DOI: 10.5061/dryad.1jwstqjxv
title: Pessimistic cognitive bias is associated with enhanced reproductive investment in female zebrafish

description: Optimistic and pessimistic cognitive biases have been described in many
 animals and are related to the perceived valence of the environment. We,
 therefore, hypothesize that such cognitive bias can be adaptive depending
 on environmental conditions. In reward rich environments an optimistic
 bias would be favored, whereas in harsh environments a pessimistic one
 would thrive. Here, we empirically investigated the potential adaptive
 value of such bias using zebrafish as a model. We first
 phenotyped female zebrafish in an optimistic/pessimistic axis using a
 previously validated judgment bias assay. Optimistic and pessimistic
 females were then exposed to an unpredictable chronic stress protocol for
 17 days, after which fish were euthanized and the sectional area of the
 different ovarian structures was quantified in both undisturbed
 and stressed groups. Our results show that zebrafish ovarian
 development responded to chronic stress, and that judgment bias impacted
 the relative area of the vitellogenic developmental stage in the stress
 treatment, with pessimists showing higher vitellogenic areas as compared
 with optimists. These results suggest that pessimism maximize
 reproductive investment, through increased vitellogenesis, indicating a
 relationship between cognitive bias and life-history organismal decisions.
description: 1.1       Fish and housing 
 All subjects used were 4
 months-old female wild-type (TU) zebrafish (Danio rerio) (n = 72) bred and
 held at the Animal house Facility at Instituto Gulbenkian de Ciência (IGC,
 Oeiras, Portugal). Fish were kept in mixed sex groups (10 adults/L) in a
 recirculation system (Tecniplast®) at 28 °C, 750 μS, 7.0pH in 14L:10D photoperiod
 and fed twice a day with freshly hatched Artemia salina in the morning and
 commercial food flakes (Gemma) in the afternoon. Details of husbandry protocols and health
 program have been described previously [28]. All procedures described in
 this study were carried out in accordance with the relevant guidelines and
 regulations for animal experimentation, reviewed by the Instituto Gulbenkian de Ciência
 Ethics Committee, and approved by the competent Portuguese authority
 (Direcção Geral de Alimentação e Veterinária; permit number:
 0421/000/000/2019). 1.2       Experimental design 
 Individual zebrafish were
 first categorized in an optimistic/pessimistic
 dimension following a
 validated protocol for measuring judgment bias in zebrafish [8,
 29]. In brief, a Go/No-go
 task was designed in a half radial maze where individual zebrafish were
 trained to approach a positive cue (P; food reward) and to avoid a
 negative cue (N; punishment).Once fish were able to distinguish between P
 and N cues (as indicated by different latencies to enter each cued arm),
 their response to an ambiguous cue (an intermediate location/colour
 cue between the P and N locations/colour cues) was then tested
 (for a detailed
 description of the judgement bias protocol see supplementary
 information). Video recordings of the judgment bias assay were
 analyzed by using multi-event recorder software (The Observer XT, Noldus
 technology, version 9). A total of 48 (out of 64; Figure 1S) individuals
 scoring lower (n = 24; optimists) and higher (n = 24; pessimists) in the
 JBS values were selected for the chronic stress
 experiment. Selected fish
 were individually tagged using a validated procedure for zebrafish
 [30]. After a recovery
 period of 4 days, tagged zebrafish (n=48) were randomly assigned to one of
 two different groups: receiving unpredictable chronic stress (UCS; stress
 group) or left undisturbed (control group). Fish assigned to each
 treatment were distributed across four tanks (replicates). JBS values were
 counterbalanced between the two treatments (stress vs. control), and each tank
 (replicate) consisted of a mixed-phenotype group of 6 fish (i.e. 3
 optimists and 3 pessimists). Four experimental treatments were therefore
 set-up: optimists control, pessimists control, optimists stressed, and
 pessimists stressed (n =
 12 individuals per group) (for a detailed  description of the statistical calculation of
 sample sizes see supplementary information). Fish were then exposed to an UCS protocol
 already validated for zebrafish [31]. In brief, the UCS group was stressed twice per day using
 ten different stressors given in a random order across 17 days
 (Table S1). All fish of the same home tank were given the
 same stressor at the same time. Stressors included: alarm substance exposure, air-exposure, chasing
 fish with a hand net, changing fish between different tanks, lowering
 water level until the dorsal part of the fish is exposed to air, crowding,
 lowering the water temperature, social isolation, heating up water, and
 restraint stress (for a
 detailed description of the UCS protocol see supplementary
 information). 1.3       Histological preparation
 The day after the
 UCS protocol ended, fish were collected from their home tank
 and euthanized using a
 lethal dose of MS-222 (1g/L; Sigma, MO, USA). Ovaries were dissected
 out and fixed for 72 hours in 10% neutral buffered formalin. After
 fixation, ovaries were dehydrated through a series of graded ethanol
 solutions (70–99.8%), cleared in xylene, and embedded in paraffin. Each
 gonad was entirely sectioned into thin sections (3 µm thick) and stained
 with haematoxylin-eosin. 1.4       Histological and quantitative
 analysis The sectional area of the different ovarian
 structures was quantified using the Visiopharm Integrator System software
 (VIS; Visiopharm A/S, Hoersholm, Denmark) and a NanoZoomer-SQ Digital
 slide scanner (Hamamatsu Photonics). For the quantitative measurements,
 ten sections corresponding to the medial zone of each ovary were selected.
 Sections were spaced 15μm apart from one another. A systematic uniform
 random sampling (meander sampling) was carried out for each
 slide. Step-lengths of
 1435 µm were used in both x-and y-directions, enabling the acquisition of
 50% of the total area using an objective of 10x. The meander sampling
 generated an average of 60 fields for each slide, which were overlapped
 using a test system. A total of 64 grid points were regularly arranged,
 covering 16095 µm2 per point (area per point; a/p). The sectional area of the ovarian structures was
 estimated by an unbiased, stereological technique based on point-counting
 [32], in which the total number of grid points in a section hitting the
 structures of interest (p structure) was
 calculated: Sectional area per structure = ∑ (p structure) *
 (a/p) * 2 Results are therefore expressed as the average
 sectional area of each oocyte stage per section. In this study, four
 follicular stages of maturation were identified and counted from the
 zebrafish ovaries: (1) primary growth stage; (2) cortical alveolus stage;
 (3) vitellogenic stage; and (4) mature stage (see Figure S2 and
 supplementary material for detailed description).
 1.5       Statistical analyses 
 For the analyses of
 the average sectional
 areas for each oocyte stage we used the R software [33] packages “lme4” [34]
 and “afex” [35] for the linear mixed effects models (GLMM). Sectional
 areas for cortical
 alveolus and mature stages were log transformed. The other variables did
 not need transformations, confirmed with the Shapiro-Wilk test of
 normality. In all models,
 the fixed effects were the judgment bias phenotype (with two groups:
 optimists and pessimists) in interaction with treatment (with two groups:
 control and stress). The random effect was the tank identity, since the
 fish of the control and stress groups were distributed in four
 tanks (replicates) each.
 This procedure allowed controlling for a possible tank
 effect. Inspection of
 model residuals showed satisfactory normal distributions. All P-values are
 two-tailed.