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.

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.