DOI: 10.5061/dryad.g79cnp5w0
title: A distinct neurogenomic response to a tradeoff between social challenge and opportunity in male sticklebacks (Gasterosteus aculeatus)

description: Animals frequently make adaptive decisions about what to prioritize when
 they are faced with multiple, conflicting demands at the same time.
 However, the proximate mechanisms of decision-making in the face of
 competing demands are not well understood. We explored this question using
 brain transcriptomics in a classic model system: threespined sticklebacks,
 where males face conflict between courtship and territorial defense. The
 outcome of decisions made by breeding males over whether to court mates or
 defend against territory intrusion are profoundly important to fitness. We
 characterized the behavior and brain gene expression profiles of males
 confronted by a tradeoff between courtship and territorial defense by
 comparing them to males not confronted by this tradeoff. When faced with
 the tradeoff, males behaviorally prioritized defense over courtship, and
 this decision was reflected in their brain gene expression profiles. A
 distinct set of genes and biological processes was recruited in the brain
 when males faced a tradeoff and these responses were largely
 non-overlapping across two brain regions. Combined, these results raise
 new questions about the interplay between the neural and molecular
 mechanisms involved in decision-making.
description: <em>Laboratory populations and
 housing</em> All
 experiments were conducted during the summer breeding season (June –
 August) of 2020 using lab-reared individuals at the University of Illinois
 Urbana Champaign. Parents of the F1 individuals were wild caught from a
 population of the “white” ecotype (Blouw and Hagen 1990; Samuk 2016) of
 threespined stickleback (<em>Gasterosteus aculeatus</em>) from
 Canal Lake, Nova Scotia, CA (44°29'54.0"N
 63°54'09.1"W). Laboratory conditions were maintained at 20°C and
 a 16:8 light / dark photoperiod to stimulate male nesting behavior. All
 individuals were fed <em>ad libitum</em> once a day with a
 mixture of brine shrimp (<em>Artemia </em>sp.), mysis shrimp
 (<em>Mysis </em>sp.), bloodworms (<em>Chironomus
 </em>sp.), and Cyclop-Eeze. Prior to experimental trials, males and
 females were housed socially (5-20/tank, 53L x 33W x 24H cm), but focal
 males were moved to individual tanks at least 24 hours prior to
 experimental trials. N=17 focal males were used in this
 experiment. <em>Experimental tank
 set-up</em> Each
 experimental tank (53L x 33W x 24H cm) was filled with a layer of gravel
 on the bottom and divided into three sections. The middle section
 contained a clump of algae and a plastic tray filled with sand to provide
 nesting material, and the outer two sections were used to present stimuli
 during behavioral trials. To ensure construction of the nest in the middle
 section of the tank, the focal male was first confined to the middle
 section using dividers. After the nest was constructed, the dividers were
 removed so trials could be conducted.<em> </em>
 <em>Overview of behavioral
 trials</em> Behavioral trials were conducted in August 2020 to
 investigate the behavioral mechanisms underlying how males managed the
 competing demands of courtship and territory defense.
 Behavioral trials consisted of four different treatments: a
 courtship opportunity, a territorial challenge, simultaneous courtship
 opportunity and territorial challenge (hereafter referred to as the
 “tradeoff” treatment), and a control. At the start of each trial, two
 flasks were introduced into the focal male’s tank. In the courtship
 opportunity treatment, a gravid female was confined to one of the flasks
 (the other flask was empty), in the territorial challenge treatment, a
 rival male (a male displaying breeding coloration) was confined to one of
 the flasks, and in the tradeoff treatment, a gravid female was confined to
 one of the flasks and a rival male was confined to the other flask. Rival
 males were size matched to focal males as much as possible, which gave an
 average ratio of intruder standard length to focal standard length of
 0.97mm (+/- 0.02 standard deviation). In the control treatment, males were
 presented with two empty flasks. Each
 focal male was exposed to each of the four treatments (courtship
 opportunity, territorial challenge, tradeoff, and control) twice in a
 randomized block design, for a total of eight trials per focal male.
 Therefore, each focal male was exposed to each of the four treatments in a
 random order once, followed by each of the four treatments in a random
 order a second time. Each male was exposed to only one trial per
 day. This study design is
 powerful, as it allowed us to investigate plasticity and repeatability in
 courtship and aggressive behaviors while controlling for order
 effects. The
 focal male was acclimated to the presence of the observer for five minutes
 before the start of each trial. For each trial, the side of the tank where
 the flask containing a stimulus was placed was determined by coin toss
 (excepting the control treatment, which consisted of an empty flask on
 each side). The two flasks were then gently placed on their respective
 sides of the tank and male behavior was scored for five minutes using
 JWatcher (Blumstein and Daniel 2007). The number of zigzag displays (a
 courtship display) and the number of bites performed by the male were
 recorded. Males occasionally performed zigzags toward the territorial
 challenge or empty flask and bites toward the courtship opportunity or
 empty flask, however it is not possible to distinguish between mistakes
 and intentional behavior, therefore the number of zigzags and the number
 of bites performed include all instances regardless of where they were
 directed. <em>Statistical
 analyses</em> All
 analyses were conducted using R version 4.2.2 (R core development team,
 2022). To investigate how individuals behaviorally responded to the
 tradeoff between courtship and territory defense, generalized linear
 models were fit using the ‘lme4’ package and p-values were calculated
 using a Satterthwaite approximation with the ‘lmerTest’ package. Two
 models were fit with 1) the number of zigzag displays when a female was
 present and 2) the number of bites when a territorial intruder was
 present, respectively, with treatment as a fixed effect. In the model for
 zigzags, treatment consisted of the courtship opportunity and the tradeoff
 treatments, since the territorial challenge and control treatments did not
 contain a female stimulus. Similarly, in the model for bites, treatment
 consisted of the territorial challenge and the tradeoff treatments.
 Individual ID was included as a random effect to control for repeated
 measures of the same individuals. Zigzags and bites are count variables,
 therefore models were fit with poisson error distributions and an
 observation level random effect was included to control for overdispersion
 (Harrison 2014). To investigate order effects, we fit trial set (the first
 or second time the set of the four treatments was presented), trial order
 (whether the stimulus was presented first, second, third, or fourth in the
 sequence), and the interaction between trial set and trial order as
 additional fixed effects in the models. We then performed likelihood ratio
 tests to determine whether each factor and their interaction significantly
 improved the fit of the model. Next, we estimated adjusted repeatability
 of zigzags and bites as the proportion of total variance accounted for by
 differences among individuals, while accounting for treatment effects,
 using the ‘rptR’ package (Nakagawa and Schielzeth 2010). We fit two models
 with zigzags in the courtship opportunity and tradeoff treatments and
 bites in the territorial challenge and tradeoff treatments as the response
 variables, with individual ID as a random effect. We fit models with
 poisson error distributions and ‘rptR’ fits an additive overdispersion
 correction.
description: # Title of Dataset: Tradeoffs *** Datasets for: Barbasch, T.A., Behrens,
 C., McLain, M., Arredondo, E., & Bell, A.M. 2023. A distinct
 neurogenomic response to a tradeoff between challenge and opportunity in
 male sticklebacks (Gasterosteus aculeatus). Biology Letters. Corresponding
 Author Information Name: Tina Barbasch Institution: University of Illinois
 Urbana Champaign Address: Urbana, IL Email: Barbasch@illinois.edu ##
 Description of the data and file structure **File List:** A)
 2020Tradeoffs.csv B) Aggression\_sampleinfo.csv C) htseq results:
 298D.txt, 298T.txt, 299D.txt, 299T.txt, 302D.txt, 302T.txt, 303D.txt,
 303T.txt, 304D.txt, 304T.txt, 306D.txt, 306T.txt, 307D.txt, 307T.txt,
 308D.txt, 308T.txt, 317D.txt, 317T.txt, 318D.txt, 318T.txt, 319D.txt,
 319T.txt, 320D.txt, 320T.txt, 321D.txt, 321T.txt, 322D.txt, 322T.txt,
 323D.txt, 323T.txt, 329D.txt, 329T.txt, 330D.txt, 330T.txt D) eFDR null
 p-values: DcephalonWhite\_nullChallengeDControlBROAD2023-02-27.txt,
 DcephalonWhite\_nullChallengeOpportunityDControlBROAD2023-02-27.txt,
 DcephalonWhite\_nullOpportunityDControlBROAD2023-02-27.txt,
 TelcephalonWhite\_nullChallengeDControlBROAD2023-02-24.txt,
 TelcephalonWhite\_nullChallengeOpportunityDControlBROAD2023-02-24.txt,
 TelcephalonWhite\_nullOpportunityDControlBROAD2023-02-24.txt E) eFDR
 empirical p-values:
 DiencephalonWhite\_contrast\_Challenge\_eFDRDControlBROAD2023-02-27.txt,
 DiencephalonWhite\_contrast\_ChallengeOpportunity\_eFDRDControlBROAD2023-02-27.txt, DiencephalonWhite\_contrast\_Opportunity\_eFDRDControlBROAD2023-02-27.txt, TelencephalonWhite\_contrast\_Challenge\_eFDRDControlBROAD2023-02-24.txt, TelencephalonWhite\_contrast\_ChallengeOpportunity\_eFDRDControlBROAD2023-02-24.txt, TelencephalonWhite\_contrast\_Opportunity\_eFDRDControlBROAD2023-02-24.txt # Behavioral Data: 2020Tradeoffs.csv Description of Variables date: Calendar date that data was collected observer: Initials of observer ID: Unique identifier for each focal male time: Time at the start of the trial bites: Number of bites performed by the focal male during the 5 minute trial zigzag: Number of zigzags performed by the focal male during the 5 minute trial trial\_type: Treatment the male was given. M = Territorial challenge, F = Courtship Opportunity, MF = Tradeoff, C = Control trial\_set: Whether the trial occurred during the first or second time the set of four trials was presented trial\_order: Whether the stimulus was presented first, second, third, or fourth in the sequence # Sample Information: Aggression\_sampleinfo.csv Description of Variables Sample: Unique identifier for each brain sample ExtractionDate: Calendar date when brain sample was taken Population: Identifier for the stickleback population from which data were taken Family: ID number of family that male originated from Experiment: Identifier for which experiment the samples belong Treatment: Treatment the male was given (as with Behavioral Data) bites: Number of bites performed by the focal male during the last trial before brain dissection zigzags: Number of zigzags performed by the focal male during the last trial before brain dissection # htseq results Count data from diencephalon (\*D.txt) and telencephalon (\*T.txt) # eFDR\_null\_pvalues Null distribution of p-values for eFDR correction. Dcephalon = diencephalon; Tcephalon = telencephalon nullChallenge = Territorial challenge treatment nullChallengeOpportunity = Tradeoff treatment nullOpportunity = Courtship opportunity treatment # eFDR\_contrast\_pvalues eFDR corrected p-values Dcephalon = diencephalon; Tcephalon = telencephalon nullChallenge = Territorial challenge treatment nullChallengeOpportunity = Tradeoff treatment nullOpportunity = Courtship opportunity treatment ## Sharing/Access information Links to other publicly accessible locations of the data: * https://github.com/tbarbasch/Tradeoffs/ ## Code/Software Code\_Tradeoffs.R Code for the statistical analysis of behavioral and gene expression data, and to generate all figures in the manuscript. All analyses were conducted using R version 4.2.2