A behavioral logic underlying aggression in an African cichlid fish

TL;DR: This research found that the relative SL of intruders played an important role in determining behavioral performance, and suggested that A. burtoni males amplify non-physical aggression to settle territorial disputes in response to differences in relative SL that were not previously considered to be behaviorally relevant.

Abstract: Social rank in a hierarchy determines which individuals have access to important resources such as food, shelter, and mates. In the African cichlid fish Astatotilapia burtoni, rank is under social control, such that larger males are more likely than smaller males to be dominant in rank. Although it is well known that the relative size of A. burtoni males is critical in controlling social rank, the specific behavioral strategies underlying responses to males of different sizes are not well understood. In this research, our goal was to characterize these responses by performing resident-intruder assays, in which aggressive behaviors were measured in territorial males in response to the introduction of unfamiliar males that differed in relative standard length (SL). We found that the relative SL of intruders played an important role in determining behavioral performance. Resident males exposed to larger (>5% larger in SL) or matched (between 0 and 5% larger or smaller in SL) intruder males performed more lateral displays, a type of non-physical aggression, compared to resident males exposed to smaller (>5% smaller in SL) intruder males. However, physical aggression, such as chases and bites, did not differ as a function of relative SL. Our results suggest that A. burtoni males amplify non-physical aggression to settle territorial disputes in response to differences in relative SL that were not previously considered to be behaviorally relevant. Highlights Relative size determines social rank in the African cichlid Astatotilapia burtoni Resident male A. burtoni respond differently to small size differences in intruder males Residents perform more non-physical aggression against larger intruders Residents do not alter physical aggression as a function of differently sized intruders Distinct behavioral strategies are used against different intruders

Summary

Ethical Note

• The protocols and procedures used here were approved by the Stanford University Administrative Panel on Laboratory Animal Care (protocol number: APLAC_9882) and followed the ASAB/ABS Guidelines for the use of animals in research.
• The authors were able to monitor the behaviours of all fish throughout each day of the study (see below).
• Throughout the whole assay, each tank was monitored in real time through a Wi-Fi-enabled camcorder remotely connected to a tablet (iPad).
• Fish in all other tanks were monitored three times daily by visual inspection, to ensure they experienced no physical harm.
• No fish were physically harmed at any point during the assay.

General approach

• The authors took several steps to control for social experience and age of both the resident and intruder, since these factors have been found to influence behavior in A. burtoni (Alcazar et al., 2014; Solomon-Lane & Hofmann, 2019) .
• To control for previous social experience, the resident and intruder were unrelated and had had no visual, physical, or chemical interaction at any point prior to the assay.
• Another step the authors took to control for social experience was to socially suppress all males before they were given the social opportunity to ascend to dominance and were provided a territory.
• The authors also physically isolated socially ascending fish from other fish, to further control for the role of social experience on behavioral responses to the intruder.
• Finally, all resident-intruder pairs were age-matched, to control for effects of age on behavior (Alcazar et al., 2014) .

Social suppression

• Two 121-liter social suppression tanks (see Fig. 2a for illustrated example) were each filled with 20 related, small suppressed males, as well as 3 large, unrelated dominant males and .
• CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
• The two tanks contained broods of the same age from different parents.
• Smaller suppressed males were housed in these conditions for at least 45 days before being transferred to a dominance inducing tank (see below).
• (d) After the assay, male residents are weighed for body and gonad mass and behavior videos are scored and analyzed.

Dominance inducing tank setup

• Thirty-liter dominance inducing tanks (see Fig. 2b for illustrated example) were set up for the isolation of previously suppressed males to allow for controlled social ascent to dominance.
• Each tank contained a shelter (half terra cotta pot) and faced a 121-liter tank filled with 10 unrelated females with which the male could interact visually but not physically or chemically.
• CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
• The copyright holder for this preprint (which this version posted July 22, 2020.
• Males were transferred from the suppression tank and isolated in a dominance-inducing tank for 2-4 weeks before entering the assay tank.

Dissections

• Immediately following the completion of the resident-intruder assay, the resident male was removed, weighed, and euthanized via rapid cervical transection (see Fig. 2d ).
• An incision was made anterior to the vent to the caudal fin and the gonads were removed and weighed.

Scoring behavior

• Based on previous work, multiple types of behavior were quantified ( Fernald & Hirata, 1977 ; see Fig. 3 for illustrated examples of behaviors): fleeing from male; physical aggression (chase male and bite male); non-physical aggression (lateral display and flexing); and pot entry, a territorial behavior.
• Chase was defined as a rapid swim directed towards a fish.
• Lateral displays were defined as aggressive displays classified as presentations of the side of the body to another fish with erect fins, flared opercula, and trembling of the body.
• Pot entry was defined as as any time a male entered the half terra cotta pot.

Aggressive behaviors

• Chase Bite Lateral display (a) (b) (c) Matched, and Larger.
• The "Smaller" group contained residents that were exposed to intruders 5% or more larger in SL; the "Matched" group included residents that were within ±5% of the size of the intruder; and the "Larger" group included residents that were exposed to intruders 5% or more smaller in SL.

Statistical analysis

• All statistical tests were performed in Prism 8.0.
• The authors used Kruskal-Wallis ANOVAs followed by Dunn's post-hoc tests for comparisons of physiological and behavioral measures across groups.
• When comparing only two groups, the authors used Mann-Whitney tests.
• Raster plots were generated using custom software packages in R (available at https://github. com/FernaldLab).
• Correlational analyses were conducted using Pearson's r. Effects were considered significant at p≤0.05.

Qualitative analysis of behavior as a function of relative SL

• The authors first visualized behavioral output for all fish in raster plots (Fig. 4 ).
• These plots showed that regardless of relative SL, residents attacked intruders at similar rates.
• Finally, most intruders performed zero aggressive behaviors towards the resident (see Fig. 5 ; results not shown in raster plots because it occurred at such low rates and only in a few fish; see below), indicating that the resident fish all maintained dominance throughout the challenge (or "won").

Correlational analyses reveal different features of resident aggression scale with relative SL

• The authors next ran correlational analyses to assess the relationship between relative SL and behavior.
• When relating the proportion of each behavior performed to the relative SL of the intruder, the authors found a significant positive relationship between lateral display proportion and relative SL (r=0.62, N=17, P=0.008) (Fig. 6c ).

No effects of gonadosomatic index or other physiological factors on resident behavior

• No significant correlations were found between resident GSI and resident behaviors (r³-0.37, N=17, P³0.13).
• CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
• The copyright holder for this preprint (which this version posted July 22, 2020.
• This overall pattern of differences does not systematically relate to their pattern of behavioral findings (see Fig. 4-8 ), suggesting that their behavioral differences are specifically related to the effects of the intruder's relative SL.

Discussion

• Specifically, when resident dominant males are exposed to an intruder who is matched or larger in relative SL, they use a behavioral strategy that emphasizes non-physical aggression.
• Recent studies have shown that physical and non-physical aggression are uncorrelated in A. burtoni, suggesting that these aggressive behaviors function differently during social interactions.
• ; https://doi.org/10.1101/2020.07.22.216473 doi: bioRxiv preprint Indeed, if it was the case that only size differences guided behavioral performance, then intruder males that were larger than the resident should have performed more lateral displays--but this was clearly not the case.
• Furthermore, testosterone, which is higher in dominant males than non-dominant males (Parikh, Clement, & Fernald, 2006) , increases the winner effect (Oliveira, Silva, & Canário, 2009) .

Full text

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Title: A behavioral logic underlying aggression in an African cichlid fish
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Short title: Aggression in a cichlid
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Authors: Beau A. Alward
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, Phillip H. Cathers
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, Danielle M. Blakkan
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, Russell D. Fernald
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Affiliations:
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University of Houston, Department of Psychology;
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University of Houston,
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Department of Biology and Biochemistry;
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Stanford University, Department of Biology
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*Corresponding author: balward@uh.edu
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Competing interest: We have no competing interests.
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Author’s contributions: Conceptualization, B.A.A., P.H.C., D.M.B., R.D.F.; Methodology,
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B.A.A., P.H.C., D.M.B.; Investigation, B.A.A., P.H.C.; Writing – Original Draft, B.A.A.; Writing –
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Review & Editing, B.A.A., P.H.C., D.M.B., R.D.F.; Supervision, B.A.A., R.D.F.
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Abstract
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Social rank in a hierarchy determines which individuals have access to important resources
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such as food, shelter, and mates. In the African cichlid fish Astatotilapia burtoni, rank is under
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social control, such that larger males are more likely than smaller males to be dominant in rank.
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Although it is well known that the relative size of A. burtoni males is critical in controlling social
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rank, the specific behavioral strategies underlying responses to males of different sizes are not
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well understood. In this research, our goal was to characterize these responses by performing
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resident-intruder assays, in which aggressive behaviors were measured in territorial males in
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response to the introduction of unfamiliar males that differed in relative standard length (SL). We
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found that the relative SL of intruders played an important role in determining behavioral
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performance. Resident males exposed to larger (>5% larger in SL) or matched (between 0 and
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5% larger or smaller in SL) intruder males performed more lateral displays, a type of non-
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physical aggression, compared to resident males exposed to smaller (>5% smaller in SL)
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intruder males. However, physical aggression, such as chases and bites, did not differ as a
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function of relative SL. Our results suggest that A. burtoni males amplify non-physical
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aggression to settle territorial disputes in response to differences in relative SL that were not
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previously considered to be behaviorally relevant.
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 22, 2020. ; https://doi.org/10.1101/2020.07.22.216473doi: bioRxiv preprint

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Keywords: Aggression, territorial, resident-intruder, social behavior, cichlid
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Highlights
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• Relative size determines social rank in the African cichlid Astatotilapia burtoni
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• Resident male A. burtoni respond differently to small size differences in intruder males
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• Residents perform more non-physical aggression against larger intruders
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• Residents do not alter physical aggression as a function of differently sized intruders
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• Distinct behavioral strategies are used against different intruders
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Introduction
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Intraspecific aggression is widespread among social animals (van Staaden, Searcy, &
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Hanlon, 2011). Aggressive behavior, either through physical attacks or non-physical signaling, is
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used to resolve conflicts related to access to resources such as food, shelter, territory, and
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mates. Extraordinary diversity exists in how different species express aggression and the rules
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that govern aggressive interactions. However, one rule seems to apply across species: physical
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or injurious behaviors are considered to be escalatory, occurring primarily in response to
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conflicts that are difficult to resolve (Holekamp & Strauss, 2016; Maynard Smith & Harper, 1988;
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van Staaden et al., 2011). The degree of conflict has been formally defined in terms of
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differences in resource holding potential (RHP). RHP can take the form of different levels of
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fighting ability as measured by body or weapon size. When large asymmetries in RHP exist
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among animals, aggressive interactions do not escalate from non-physical to physical; however,
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when asymmetries in RHP among animals are smaller, aggressive interactions are more likely
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to escalate, involving more physical and injurious forms of aggression.
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 22, 2020. ; https://doi.org/10.1101/2020.07.22.216473doi: bioRxiv preprint

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Evolution has shaped social dynamics across species to resolve aggressive interactions
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with as little physical fighting as possible, as this ensures individual and species survival
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(Holekamp & Strauss, 2016; Maynard Smith & Harper, 1988; van Staaden et al., 2011). This is
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abundantly clear in social animals that exist in a hierarchy, where rank determines which
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individuals possess a territory and the behaviors they perform. This is the case for the African
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cichlid fish Astatotilapia burtoni, where males stratify along a dominance hierarchy and exist as
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either non-dominant or dominant (Fernald, 2012). Dominant males possess a territory which
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they defend through aggressive interactions and in which they mate with females, while non-
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dominant males do not perform these behaviors. Dominant males also possess larger testes
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and brighter body coloration compared to non-dominant males. Social hierarchies in A. burtoni
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remain in flux, however, as non-dominant males constantly survey the environment, searching
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for a social opportunity to ascend in social rank to dominance. Social opportunity for a non-
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dominant A. burtoni male typically occurs when a larger male is absent from the environment,
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which a given smaller non-dominant male perceives as an opportunity to ascend to dominant
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rank. Within minutes of the opportunity, the non-dominant male increases aggressive and
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reproductive behavior in an attempt to establish a territory. Dominant males who encounter a
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larger dominant male in their environment will begin to descend in social rank by reducing
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aggressive and reproductive behavior (Maruska, Becker, Neboori, & Fernald, 2013).
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Size-induced social control of social status in A. burtoni has been shown in several
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studies. The reliable occurrence of this phenomenon makes size an excellent tool for
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controlling social environments in the laboratory, with the goal of generating fish with a given
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social status and studying the associated physiological underpinnings (for examples, see
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Alward, Hilliard, York, & Fernald, 2019; Maruska, Becker, Neboori, & Fernald, 2013; Maruska &
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Fernald, 2010). Although it has been shown repeatedly that size influences social status in male
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A. burtoni, a precise understanding of the relationship between size and behavior has not been
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 22, 2020. ; https://doi.org/10.1101/2020.07.22.216473doi: bioRxiv preprint

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established. For instance, while size is something that modifies social decisions in male A.
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burtoni, it is unclear what size difference males actually perceive as different and how they
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modify their behavior accordingly. Previous work has defined male A. burtoni as “matched” in
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size within a large range of standard length (e.g., 0-10% larger or smaller in standard length
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(SL=measured from the most anterior portion of the mouth to the most anterior portion of the
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caudal fin); see Alcazar, Hilliard, Becker, Bernaba, & Fernald, 2014; Desjardins & Fernald,
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2010)). However, recent work suggests that very small size differences between male A. burtoni
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can affect social interactions. For example, Alcazar et al ( 2014) found that males that were 2.1-
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4.9% larger in SL than their competitor consistently won during a contest, suggesting that size
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differences previously regarded as “matched” may actually be behaviorally relevant. However,
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this study was focused on which fish won each contest and not on the specific behavioral
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strategies underlying responses to differently sized males.
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Characterizing the specific behavioral patterns in A. burtoni that occur in response to
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differently sized males may yield insight into the capacity of A. burtoni to discern different levels
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of social opportunities, which would allow for a deeper understanding of the cognitive abilities
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required to successfully navigate a social hierarchy. In the present study we characterized
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behavioral responses in male A. burtoni as a function of differently sized male competitors
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during resident-intruder assays, in which a dominant male with a territory (i.e., the resident) was
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exposed to an unfamiliar, non-dominant male intruder that differed in relative standard length
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(SL). (illustrated in Fig. 1). The results of this study could shed light on the rules of engagement
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during social interactions in male A. burtoni.
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 22, 2020. ; https://doi.org/10.1101/2020.07.22.216473doi: bioRxiv preprint

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Figure 1. The behavioral patterns in male Astatotliapia burtoni underlying responses to
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differently sized males have not been characterized. Male A. burtoni change their social
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status depending on the social environment. Large males socially suppress smaller males and
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large males are more likely to be dominant. The specific behaviors males perform in response
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to differences in relative size, however, have not been determined. We asked during a resident-
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intruder assay what behavior patterns resident males use when presented with male intruders
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that were smaller, larger, or matched in size.
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Methods
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Ethical Note
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The protocols and procedures used here were approved by the Stanford University
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Administrative Panel on Laboratory Animal Care (protocol number: APLAC_9882) and followed
125
the ASAB/ABS Guidelines for the use of animals in research. We were able to monitor the
126
behaviours of all fish throughout each day of the study (see below). Throughout the whole
127
assay, each tank was monitored in real time through a Wi-Fi-enabled camcorder remotely
128
connected to a tablet (iPad). Fish in all other tanks were monitored three times daily by visual
129
inspection, to ensure they experienced no physical harm. No fish were physically harmed at any
130
point during the assay.
131
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 22, 2020. ; https://doi.org/10.1101/2020.07.22.216473doi: bioRxiv preprint