DOI: 10.5061/dryad.dfn2z35cn
title: Male activity under risk predicts paternal care and reproductive success in a bi-parental cichlid

description: Here, we explored a hypothesis from a framework proposing that sexual
 selection plays a role in shaping and maintaining consistent
 among-individual behavioral variation, commonly referred to as personality
 differences. This framework suggests that parental care may be a mechanism
 linking personality differences with reproductive fitness. To test this
 hypothesis, we repeatedly measured boldness as activity under simulated
 predation risk in male and female rainbow kribs, Pelvicachromis pulcher, a
 bi-parental West African cichlid. We then formed breeding pairs with
 varying degrees of behavioral contrast, recorded parental care (parental
 activity under risk and brood guarding) for both parents, and assessed key
 reproductive outcomes: the likelihood to reproduce, clutch size, offspring
 survival, and offspring size at the end of breeding. We observed
 consistent among-individual variation in all three behaviors. Males with
 higher pre-breeding activity under risk were less likely to reproduce.
 However, when these males did breed successfully, they achieved higher
 offspring survival rates compared to their less active counterparts. This
 relationship was mediated by parental care: males with higher pre-breeding
 activity under risk spent more time guarding their brood, and increased
 brood guarding was associated with higher offspring survival. Female
 pre-breeding activity under risk was not associated with parental care or
 reproductive success. These results suggest that male, but not female
 boldness might be subject to sexual selection. The observed pattern of
 bold males siring fewer but larger broods than less bold males could
 maintain among-individual variation in male boldness if it results in
 equal average fitness (e.g., due to environmental heterogeneity).
description: All animal care and experimental protocols complied with local
 and federal laws and guidelines and were approved by the appropriate
 governing body in Germany, the Landesamt für Gesundheit und
 Verbraucherschutz (LaGeSo 52/16).   
 <strong>Experimental
 procedures</strong>  
 <em>Study animals and housing
 conditions</em>   The
 rainbow krib is a small cichlid originating from rivers and streams in
 West Africa (Heiligenberg, 1965; Nwadiaro, 1985). Females are shorter and
 deeper bodied, and often more intensely colored, compared to the more
 streamlined, less colorful males (Heiligenberg, 1965; Martin &
 Taborsky, 1997; Reddon & Hurd, 2013). Males occur in two color
 morphs with red versus yellow opercula (Heiligenberg, 1965). Both sex and
 male color morph determination are influenced by the pH during early
 development (Martin & Taborsky, 1997; Reddon & Hurd, 2013;
 Rubin, 1985).   Rainbow
 kribs used in this study were obtained from a house breed at the
 Universität Hamburg and local suppliers; and were maintained in same-sex
 groups matched for family and origin (100-200l tanks), at 25±1°C, and a
 12:12 hours light:dark period. Tanks were endowed with a layer of sand
 (approx. 1 cm thick), an internal filter and plastic plants. Water changes
 (50%) were done once a week and fish were fed daily with live Artemia
 spp.. Water changes were done using pre-heated, aged tap water. All tanks
 were supplied with the same water from a common supply container, ensuring
 standardized water conditions for all tanks. Four days before the first
 boldness test, individuals were transferred to individual housing tanks
 (25L, 50 x 25 x 25 cm, water level = 20cm, same holding conditions as
 above). On the day of transfer, individuals were photographed and we
 measured their standard length (i.e., the length from the tip of the snout
 to the end of the spine) from photos using ImageJ (Schneider et al. 2012)
 (males: mean ± SE standard length = 5.42 ± 0.05 cm, range = [4.52, 6.25
 cm], N = 54; females: mean ± SE standard length = 4.39 ± 0.04 cm, range =
 [3.90, 5.17 cm], N = 54). All test fish were uniquely VIE-tagged for
 identification (Schuett et al., 2017).
   <em>Experimental
 Outline</em>   We
 measured boldness before breeding as activity in the presence of a
 computer-animated predator (see ‘Boldness tests’ for experimental details
 and see below for an explanation of our choice of boldness measure). We
 then created breeding pairs that varied regarding their similarity in
 average activity under simulated risk (see ‘Pairing and breeding’) and
 during breeding, we performed six parental care tests to assess parental
 activity under risk and brood guarding for each successfully reproducing
 breeding pair (see ‘Parental care tests’). We measured reproductive
 success as the likelihood to reproduce, clutch size (number of eggs laid),
 offspring survival and the size of offspring produced after approx. one
 month of care (see ‘Pairing and breeding’ for details). Boldness tests and
 pair formation were carried out in 5 experimental blocks with 12 males and
 12 females per block, amounting to N = 60 males and females, respectively.
 Each experimental block took up to 10 weeks (including pre-breeding
 behavioral typing, pair formation, and the parental care period); and
 blocks were started with 2-3 weeks in between successive blocks. The total
 number of fish tested exceeded the number of fish used for pairing (N = 54
 breeding pairs), giving us more options for the pair
 formation.   Prior to the
 experiment, we conducted a pilot study (Scherer, Godin, et al., 2017) to
 test how individuals react towards a predator under our specific
 experimental conditions and to test for the suitability of using virtual
 stimuli instead of live stimuli. We used a natural predator of rainbow
 kribs, the African obscure snakehead, Parachanna obscura. In short, we
 tested behavioral responses of 36 rainbow kribs towards both live
 predators and animated color-photographs thereof. For each focal
 individual, we measured the activity and mean distance to the stimulus.
 Both decreasing activity and keeping distance to a potential threat are
 well-known fear responses in fish (Broom & Ruxton, 2005; Cooper
 & Martín, 2016; McLean & Godin, 1989; O’Connor et al.,
 2015), although some fish may also cautiously “inspect” a predator to
 gather information (Godin & Dugatkin, 1996; Hesse et al., 2015;
 Pitcher et al., 1986). Time spent in an “inspection zone”, i.e., near the
 predator, was also recorded but it was highly correlated with the mean
 distance to the predator (Spearman rank correlation: rs = 0.84-0.93) and
 was therefore not analysed to avoid redundancy. Compared to control trials
 (empty aquarium and blank computer screen), individuals decreased their
 activity when presented with both the animated and the live predator with
 no difference in response between these two stimuli. Individuals decreased
 their distance towards the live predator compared to the empty predator
 tank control. But no difference in distance towards the animated predator
 vs. blank computer screen was observed and individuals kept a
 significantly larger distance to the animated predator compared to the
 live predator. The difference in inspection behavior may be caused by live
 predators having been rather motionless (and therefore potentially safe to
 inspect), while animated predators were swimming back and forth (and were
 therefore behaving potentially more suspiciously).
   The results of our pilot study
 demonstrate a similar fear response towards both an animated and live
 predator, but they also highlight nuanced differences in how these two
 stimuli were perceived. Specifically, individuals decreasing activity in
 response towards both the animated and live predator indicates that the
 predator was perceived as potentially dangerous in both scenarios. On the
 other hand, individuals exhibited inspection behavior when presented with
 the live predator (as indicated by a lower distance to the live predator
 compared to the empty tank control), while no such tendency was observed
 when individuals were confronted with the animated predator. Taken
 together, activity under simulated risk seems the most robust measure to
 quantify boldness when using animated predators in our study
 species.  
 <em>Boldness tests</em>
   Before breeding, all males (N = 60)
 and females (N = 60) were tested for their activity under simulated risk
 twice with five days in between trials. Boldness tests were performed
 following Scherer, Godin, et al. (2017). In short, individual test fish
 were exposed to a photograph of a predator specimen (P. obscura), which
 was animated to swim back and forth on a computer screen (see Scherer,
 Godin, et al., (2017) for details on the animation production). Test
 individuals were granted a 10 min acclimation period in the test tank,
 followed by 11 minutes of predator exposure. Recording from above (with a
 Sony HD-CX405), we assessed individual activity in the presence of the
 predator as the total number of squares visited (including revisits) for
 10 min (no tracking of the first minute) using the tracking software
 Ethovision XT 11 (Noldus, Wageningen, The Netherlands). Therefore, test
 tanks (50 x 25 x 25 cm, water level = 10 cm) were divided into 8 squares
 each measuring 12x12 cm squares. For all trials, we used a predator
 specimen the test fish had not seen before (N = 4 predators, mean ± SE
 standard length = 19.3 ± 0.3 cm). To create breeding pairs and for
 analyses (excluding repeatability analysis), we used the average activity
 under simulated risk an individual showed over both boldness tests (mean ±
 SE number of squares visited: males = 63.56 ± 4.26, females = 41.07 ±
 3.64). Prior to the experiment, individuals were habituated to test tanks
 twice for 30 min, respectively (in groups of approximately ten
 fish).   <em>Pairing
 and breeding</em>  
 Four days following the boldness tests, we set up breeding
 pairs (N = 54) of varying behavioral contrasts in activity under simulated
 risk. We calculated male-female behavioral contrast as average male
 behavior minus average female behavior, i.e., positive values indicate
 that the male is more active under risk than the female and vice versa.
 Pairs were formed in a way that we minimized the behavioral contrast for
 half of the pairs, whereas the other half was maximized for within-pair
 behavioral contrast (varying sex of the bolder individual). To initiate
 breeding, we introduced the respective male and female into a breeding
 tank (50 x 50 cm, water level = 25 cm), equipped with half a clay pot as
 breeding cave, a plastic plant, a layer of sand, and an internal heater
 (all in a standardised position). We monitored the breeding cave for eggs
 daily using a small dentist mirror (diameter = 3 cm) and took a photo of
 clutches laid when first seen. The number of eggs was counted from the
 photo (mean ± SE = 190 ± 14 eggs, N = 18 out of 20 clutches, no photos for
 two clutches). Breeding pairs that did not produce fry within 28 days were
 transferred back to their home tanks and were not further used in this
 experiment (N = 34 males and females, respectively). Breeding pairs that
 did produce fry were allowed to raise their brood for 33 days (spawning =
 day 1). During this breeding period, we assessed parental care behavior as
 outlined below. Different to housing conditions, we increased the number
 of water changes to three times a week and reduced the amount of water
 being changed to approx. 30% during the breeding period (no water changes
 on parental care test days or the day prior to tests).
   All but two breeding pairs spawned at
 least once, some pairs spawned two or three times (N total number of
 clutches laid = 83), indicating that the pairing duration of 28 days was
 sufficient for reproduction to occur. For breeding pairs that produced and
 successfully raised fry (N = 20 breeding pairs), we counted all offspring
 raised (mean ± SE = 68.7 ± 9.4 offspring per brood) and measured their
 size (mean ± SE = 1.56 ± 0.03 cm, standard length measured from photos
 using ImageJ, Schneider et al. (2012)) at the end of the breeding period
 (day 33). For statistical analyses, we averaged offspring size per brood
 and calculated offspring survival as the proportion of offspring raised
 relative to the clutch size (mean ± SE = 0.41 ± 0.05, N = 18 breeding
 pairs). For two breeding pairs, we found no eggs prior to fry appearing in
 the tank. Consequently, were not able to fully assess reproductive success
 for these two breeding pairs and removed them from all analyses performed
 on successful breeding pairs (N (final) = 18 breeding pairs). Notably,
 offspring survival and the absolute number of offspring raised were highly
 correlated (linear mixed-effect model with offspring survival as response,
 number of offspring raised as predictor, and male family, female family,
 male color morph, and experimental block as random terms; χ2 = 32.569, p
 &lt; 0.001, R2 = 0.903).  
 <em>Parental care tests</em>
   During the breeding period, we
 quantified brood guarding and parental activity in the presence of an
 animated intruder. We used three different intruder types: a predator
 (same predators as during boldness tests), a conspecific male, and a
 conspecific female (conspecifics are brood predators in the species). Each
 intruder type was used twice, i.e., each breeding pair was tested for
 their parental care behavior six times with three days elapsing between
 successive trials. For each breeding pair, we randomised the testing order
 for the first time each intruder type was used (first, second and third
 parental care test) and then repeated parental care tests in the same
 order (six tests in total, e.g., a breeding pair was tested in the
 following order: male intruder, female intruder, predator, male intruder,
 female intruder, predator). Parental care observations started one day
 after offspring became free-swimming, i.e., day 10 post spawning.
 Fertilised P. pulcher eggs take three days to develop into wrigglers (free
 embryos), which stay in the breeding cave for approximately another five
 days. On day 9 following fertilisation, offspring become free-swimming fry
 and leave the breeding cave to search for food.
   To start a parental care test, we
 introduced a tablet (Surftab Theatre, 13.3" Full-HD-IPS display;
 Trekstar, Bensheim, Germany) on a standardised side of the breeding tank
 showing one of the three intruder types for 11 min (Scherer, Godin, et
 al., 2017). We video-recorded the breeding pair's response and
 manually assessed the activity of each parent from the videos (duration of
 video analysis was 10 min, starting 1 min after the start of the video).
 The camera (Sony HD-CX405) was positioned in front of the tank, filming
 from diagonally above at a distance of 1 meter. The camera angle was
 chosen to capture the 3-dimensionality of the tank. Prior to starting
 trials, fish were allowed to acclimate to the camera for 10 min. Similar
 to the above boldness tests, parental activity under simulated risk was
 assessed as the total number of squares visited (including revisits).
 Therefore, the breeding tank was divided into 16 squares (each measuring
 12 x 12 cm) using markings alongside the vertical tank walls. Further,
 male and female brood guarding was quantified from snapshots, i.e., over
 the 10 min test period, we scored every 30 sec (21 frames in total)
 whether a parent was within a 6 cm distance to the brood (half a square,
 referring to approx. one total fish length). If so, this was scored as
 brood guarding (Thünken et al., 2010). Distance to the brood was assessed
 in 3-dimensional space, encompassing both horizontal distance on the
 ground and vertical distance in the water column. However, in practice,
 parental fish did not swim more than 6 cm above or below their offspring
 but all fish were mostly swimming close to the ground. Individual brood
 guarding was then calculated as the numb­er of frames where the individual
 was attending the brood divided by all frames analysed (resulting in
 values ranging from 0 to 1: 0 = the parent was not attending the brood at
 all, 1 = the parent was with the brood throughout). All videos were
 analysed by the same observer (US). During video analyses, the observer
 was unaware of the test fish’s pre-breeding activity under simulated
 risk.   Male intruder sizes
 were matched to the male's standard length and, similarly, female
 intruder sizes were matched to the female's standard length (size
 difference ≤ 2 mm). Predator sizes were as described in the boldness test.
 Test fish were presented with unfamiliar intruder specimen only. The
 intruder type (male conspecific, female conspecific, predator) did not
 affect parental care behavior, regardless of focal individual personality
 (Supplementary information 2 “Intruder type did not affect parental care
 behavior”). Consequently, we averaged individual parental care behavior
 over all six parental care tests for statistical analyses (not
 repeatabilities).
description: # Male activity under risk predicts paternal care and reproductive success
 in a bi-parental cichlid
 [https://doi.org/10.5061/dryad.dfn2z35cn](https://doi.org/10.5061/dryad.dfn2z35cn) ## Description of the data and file structure The experiment investigated the role of personality differences in boldness on parental care and reproductive success in rainbow kribs. Before breeding, individual fish were assessed for boldness using an animated predator stimulus, measuring their activity under simulated risk. Breeding pairs were then formed based on their behavioral contrast in boldness, and their reproductive success - clutch size, offspring survival, and offspring size - was monitored. During the breeding period, parental care behavior was tested under different simulated intruder conditions (predator, male conspecific, female conspecific), evaluating both brood guarding and boldness levels. ### Files and variables #### File: boldness\_tests.csv **Description** Results of boldness tests for all individuals, conducted prior to pair formation and breeding. Two boldness test per individual with five days in between trials. Boldness was measured as number of squares visited (including revisits). ##### Variables * experimental_block: the experiment was run in 5 experimental blocks. * pair_ID: pair identifier * sex: male or female * fish_ID: fish identifier * family_ID: family identifier * boldness_test_day: trial number (1 or 2, as all individuals were tested for their boldness twice) * prebreeding_boldness_total_number_of_squares_visited: individual boldness level assessed prior to breeding, measured in number of squares visited (including revisits) * color_morph: male color morph (red or yellow) #### File: likelihood\_to\_reproduce.csv **Description:** The data set comprises information on whether a breeding pair successfully reproduces. ##### Variables * experimental_block: the experiment was run in 5 experimental blocks. * pair_ID: pair identifier * male_ID: fish identifier * female_ID: fish identifier * male_family: family identifier * female_family: family identifier * male_standard_length: fish size in cm * female_standard_length: fish size in cm * male_prebreeding_boldness_total_number_of_squares_visited_average: male boldness level assessed prior to breeding, measured in number of squares visited (including revisits), averaged over 2 boldness tests * female_prebreeding_boldness_total_number_of_squares_visited_average: female boldness level assessed prior to breeding, measured in number of squares visited (including revisits), averaged over 2 boldness tests * fry: 1 = breeding pair successfully reproduced, 0 = breeding pair did not reproduce * fry_final_number: total number of offspring raised at the end of breeding (after 33 days of care). #### File: parental\_care\_tests.csv **Description:** Results of parental care tests for all successfully reproducing individuals. Each pair was tested six times over 30 days. ##### Variables * experimental_block: the experiment was run in 5 experimental blocks. * pair_ID: pair identifier * male_ID: male identifier * female_ID: female identifier * male_family: family identifier * female_family: family identifier * male_standard_length: fish size in cm * female_standard_length: fish size in cm * test_series: trial number * stimulus_type: intruder type (male or female conspecific, predator) * male_brood_attendance: relative time spent guarding the brood * female_brood_attendance: relative time spent guarding the brood * male_parental_boldness_number_of_squares_visited_total: male parental boldness level assessed during breeding, measured in number of squares visited (including revisits) * female_parental_boldness_number_of_squares_visited_total: female parental boldness level assessed during breeding, measured in number of squares visited (including revisits) * eggs_laid_time_after_pairing_days: time elapsed between pairing and laying the clutch that was raised, NAs for two breeding pairs where eggs where not discovered prior to wrigglers appearing in the tank * eggs_number: clutch size, NAs for two breeding pairs where eggs where not discovered prior to wrigglers appearing in the tank * fry_final_number:  total number of offspring raised at the end of breeding (after 33 days of care). * fry_mean_standard_length: average offspring size in mm * male_prebreeding_boldness_number_of_squares_visited_AVERAGE: male boldness level assessed prior to breeding, measured in number of squares visited (including revisits), averaged over 2 boldness tests * female_prebreeding_boldness_number_of_squares_visited_AVERAGE: female boldness level assessed prior to breeding, measured in number of squares visited (including revisits), averaged over 2 boldness tests * prebreeding_boldness_similarity: male-female behavioral contrast in pre-breeding boldness, measured in number of squares visited (including revisits) * color_morph: male color morph (red or yellow)