DOI: 10.5061/dryad.rfj6q57g0
title: Fighting investment of Neolamprologus pulcher after receiving single, reinforcing, and contradictory contest experiences

description: When animals live in long-term groups, the potential for conflict is high.
 Conflict is costly, so an individual’s decision to engage depends on the
 information it has about the costs and benefits of fighting. One source of
 information could be past contest experience, where previous
 winners/losers typically become more likely to win/lose in the future.
 However, repeated interactions can familiarize individuals with conflict
 and provide opportunities to learn to become better fighters, regardless
 of outcome. We explored how individuals integrate information from
 previous contests to inform future encounters in a group-living fish,
 Neolamprologus pulcher. We gave contestants single, reinforcing, and
 contradictory experiences and measured behavior and post-fight water-borne
 levels of androgenic steroids (testosterone, 11-ketotestosterone).
 Contradictory outcomes were associated with reduced investment in
 fighting. More fighting experience did not lead to greater investment in
 fighting, as consecutive losses resulted in reduced aggression. Also,
 there was no effect of fighting treatment on water-borne androgen
 concentrations. Interestingly, there were sex differences in which
 behaviors were influenced by experience, and in whether body mass was
 associated with androgen concentrations, which could indicate that males
 and females vary in how perceived fighting ability changes with contest
 experience. Our data reveal the complex ways in which repeated experiences
 can alter an individual’s propensity to invest in conflict. Repeated
 interactions associated with predictable changes in behavior can
 contribute to rank stability in groups and our results indicate that
 whether and how they do depend on the quality and quantity of interactions
 plus individual factors such as sex.
description: <em>Neolamprologus pulcher</em> were purchased from
 Old World Exotic Fish, Inc. in Miami, FL in December 2018, or were F1
 offspring of these fish. Contestants were housed in well-established
 social groups that maintained consistent group membership for at least two
 years prior to the start of this experiment. No contestant/opponent pair
 had been housed together or in adjacent tanks for at least two years prior
 to this experiment to reduce the effects of familiarity. Two weeks prior
 to the first contest trial, each fish was measured for total length (TL,
 to the nearest 0.01 mm), standard length (SL, to the nearest 0.01 mm), and
 mass (to the nearest 0.001 g). Each fish also was sexed by examination of
 the external genital papilla (Stiver et al. 2005) and received a unique
 visible implant elastomer tag (a subcutaneous injection of a
 silicone-based colored liquid) (Jungwirth et al. 2019) at this time. Only
 sexually mature animals (more than 30 mm in length) (Hellmann, Ligocki, et
 al. 2015; Antunes and Taborsky 2020) were used in this experiment.
 Individuals were randomly assigned ‘focal’ or ‘opponent’ status. A total
 of 43 contestants and 72 opponents were used for this experiment. Focal
 fish occupied a range of social ranks in their home tanks (n = 10 breeder
 females, 10 helper females, 11 breeder males, 12 helper males). Body size
 is a strong predictor of contest outcomes in N.pulcher (Reddon et al.
 2011), so same-sex opponents were haphazardly selected to be 8.05 ± 2.53
 mm smaller or larger in absolute SL than the focal fish to create a
 winning or losing experience, respectively. All focal fish were naïve to
 experimental procedures. If an opponent was to be used in more than one
 trial (n=24 opponents used in more than one trial), they had at least one
 week between trials (Miklósi et al. 1997). Focal fish sometimes were used
 as opponents (n=6) after they experienced contest trials as the focal
 contestant; at least one week elapsed between trials before a focal fish
 served as an opponent. <strong>Contest
 trials</strong> Contests took place November -
 December 2021. Focal fish were randomly allocated to one of six
 experimental treatments; a single contest (win, lose), reinforcing contest
 experiences (win-win, lose-lose), or contradictory contest experiences
 (win-lose, lose-win). All contests were conducted between same-sex pairs
 prior to feeding in experimental tanks detailed in Figure 1, similar to
 (Reddon et al. 2011; Hick et al. 2014). Contestants competed for access to
 a single terracotta flowerpot placed in the middle of the experimental
 tank, which we refer to as the ‘central contest zone.’ N.pulcher use these
 pots for shelter and breeding in our laboratory and will fight for access
 to this resource (Reddon et al. 2011; Hick et al. 2014; Pisanski et al.
 2015). Contestants that experienced more than one contest were immediately
 moved from their first contest to a 30-minute acclimation period for their
 second contest with a different opponent. Experimental tanks were
 sanitized with ethanol and the water was replaced between each
 trial.  Mirror tests are an appropriate proxy for
 aggression toward live opponents in N.pulcher (see Electronic
 Supplementary Material A, (Balzarini et al. 2014)). After their final
 contest (after the second contest for individuals that received a
 reinforcing or contradictory experience and after the only contest for
 individuals that received a single experience), contestants had a
 30-minute acclimation period before a 20-minute mirror test. Mirror tests
 took place in an independent arena set up in the same way as the
 experimental tanks but with a mirror attached to a randomly selected
 barrier that was not removed and gave contestants access to 2/3 of the
 tank. <strong>Behavioral
 observations</strong> A high-definition camcorder
 was used to record each contest and mirror test.  The number of aggressive
 behaviors performed by the focal contestant and duration of time the focal
 fish spent in the central contest zone were scored continuously using
 Behavioral Observation Research Interaction Software (BORIS) (Table B.1). 
 Displayed acts of aggression such as spreading of the fins or swimming
 parallel to the mirror or opponent were scored as restrained aggression
 whereas aggressive acts that involved contact with the mirror or opponent
 were scored as overt aggression (see Electronic Supplementary Material B).
 The scorer was blind to experimental treatment during scoring.
 All contest trials were observed live from behind a blind.
 Contests lasted for 20 minutes or until a ‘winner’ and a ‘loser’ could be
 determined. A contestant was declared a ‘loser’ when it displayed three
 consecutive submissive behaviors toward its opponent, or when it held a
 submissive posture while avoiding the other fish (Reddon et al. 2011; Hick
 et al. 2014). A contestant was declared a ‘winner’ when it chased its
 opponent three consecutive times or when it prevented its opponent from
 approaching the central shelter (Riebli et al. 2011; Dijkstra et al.
 2012). When one fish was declared a ‘winner,’ its opponent was
 automatically considered a ‘loser’ and vice versa. Contests were
 terminated as soon as a ‘winner’ and a ‘loser’ were determined. If there
 was no clear winner or loser at the end of the 20-minute observation
 period, then the contest was declared a tie. Any focal individual that
 experienced a tie or did not experience their assigned contest outcome
 were returned to their home tank without completing a mirror
 trial.  <strong>Hormone
 measurements</strong> Due to the small size of
 N.pulcher, it is not possible to consistently collect a large enough blood
 sample to measure steroid hormone concentrations without sacrificing the
 individuals. Therefore, steroid hormones were extracted from fish holding
 water so these individuals can be used for future studies and to provide a
 large enough sample volume to measure multiple hormones from a single
 sample (Bender et al. 2006; Sebire et al. 2007). This method extracts
 free, unconjugated hormones that have diffused from the bloodstream,
 through the gills, into the holding water and reliably reflect circulating
 hormone levels for N.pulcher (Scott et al. 2008). With passive diffusion,
 there is a time lag between maximum release rates reflected in water-borne
 hormone samples and maximum circulating hormone concentrations. Therefore,
 hormones were extracted two days after the focal fish experienced the
 contest trials.   Fish were placed in individual
 beakers with 300 ml of distilled water in separate boxes for exactly one
 hour before they were returned to their original tanks. This water was
 kept at the same pH and temperature as water in the fish tanks, but no
 fish were housed in it prior to hormone extractions. Fish were exposed to
 this procedure three times prior to their contest trials to habituate them
 to the process so the procedure itself (e.g., transferring from original
 tank to beaker) would not affect hormone release rates (Ligocki et al.
 2015). After this time, the holding water was passed through Grade 1
 filter paper to remove any solids before being drawn through Sep-Pakâ
 solid-phase C18 extraction cartridges (500 mg sorbent, 3 ml cartridge;
 Watersä Corporation, Milford, MA) using a vacuum manifold. These samples
 were frozen at -80oC and shipped to the University of Alabama. Salts were
 purged from the columns with 2 ml distilled water, and free, unconjugated
 steroids were eluted with 3 x 2 ml of ethyl acetate into 13 x 100 mm
 borosilicate vials. A gentle stream of ultra-pure nitrogen (~7 bar) was
 passed over the samples in a 37ºC water bath to facilitate evaporation of
 the solvent, leaving a hormone residue. This residue was resuspended in 25
 µl EtOH and vortexed for 1 min before adding 475 µl EIA buffer and
 vortexing again for 1 min (500 µl total resuspension volume).  A pooled
 sample of hormone was generated by combining 30µl from each experimental
 sample, representing the “average” water-borne hormone concentration
 across all animals. 500 µl of this pool was then serially diluted 1:1 to
 1:128 with EIA buffer and assayed using enzyme-linked immunosorbent assays
 (ELISA) kits from Cayman Chemical (Ann Arbor, Michigan) for
 11-ketotestosterone (11KT) and testosterone (T). Comparisons of the serial
 dilution curves to the standard curves indicated that the best dilution
 factor (i.e., that which was most likely to position samples on the linear
 phase of the standard curve) was 1:4 for 11KT and 1:50 for T. These
 dilutions were achieved by combining 100 µl of each experimental sample
 with 300 µl of EIA buffer (for 11KT) and 40 µl of each experimental sample
 with 1.96 ml EIA buffer (for T), followed by vortexing. Serial dilution
 curves also were parallel to the standard curve (11KT: t12 = 0.075, P =
 0.94; T: t12 = 0.039, P = 0.97), validating the use of these ELISAs for N.
 pulcher. Assays were conducted on two 96-well plates per hormone with
 intra-assay coefficients of variation being 6.9% and 4.2% for T, and 3.7%
 and 4.5% for 11KT. Inter-assay coefficients of variation were 5.3% for T
 and 3.8% for 11KT. <strong>Statistical
 analysis</strong> Generalized linear models
 (GLMs) were used to measure associations between steroid hormone
 concentrations, behavioral response, and experimental treatment. Counts of
 restrained and overt aggression, duration of time spent in the central
 contest zone, and concentrations of T and 11KT were analyzed in five
 separate GLMs. For behavior measures, fixed effects of the full models
 included the three-way interaction between sex, type of experience
 (single, reinforcing, contradictory), and most recent experience (win or
 loss) plus focal animal mass as a covariate. Although
 previous studies on N.pulcher found individuals of different sizes release
 similar amounts of steroid hormones over the same time interval (Bender et
 al. 2006; Bender et al. 2008; Scott et al. 2008), it is possible that
 hormone release rates may vary depending on the size of the individual
 (Sadoul and Geffroy 2019). To account for the potential effect of body
 size on hormone release rates, fixed effects of the full models for
 hormone concentration response variables included the three-way
 interaction between sex, type of experience, and most recent experience
 plus the two-way interaction of each of these variables with
 mass. The best fitting model for each response variable
 was selected by likelihood ratio test using the step function in R. After
 running the generalized linear model, a Tukey post-hoc test was used to
 compare treatment effects. All statistical analyses were performed using R
 4.1.1. We used the car and emmeans statistical packages (Fox et al. 2022;
 Lenth et al. 2022) and RStudio. The significance level for all tests was α
 = 0.05.
description: All statistical analyses were performed using R 4.1.1. We used
 the car and emmeans statistical packages and RStudio.