences across these binary changes in status may contribute to the maintenance of trait variation over evolutionary time. This may occur if females are maximizing fitness at different points in their lifetime. Less aggressive females may make better mate choices, possibly by being more choosy or less likely to cannibalize the male (Johnson and Sih 2005), while more aggressive females may instead maximize energy intake and reproductive output postmating. A similar parallel can be found in other contexts that are demarcated by binary events, such as dispersal. In the case of western bluebirds, males vary in aggression, and aggressive males are more likely to disperse (Duckworth and Badyaev 2007). These aggressive, dispersing males have higher fitness when colonizing a new habitat rather than when staying in an existing one (Duckworth 2008) and thus experience an increase in fitness after the dispersal decision. Alternatively, there may be context-dependent trade-offs occurring (Dingemanse et al. 2004; DiRienzo et al. 2013; Belgrad and Griffen 2016), whereby when male density is high, increased female choice is favored; yet, under low-resource conditions, individuals who display increased foraging effort obtain higher fitness. Overall, differential fitness benefits across binary events, such as mating, dispersal, and metamorphosis, as well as context-dependent trade-offs, may be an important mechanism for maintaining behavioral variation.
While this study focused on standard measures of web structure such as the number of gumfooted lines and structural lines and web weight (Blackledge and Zevenbergen 2007; DiRienzo and Montiglio 2016a; DiRienzo and Montiglio 2016b), in an effort to create a simple measure of three-dimensionality, we also incorporated the number of lines connected to the top of the web building structure (termed “rim lines”). Interestingly, we found that virgin females built significantly more of these rim lines relative to the other groups and frequently few gumfooted or structural lines, suggesting that they are allocating increased web mass higher off the ground. Observationally, these webs also appeared to be formed such that the female built an elevated sheet, yet one that is much higher in tension than the sheet typically associated with foraging webs, although this was not tested directly (ND, pers. obs.). There are several explanations for this web that appears unique to virgin females. One is that virgin females are building webs that act as sexual signals. There is evidence that females deposit pheromones that convey aspects of state (Baruffaldi and Andrade 2015), and females release volatile pheromones from their webs that attract males (Kasumovic and Andrade 2004). Building an elevated web may aid in the distribution of the volatile compounds and increase the probability of attracting a male. Alternatively, as males use vibratory sexual signals (Ross and Smith 1979; Vibert et al. 2014), the apparently higher tension may aid in signal transmission. This may increase the female’s ability to accurately perceive male signals, thus aiding in mate choice. Although speculative, these results highlight the potential role of web structure in sexual selection and deserve further attention.
Biogenic amine levels, specifically that of DA and its catabolite, were shown to be greater in mated vs. virgin females. Yet, these amines, and others, were also shown to be correlated with both behavior and web structure, even after accounting for differences in mating status. To date, there are few studies demonstrating that biogenic amines correlate with individual differences in behavior. Nishi et al. (2010) demonstrated that injections of DA and OA decrease death feigning behavior in red flour beetles, suggesting that these amines are related to risk aversion (Nishi et al. 2010). Similarly, DA and OA have been shown to relate to aggressive behavior in field crickets (Adamo et al. 1995; Stevenson et al. 2005). Thus, DA may play a similar role in regulating individual differences in aggressive behavior in redback spiders, where aggressive individuals have consistently higher CNS DA levels than their non-aggressive counterparts. Alternatively, in Polistes wasps, increases in brain DA levels are linked to egg laying behavior (Sasaki et al. 2007). It is possible that the differences in DA are more related to mating status and that although aggression increased with mating status, it is regulated by a different neural or physiological mechanism. The lack of effect of OA is surprising given its known relationship with aggression in a variety of invertebrate taxa (Adamo et al. 1995; Roeder 2005; Dierick 2008) and even male mating behavior in arachnids (Hebets et al. 2015). We did find a trend indicating that mated females had higher OA (p = 0.08, Tables S1), but OA did not predict aggressive behavior after controlling for differences in mating status. Thus, OA may be related to behavioral differences, but the main effects seen here appear to be driven by DA. Manipulative studies are needed to assess the true relationship between DA, OA, and aggressive behavior. Interestingly, we found that the catabolite Nac-TA was related to several aspects of web structure, although its associated amine, TA, was not. Specifically, those with webs less structured for mating and prey capture (who were also more aggressive) had higher Nac-TA levels. This suggests that individual differences in web structure may not be a result of different basal TA levels, but instead the rate at which they are metabolized by the organism. It is important to note that three of the mated spiders had rather high DA (e.g., >300 ng/CNS) levels relative to the rest of the spiders, although these same spiders did not necessarily have higher levels of the other amines, suggesting that the high DA levels were intrinsic to the individuals. One potential explanation is that we are unaware of the latency of the last mating event in the field-caught spiders. If DA also increases immediately after mating, then our observations could be driven by having several spiders who mated more recently than the rest. Collectively, these results provide evidence for the biogenic amines in mediating among-individual differences in behavior and web structure. One issue was that given the destructive nature of our sampling method, we were unable to determine if individuals show consistent differences in amine expression, across either state changes or time. Nonetheless, the correlation between consistent behavioral and web tendencies and biogenic amine and catabolite levels, and that these traits are repeatable across state changes, suggests that differences in the aminergic system may drive stable differences after mating. Future efforts will be directed at attempting repeated non-destructive hemolymph sampling, although this can be problematic in arachnids (DiRienzo et al. 2015). From a practical perspective, these results suggest that aspects of individual differences may more strongly be related to the metabolic pathways associated with these amines and thus need to take a broader approach in their HPLC measurements.
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We thank Dr. Katrina Dlugosch for the use of her microbalance, as well as Pierre-Olivier Montiglio and Anna Dornhaus for comments on the original version of this manuscript.
ND was supported by the Japanese Society for the Promotion of Science Postdoctoral Fellowship and the University of Arizona Postdoctoral Excellence in Research and Teaching Fellowship (NIH No. 5K12GM000708-17). HA was supported by grants-in-aid for scientific research (KAKENHI) from the JSPS (15F15084).
The data from this study have been deposited in the Dryad data repository: doi:10.5061/dryad.8tf05.
Communicated by W. Hughes
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DiRienzo, N., Aonuma, H. Individual differences are consistent across changes in mating status and mediated by biogenic amines. Behav Ecol Sociobiol 71, 118 (2017). https://doi.org/10.1007/s00265-017-2345-x
- Animal personality
- Widow spider
- State-dependent behavior